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Genomics Revolution

Guest Hosts: Matthew Hecker & Miranda Mordue

Episode 54: MERS Coronavirus- Middle East Respiratory Syndrome-related coronavirus

Hello, and welcome to Genomics Revolution. This is Matthew Hecker, and this is Miranda Mordue, bringing you in from the Hiram College Genetics course of 2020.

As we stand in April 2020, the world is currently in a state of flux with COVID-19, a novel coronavirus that is taking the world by storm, and not in a good way. In light of COVID-19, our topic for today is another variety of novel coronavirus, MERS-Coronavirus. This stands for Middle East Respiratory syndrome coronavirus, but we’ll be calling it MERS from here on out.

MERS was first reported in 2012 in Saudi Arabia in the respiratory tract of a businessman who had died from viral pneumonia. It was the first highly pathogenic coronavirus since the SARS coronavirus in 2003. This virus had a very high mortality rate of greater than 35%.

The MERS genome is single stranded RNA and is made up of about 30,000 bases. Though sometimes it is difficult to think about the scale of genetics, this is a relatively small genome size that contains only 10 Open Reading Frames, or ORFs. Each of these ORFS is a section in which transcription can occur. One of these ORFs encodes for a “polyprotein.” This is a large encoded structure that can be cleaved in order to serve different purposes, basically a conglomeration of different proteins that can be broken up and used as necessary. In the case of MERS, this polyprotein encodes viral replicase and the methods of interaction with ribosomes. Basically, this polyprotein is what allows for the infection and takeover of host cells to replicate itself, while the rest of the ORFs translate to structural and functional proteins. These other proteins are responsible for getting the polyprotein into host cells.

Knowing this, what else can genetics tell us about MERS? Based on genomic comparison, it is very likely that the strain of MERS that eventually came to affect humans began development in the dromedary camel. With MERS, the evidence of human to human transmission is differing based on location the virus was found. In Riyadh, there were cases of human transmission, but in other places, it was not observed. This means that some of the infections of humans beyond the initial case are a result of contact with livestock.

Because of this discreet infection from various sources, it was actually discover that that are many different strains of MERS. In a study from 2013, 21 samples of MERS were examined from different patients, and 10 different genomes were produced for the virus. This means that MERS infections are not limited to human contact and this will obviously affect the manner in which the virus must be addressed.

MERS has also shown some resistance to the innate immune response of the body. The codon selectivity in MERS is variable in three genetic clusters, which have developed a codon bias to survive inside the host. By using different codon sequences, the immune system is not able to recognize the virus as effectively. In different strains, this leads to adaptation to resist the human immune system, making MERS a more dangerous virus.

As of now, there is no specific “cure” for MERS, but levels of infection remain relatively low due to low human to human transmission in strains, as well as potentially lethal symptoms from the onset which cause quarantining very quickly.

I’d hate to end on too negative a note, so let me just say this: Scientists all over the world are very capable at what they do, and they will be able to figure out a way to defeat MERS, as well as the current COVID-19. Just remember to continue listening and learning! Thank you all for listening! This has been Genomics Revolution.

Sources:

1) Cotten et al., 2013. The Lancet Vol 382, pp 1993-2002. Transmission and evolution of the Middle East Respiratory syndrome coronavirus in Saudi Arabia: A descriptive Genomics Study.

2) Alnazawi et al., 2017. Biol. Pharm. Bull. Vol 40 pp 1289-1298. Comparative Genomic Analysis MERS CoV Isolated from Humans and Camels with Special Reference to Virus Encoded Helicase.

3) Shapiro et al., 2016. Disaster and Military Medicine 2:9. Middle EAst Respiratory Syndrome Coronavirus: A Review of the Current Situation in the World.

4) Chafekar, A., & Fielding, B. C., 2018. Viruses vol. 10,2 93. MERS-CoV: Understanding the Latest Human Coronavirus Threat.

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Genomics Revolution

Guest Hosts: Denise Hart & Madyson Morris

Episode 53: SARS Coronavirus

Welcome to Genomics Revolution. This is Denise Hart and Madyson Morris from the 2020 Hiram College Genetics course hosting this episode on the SARS coronavirus. The acronym SARS stands for Severe Acute Respiratory System.1 To distinguish between the virus and the disease it causes, we will call the virus the SARS coronavirus and the disease SARS from here on out. Today, we will discuss the SARS coronavirus genome, as well as the outbreak that occurred across the globe from 2002 to 2003.

Did you know that SARS coronavirus was once the largest RNA virus genome? This genome is also single-stranded and it has 29,751 RNA molecules. The genome has 14 genes and 29 mature proteins. The largest gene found within the SARS coronavirus genome encodes a polyprotein. This polyprotein gets cut into 16 mature proteins. In addition to these mature proteins, the genome also shows that hypothetical proteins, unique to SARS-coronavirus, exist. Researchers used BLAST, along with other function prediction programs, to determine the function of these proteins.3Their research concluded that the genome has carbon-oxygen lyase, oxidoreductases that act on CH-OH groups, an ATP-binding cassette transporter, structural proteins, and a voltage-gated ion channel.3

So, why should we care about SARS coronavirus and the disease it causes? The outbreak started almost 20 years ago!2 We should care about understanding this virus because it causes most patients to develop pneumonia, hence the “Severe Acute Respiratory System” title. Pneumonia can be a very deadly disease if not treated right away. We should also care about educating ourselves on SARS coronavirus because during the outbreak, 8,098 people suffered from SARS as well as pneumonia or respiratory distress syndrome. 774 of those people died.1,2 If contracted, SARS coronavirus can have detrimental side effects, and having a good understanding of this virus could potentially prevent future outbreaks and pandemics caused by it.

SARS coronavirus originally started out in bat species, but is able to be contracted by humans. The symptoms of SARS coronavirus in humans include fever, dry cough, headache, muscle aches, and difficulty breathing. These symptoms are very typical of Coronavirus- and true for most of the 36 types of coronaviruses.6 While it is good to be aware of these symptoms, 1 symptom really sets SARS coronavirus apart from other strains: urinary abnormalities. For the first patients with SARS, they were diagnosed with other ailments due to these unique symptoms. It wasn’t until the genome was sequenced that researchers learned that SARS not only turns cells along the respiratory tract into host cells, but also cells in the intestines, liver, heart, vascular endothelium, testis, and the kidneys!6

Not only did some of the patients with SARS get treated for other ailments due to having urinary abnormalities, some of the patients with SARS received treatment but nothing happened!6,8 These were patients with the common symptoms. They received treatment but they did not get better. This was because the SARS coronavirus had mutated 14 times.6,7 Some patients had the original strain and others had one of the mutated strains. The treatment of the original strain did not help patients with a mutant strain. Researchers used high density sequencing arrays to find the places in the SARS coronavirus genome that had mutated.5,6 The scientists found that in mutant strains, 5-6 nucleotides were inserted or deleted. 5,6 This small change caused the virus to be harder to treat because of the variations between each mutation.

As these mutations were being discovered, the need to understand this virus was vital because of the global outbreak. Although there were not enough cases to consider it a pandemic, a virus causing a global outbreak still needs to be understood to prevent the outbreak from turning into a pandemic! In order for a disease to be considered a pandemic, it must affect several countries and a very large amount of people. The CDC does not define how many countries or people, but a pandemic does affect more people than an outbreak.4

It’s about time to wrap up. Today we learned about the SARS coronavirus and the global outbreak of SARS from 2002 to 2003. What we want you to take away from this episode of Genomics Revolution are four things:

Number 1: SARS coronavirus was one of the largest RNA virus genomes at the time. The size of the genome increased the amount of time needed for geneticists to understand the virus to create presentation education and control measures for the public.

Number 2: Whenever the SARS coronavirus genome experiences a mutation, the mutation is an insertion or deletion of 5 or 6 nucleotides.

Number 3: While SARS is an acronym for Severe Acute Respiratory System, the virus targets not just the respiratory tract, but also other organs such as the kidney and heart.

Number 4: Understanding viruses like this one is important to the future of science so that when a new strain of virus arises, we may be able to fight it quicker than the last one!

Thanks for listening to Genomics Revolution. Bye guys! Bye!

References:

1. SARS. Centers for Disease Control and Prevention. 2017 Dec 6 [accessed 2020 Apr 9]. https://www.cdc.gov/sars/about/fs-sars.html

2. SARS (Severe Acute Respiratory Syndrome). World Health Organization. 2012 Apr 26 [accessed 2020 Apr 9]. https://www.who.int/ith/diseases/sars/en/

3. Cai CZ, Han LY, Chen X, Cao ZW, Chen YZ. Prediction of Functional Class of the SARS Coronavirus Proteins by a Statistical Learning Method. Journal of Proteome. 2005 Aug 10 [accessed 2020 Mar 28]. https://doi.org/10.1021/pr050110a

4. Caceres V. What's the Difference Between an Epidemic and Pandemic? U.S. News & World Report. [accessed 2020 Apr 9]. https://health.usnews.com/conditions/articles/whats-the-difference-between-an-epidemic-and-pandemic

5. Wong CW. Tracking the Evolution of the SARS Coronavirus Using High-Throughput, High-Density Resequencing Arrays. Genome Research. 2004;14(3):398–405. doi:10.1101/gr.2141004

6. Cheng VC, Lau SK, Woo PC, Yuen KY. Severe Acute Respiratory Syndrome Coronavirus as an Agent of Emerging and Reemerging Infection. American society for microbiology. 2007;20(4):660–694.

7. Graham RL, Sparks Jifr S, Eckerie LD, Sims AC, Denison MR. SARS Coronavirus replicas proteins in pathogenesis. Virus Res. 2008;133(1):88–100.

8. Hung LS. The SARS epidemic in Hong Kong: what lessons have we learned? Jrsm. 2003;96(8):374–378. doi:10.1258/jrsm.96.8.374

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Genomics Revolution

Guest Hosts: Keegan Rankin and Torey Coward

Episode 52: Zika

Script:

Keegan: Hello welcome to the podcast! I'm Keegan!

Torey: And I’m Torey Coward!

Keegan: And we are here today to talk to you about the Zika Virus. I’ll start us off with some general information. The Zika Virus belongs to a group of viruses known as flaviviruses. Flaviviruses are single-stranded RNA viruses encapsulated by a protein coat. Some of Zika Virus’ closest relatives include Yellow Fever Virus and West Nile Virus. They replicate in the cytoplasm of host cells. All zoonotic flaviviruses are rely on arthropods as vectors. In the case of Zika, its two primary vectors are Aedes aegypti (Yellow Fever Mosquito) and Aedes albopictus (Asian Tiger Mosquito). The reservoir of the virus are primates, including humans. This virus is usually spread to a host through the bite of a mosquito, but it can also be transmitted by coming into contact with infected blood and saliva. Common symptoms include fever, headache, rash, joint and muscle pain and conjunctivitis. Symptoms usually last for several days to a week.

Torey: I’ll speak briefly about the genome of the Zika Virus. As Keegan stated before, the Zika Virus is a single-stranded RNA, and since it has been sequenced, we know today that it consists of nearly 10.8 thousand bases. There are 3424 amino acids that generate the polyprotein that the virus encodes for. The polyprotein is made up of 10 proteins, one capsid, a precursor membrane protein, an envelope protein, and 7 non-structural proteins.

Keegan: The Zika Virus was first isolated from a Macaque in 1947 obtained from the forest of Uganda. Though 80% of those infected are asymptomatic and 20% of patients contract mild, non-lethal symptoms, the real danger arises when the infected patient is pregnant. Getting Zika Virus while pregnant puts the child at risk of lethal birth defects such as microcephaly. Microcephaly is a genetic defect that causes an infant’s brain and head to be smaller than normal, healthy infants. This can result in seizures, intellectual impairment, hearing loss, visual problems, and even infant mortality. Prevention of the spread and contraction of Zika Virus is imperative to prevent infant mortality.

Studying Zika Virus and other flaviviruses on a genetic level has given use crucial revelations as to how Zika works and can spread. One such revelation is that many of its genes can be successfully targeted, which brings about the possibility of new treatments and vaccines. Another revelation is that certain mRNAs coding for viral replication have been isolated and can be targeted, yielding the possibility of developing treatments that suppress the virus further, stopping its growth. Another discovery in researching Zika virus is that it also can use other organisms as reservoirs. Research suggests that Zika Virus can possibly be transmitted by birds, horses, goats, cattle, and bats.

Torey: Hopefully the information that we have provided for you today had been insightful and helped to foster a greater understanding of the Zika Virus. From the genus and sequence, to the pathway and effects of this intriguing virus.

Keegan: And that concludes our talk for today! Thank you so much for listening to us and stay safe out there!

Works Cited:

Facts about Microcephaly. Centers for Disease Control and Prevention. 2020 Feb 18 [accessed 2020 Apr 9].

Flavivirus. Flavivirus - an overview | ScienceDirect Topics. [accessed 2020 Apr 9].

Malone RW, Homan J, Callahan MV, Glasspool-Malone J, Damodaran L, Schneider ADB, Zimler R, Talton J, Cobb RR, Ruzic I, et al. Zika Virus: Medical Countermeasure Development Challenges. PLoS neglected tropical diseases. 2016 Mar 2 [accessed 2020 Apr 9].

Zika Virus. Centers for Disease Control and Prevention. 2019 Nov 20 [accessed 2020 Apr 9].

Molecular cloning and characterization of the genes encoding the proteins of Zika virus. NCBI-PubMed. [Accessed 2020 April 9]

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Genomic Revolution

Guest Hosts: Alexus Acton & Rachna Prasad

Episode 51: Ebola

Script:

Rachna: Welcome to Genomics Revolution. This is Alexus Acton and Rachna Prasad from the 2020 Hiram College Genetics course hosting this episode on the Zaire Ebolavirus. This virus causes the disease ebola that originated from human animal contact, most likely from a bat (1). The Ebola Virus Disease, or EVD for short, was first discovered in 1976 with 2 consecutive outbreaks of fatal hemorrhagic fever in Central Africa. The first outbreak was in the Democratic Republic of Congo, which was formerly called Zaire, in à village near the Ebola river, which accounted for nearly 2700 deaths. The second outbreak was in South Sudan. Originally scientists believed it was spread by à single infected person travelling between the two areas, but it was later discovered they were two genetically distinct viruses - the Zaire ebolavirus and the Sudan ebolavirus.(4) The most recent, and familiar Ebola outbreak occurred in 2014-2016, originating in Southeastern Guinea. It rapidly spread to urban populations within weeks, and soon turned into à global epidemic. (4)

Lexi: With this virus going many months without detection we should care about knowing about this virus because the human-human transmission chain was growing exponentially. Before the World health Organization declared it an outbreak, it had already spread over country borders infecting thousands of people (1). Ebola virus is a negative- sense single strand RNA ((-)ssRNA) that has a 19 kilobase genome (1). There are several encoded proteins from EVD which are nucleoprotein (NP), viral proteins (VP) as well as RNA polymerase (L), and Glycoprotein (GP) (2). VP24 is a membrane associated protein, VP30 and VP35 are polymerase matrix proteins, and VP40 is a matrix protein (2). VP40 is the primary EVD matrix protein and regulated assembly and progress of infectious particles (2). It assembles on the inner leaflet of the plasma membrane in human cells to regulate viral budding. Each of these genes encodes for a single protein product with the exception of GP. Gp encodes three proteins of different sizes with a full length of 676 residues. Glycoprotein 1 and 2, mediates viral-host cell attachment and fusion (2). Like other RNA viruses ebola quickly generates mutations through error prone replication. The various glycoproteins are produced from frameshift as a result of mRNA editing (1).

Rachna: Ebolavirus belongs to à group of viruses called filoviruses. A phylogenetic analysis revealed that the Sudan Ebola virus diverged early from the other strains, showing that the Bundibugyo and Tai Forest were closely related to Zaire Ebola Virus (2). EBOV is à single stranded RNA virus. This gives it a higher likelihood of acquiring meaningful genetic adaptations and evolving into different strains, when compared to other DNA viruses. (5) À 2017 study, by Tao Li et al., conducted on 514 different EBOV genome sequences from patients with confirmed EVD cases showed that 11 different lineages of EBOV arose from one outbreak in Sierra Leone alone. It also showed that different lineages of EBOV had different fatality rates and certain strains with specific SNPs correlated with higher fatality rates. Variation in nucleotide sequences can help target each Ebolavirus strain from one another, ultimately leading to better diagnosis and therapeutics. This is important to note as the divergence of diseases from other common viruses could potentially be headway in targeting vaccines and treatments to those infected.

Lexi: Evidence demonstrates cooperative dimeric binding of double stranded RNA by ebolavirus VP35. The C-terminal domain of viral protein 35 dimerizes upon binding to double stranded RNA, showing coppertivity (3). Reston ebolavirus is named to show where it was derived from, which has previously been shown to be critical for RNA binding, but is also important for VP35 dimeric interface binding (3). These researches mutated R312/301 which likely abolished dsRNA binding which disrupted the formation of the viral protein dimer leaving it in unstable formation (3). This is important as the binding mechanism permits the ebola virus from avoiding the innate immune response and enhancing harmfulness to human cells. (3). This alone can help researchers target the structure based conformational states or protein targeting drugs for drug development and biodefense mechanisms.

Rachna: One study showed that multi sequence alignment generated several conserved sequences from each protein mentioned above. Using an Ebola strain from 1976 and a recent strain of 2014, the two sequences were 100% identical (6). The conservation allowed for detection of B and T cell epitopes which covered between 25.37 and 61.51% of the population (6). B cell epitope is the portion of the antigen which interacts with B lymphocytes to trigger immune responses (6). The importance of this is to understand the efficacy in eliciting immunity through humoral and cell-mediated immune responses. T cell immune response usually promises long lasting immunity, and here the prediction of T and B cell epitopes provides two alternative but effective immune response mechanisms.

Lexi: Thank you for tuning in on this podcast of Genomics Revolution. We hope you enjoyed your time and learned something new about Ebola.

References:

1. Holmes, Edward C., et al. “The Evolution of Ebola Virus: Insights from the 2013–2016 Epidemic.” Nature, vol. 538, no. 7624, 13 Oct. 2016, pp. 193–200., doi:10.1038/nature19790.

2. Jun, Se-Ran et al. “Ebolavirus comparative genomics.” FEMS microbiology reviews vol. 39,5 (2015): 764-78. doi:10.1093/femsre/fuv031

3. Kimberlin, C. R., et al. “Ebolavirus VP35 Uses a Bimodal Strategy to Bind DsRNA for Innate Immune Suppression.” Proceedings of the National Academy of Sciences, vol. 107, no. 1, 14 Sept. 2009, pp. 314–319., doi:10.1073/pnas.0910547107.

4. Li T, Yao HW, Liu D, et al. Mapping the clinical outcomes and genetic evolution of Ebola virus in Sierra Leone. JCI Insight. 2017;2(15):e88333. Published 2017 Aug 3. doi:10.1172/jci.insight.88333

5. Regnery, RL., Johnson, KM., and Kiley, MP. Virion nucleic acid of Ebola virus. J. Virol. 1980; 36(2): 465-469.

6. Yasmin, T., and A. H. M. Nurun Nabi. “B And T Cell Epitope-Based Peptides Predicted from Evolutionarily Conserved and Whole Protein Sequences of Ebola Virus as Vaccine Targets.” Scandinavian Journal of Immunology, vol. 83, no. 5, 2016, pp. 321–337., doi:10.1111/sji.12425.

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Genomics Revolution

Guest Hosts: Alysa Giudici & Rachel Jerkins

Episode 50: West Nile Fever

Script:

Rachel: Hey everybody, and welcome to another episode of Genomics Revolution. This is Rachel Jerkins and Alysa Giudici (Guh-Dee-Cee), here to talk about the West Nile Virus.

Rachel: The West Nile Virus comes from the flavivirus genus and the family flaviviridae. In 1937, the virus was first discovered in the West Nile area of Uganda in Africa. It is a single-stranded RNA virus around 11kbp in size with stem loops on the 5’ and 3’ ends. The genome codes for 10 proteins— 3 for structure in the coding region, plus seven not in the new virus structure from the non-coding region.

Rachel: The West Nile Virus causes a disease called West Nile fever (Richter et al. 2017). It is believed to spread when a mosquito bites an infected bird and then bites a person. It wasn’t until 1999 that the virus made its first appearance in the western hemisphere (White et al., 2001). It is crucial to study the disease because it can be a fatal neurological disease and has now spread across a majority of the globe.It is believed to be the main cause of viral encephalitis around the world (Chancey et al. 2015)

Alysa: Thanks Rachel, Since the sequencing of the genome, there are many key findings that have emerged. The virus thrives utilizing a vector-virus relationship. The entry of the WNV is through receptor mediated endocytosis once the virus attaches to the cell surface (Colpitts et al.) Interestingly, the virus was able to be tracked through an enzootic cycle involving Culicidae mosquitoes and birds. The birds act as a form of host reservoirs allowing the virus is amplified through the bird – mosquito – bird cycle, until the fall when female mosquitoes begin to “bite” humans. Although many external factors can contribute to the amplification cycle, the disease does exist in multiple habitats (Peterson, 2002). This form of transmission causes the virus to transmit quickly and effectively.

Alysa: The apparent symptoms appear to be anorexia, nausea, vomiting, eye pain, headache, etc. that last roughly 3-6 days (Peterson, 2002). These symptoms eventually, if untreated became neurological and possibly deadly. After research, it was determined that there are, however, two lineages of the West Nile Virus. The 1st lineage is the one that is known to affect humans. Not only is the West Nile Virus detrimental to humans, but it is also a leading neurologic disease in many animals such as the equine population. Further sequencing of this genome and Reverse transcription-PCR has further educated the veterinary world as well. Recent evidence acquired by Venter et al. in horses suggests that the lineage 2 strains are highly neuroinvasive in humans and mice. A disease that we continue to fight in humans is also a disease we will continue to fight in animals as well. Who would have guessed that? Thanks for listening.

Works Cited:

Richter, J., C. Tryfonos, A. Tourvas, D. Floridou, N. Paphitou, and C. Christidoulou (2017).

Complete genome sequence of West Nile virus (WNV) from the first human case of neuroinvasive WNV infection in Cyprus. Amer. Soc. for Microbio. 5(43) 1-2. Doi: 10.1128/genomeA.01110-17

Chancey, C., A. Grinev, E. Volkova, and M. Rios. (2015). The global ecology and epidemiology of West Nile virus. BioMed Res. Int. 376230. Doi: 10.1155/2015/376230

White, D. J., Kramer, L. D., Backenson, P. B., Lukacik, G., Johnson, G., Oliver, J., … Campbell, S. (2001). Mosquito Surveillance and Polymerase Chain Reaction Detection of West Nile Virus, New York State. Emerging Infectious Diseases, 7(4), 643–649. doi: 10.3201/eid0704.017407

Petersen, L. R., & Marfin, A. A. (2002). West Nile Virus: A Primer for the Clinician. Annals of Internal Medicine, 137(3), 173. doi: 10.7326/0003-4819-137-3-200208060-00009

Colpitts, T. M., Conway, M. J., Montgomery, R. R., & Fikrig, E. (2012). West Nile Virus: Biology, Transmission, and Human Infection. Clinical Microbiology Reviews, 25(4), 635–648. doi: 10.1128/cmr.00045-12

Venter, M., Human, S., Zaayman, D., Gerdes, G. H., Williams, J., Steyl, J., Leman, P. A., Paweska, J. T., Setzkorn, H., Rous, G., Murray, S., Parker, R., Donnellan, C., & Swanepoel, R. (2009). Lineage 2 west nile virus as cause of fatal neurologic disease in horses, South Africa. Emerging infectious diseases, 15(6), 877–884. https://doi.org/10.3201/eid1506.081515

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Genomics Revolution

Guest Hosts: Giselle Bahena & Diamond Johnson

Episode 49 – Rabies Lyssavirus

Script:

Welcome to Genomics Revolution. This is Giselle Bahena and Diamond Johnson from the 2020 Hiram College Genetics course hosting this episode covering the Rabies lyssavirus. As the scientific name of this virus implies, the disease that results from such infection is commonly known as Rabies. This disease has been around since antiquity and the earliest writings about it was found in 300BC in Mesopotamia.2 It was discovered through the infectious bite from one animal to the other. The biggest red flag that indicated a rabid animal was excessive salivation which then required preventative actions to take place in order to protect against the virus being transmitted elsewhere. It is important to understand the virus as transmission does not only occur between one animal and the other, rather humans are also at risk from such infectious bites as well. Thousands of people in third would countries continue to die of Rabies and if one is not educated nor treated for the disease, its impact on the central nervous system will take place and result in death.

The Rabies virus genome is a single stranded, antisense, non-segmented, negative stranded RNA of approximately 12kb.1. There is a 50 nucleotide leader sequence that is followed by the the five genes in the genome. The proteins encoded by these five genes are nucleoprotein(N), phosphoprotein(P), matrix protein(M), glycoprotein(G) and polymerase(L), all of which make up the structure of the bullet-shaped virion.1 Fusion of the rabies virus envelope to the host cell membrane initiates the infection process and from this point the bullet-shaped virion, with 10nm spike-like glycoprotein peplomers covering its surface, penetrates and enters the host cell cytoplasm via pinocytosis.1 Next, the viral RNA is uncoated and the transcription process of producing messengers RNAs(mRNAs) begins. Since the lyssavirus is a negative single stranded RNA genome, these mRNAs must be transcribed as they are needed to permit virus replication later on in its cycle of infection and replication.1 Now, the synthesized mRNAs are translated into the genomes structural proteins. As G protein glycosylation is processing, the first step in viral replication occurs by synthesizing full length positively stranded copies of the genome that serve as templates for the final synthesis of the negatively stranded genome.1 When this switch to replication occurs, RNA transcription then becomes non-stop as stop codons are ignored. Finally, the assembly process of the bullet-shaped virion takes place and proceeds to its budding formation.

There has been an unrecognized member of the lyssavirus genus found in bats that is similar to the one found in dogs, both of which have been seen to be transmitted in humans.3 This is important because with the genome sequence of the lyssavirus, another member in the family was able to be identified along with other animals who would not be typically associated with caring the such virus. On a similar note, sixty nine rabies virus isolates from various parts of the world were partially sequenced and compared to thirteen representative isolates of the six lyssavirus genotypes in order to analyze their genetic diversity.4 The analysis was performed on each of their complete nucleoprotein coding gene and it was discovered that all of the rabies virus isolated belonged to genotype 1, most likely diverging by the accumulation of synonymous mutations.4 With this being said, having the knowledge that the nucleoprotein is highly conserved among all the isolates is important as it can be used as a potential target for preventing the viral Rabies infection. For example, a complementary RNA can can be created to hybridize with the nucleoprotein mRNA and prevent its translation by targeting it for degradation. This would ultimately prevent one of the key structural proteins of the bullet-shaped virion to be made and prevent the virus from being transmitted. It has also been discovered that the human monoclonal antibody (HuMAbs) may serve as an alternative treatment against less affordable treatments. HuMAbs was found to be the best monoclonal antibody as it neutralized all the rabies viruses it was tested against, it recognized both minor site A and antigenic site III and was able to protect hamsters from the most lethal dose of the virus.5 This is another important finding as even if an individual is infected, HuMAbs can be applied for post-exposure protection against the viral genome.

Thank you for listening to this episode of Genomics Revolution, we hope you enjoyed your time and were able to learn something new from this talk.

References:

1. What is Rabies? Centers for Disease Control and Prevention. 2019 Jun 11 https://www.cdc.gov/rabies/about.html

2. A brief history of rabies: Microbiology. 2017 Apr 12 https://www.labroots.com/trending/microbiology/5761/brief-history-rabies

3. A brief history of rabies: Microbiology. 2017 Apr 12 https://www.labroots.com/trending/microbiology/5761/brief-history-rabies

4. Kissi B, Tordo N, Bourhy H. Genetic Polymorphism in the Rabies Virus Nucleoprotein Gene. Virology. 1995;209(2):526–537.

5. Sloan SE, Hanlon C, Weldon W, Niezgoda M, Blanton J, Self J, Rowley KJ, Mandell RB, Babcock GJ, Thomas WD, et al. Identification and characterization of a human monoclonal antibody that potently neutralizes a broad panel of rabies virus isolates. Vaccine. 2007;25(15):2800–2810.

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Brad sets the table for the last set of 2020 Hiram College Genetics course guest podcasts.

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Some More Information on How HIV Causes AIDS & on New Drugs:

https://m.youtube.com/watch?v=BADDj82oces (Battle between HIV & Immune System video from Nature Reviews)

https://www.npr.org/sections/health-shots/2019/05/30/727731380/old-fight-new-front-aids-activists-want-lower-drug-prices-now (National Public Radio (NPR) segment “AIDS Activists Take Aim at Gilead to Lower Price of HIV Drug PrEP”

https://www.npr.org/sections/health-shots/2019/06/11/731350223/expert-panel-recommends-wider-use-of-daily-pill-to-prevent-hiv (NPR segment “Expert Panel Recommends Wider Use of Daily Pill to Prevent HIV”

https://www.npr.org/sections/health-shots/2019/12/04/784733337/hiv-prevention-drugs-are-available-for-free-how-do-you-get-them (NPR segment “HIV Prevention Drugs are Available for Free: How Do You Get Them”

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Genomics Revolution

Guest Hosts: Emily Harris & Tim Murton

Episode 48: HIV & AIDS

Script:

Welcome to Genomics Revolution! This is Emily Harris and this is Tim Murton. We are from the 2020 Hiram College Genetics course, and we are hosting this episode on the genome of Human Immunodeficiency Virus, or HIV. HIV targets a host’s immune system and causes it to fail. This complication is referred to as HIV infection, and can eventually develop into acquired immunodeficiency syndrome, or AIDS, which is the most advanced stage of HIV infection (1).

This virus is in the genus lentivirus, the family of Retrovirdae, and the subfamily Orthoretrovirinae (1). HIV is typically divided into two types, HIV-1 and HIV-2, and each type can be subdivided into several smaller groups based on differences in viral antigens, and from where each strain evolved (3). HIV was recognized on a wide scale during an outbreak in the 1980s, but was actually first discovered in humans between 1920 and 1940 (1). After years of studying the virus, it was discovered that it is spread through contact with infected bodily fluids like blood, semen, breast milk, or several others. It was also discovered that the virus was very similar, genetically, to simian immunodeficiency-deficiency virus, or SIV, which is a non-human primate immunodeficiency virus (1). HIV-1 appears to have evolved from SIV strains in chimpanzees in Central Africa, and HIV-2 likely evolved from a strain in West African mangabeys (1). So it is believed that the virus was transmitted to humans when these primates were hunted for meat and their infected blood was ingested. The virus then mutated and evolved in humans into HIV (3).

Now let’s talk about the HIV genome. The genome of this virus consists of two single stranded RNA molecules and is roughly 9,200 bases in size (2). After sequencing the genome, it was found that it contains 9 genes and encodes 15 viral proteins, which is relatively small when we consider how powerful of a virus it is (3). HIV is also classified as an enveloped retrovirus (1). This means the virus uses a special enzyme called reverse transcriptase, which turns its RNA into DNA, then uses that DNA to infect a host (1). They literally insert a copy of their own genome into a host’s genome!

So this virus works in a very intelligent way making treatment for infection extremely difficult, especially before the virus was understood. This is why it was so important to sequence the HIV genome. By learning more about the genetic makeup of HIV, it became easier to understand how the virus operates, how it evolves and what it evolved from, how to prevent possible outbreaks, and what types of treatment may work. Sequencing the genome even opened the door to possible gene therapy that can be used to treat or hopefully even cure the disease someday!

Sequencing the HIV genome told us a lot about the virus, so let’s highlight a few of the key findings. Sequencing the genome is how we found out that HIV is closely related to SIV (5). This information was crucial because then we were able to use our understanding of SIV to help come up with a better treatment for HIV.

Another finding was that there are subtypes of HIV-1 such as the CG-0018a-01 HIV-1 genome (7). This subtype-L was found in the Democratic Republic of the Congo. The research showed that this subtype-L was found to be transmitting in the DRC and that there could be more strains circulating (7). Knowing this was extremely important because it shows the dangers of mutations, and how easily the virus can evolve and create new strains. This let scientists know to look out for new strains of HIV that could be more easily transmitted and harder to combat than the original strain.

Sequencing the HIV genome also showed us that HIV-1 genetic material is damaged by hypermutation (6). G-to-A hypermutation, for example, damages the virus by producing abnormal amounts of transitions from guanine to adenine. These mutations are thought to be caused by HIV’s reverse transcriptase enzyme, which has the ability to hypermutate in the presence of unbalanced nucleotide pools during the cell cycle (6). This is important because it shows that the virus has a weakness that is possibly being caused by a host mechanism that can decrease virus replication. This finding implies that if we can promote hypermutation states in HIV, we may be able to induce non-reversible mutagenesis of the viral DNA. This strategy may pave the way to discovering a cure for HIV! Thanks for listening!

References:

[1] Arbeitskreis Blut, Untergruppe ‘Bewertung Blut- assoziierter Krankheitserreger’: Human

immuno- deficiency virus (HIV). Transfus Med Hemother 2004;31:102–114.

[2] Feinberg Mark B, Greene Warner C (1992). "Molecular Insights into human immunodeficiency virus type1 pathogenesis". Current Opinion in Immunology. 4 (4): 466–474. doi:10.1016/s0952-7915(06)80041-5. PMID 1356348.

[3] Li G, Piampongsant S, Faria NR, Voet A, Pineda-Peña AC, Khouri R, Lemey P, Vandamme AM,

Theys K (February 2015). "An integrated map of HIV genome-wide variation from a population

perspective". Retrovirology. 12 (1): 18. doi:10.1186/s12977-015-0148-6. PMC 4358901. PMID 25808207.

[4] German Advisory Committee Blood (Arbeitskreis Blut), Subgroup ‘Assessment of Pathogens Transmissible by Blood’ (2016). Human Immunodeficiency Virus (HIV). Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie, 43(3), 203–222.

[5] Janini, M., Rogers, M., Birx, D. R., & McCutchan, F. E. (2001). Human immunodeficiency virus type 1 DNA sequences genetically damaged by hypermutation are often abundant in patient peripheral blood mononuclear cells and may be generated during near-simultaneous infection and activation of CD4(+) T cells. Journal of virology, 75(17), 7973–7986.

[6] Williams, K. C., & Burdo, T. H. (2009). HIV and SIV infection: the role of cellular restriction and immune responses in viral replication and pathogenesis. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, 117(5-6), 400–412.

[7] Yamaguchi, Julie BS; Vallari, Ana MS; McArthur, Carole MD, PhD; Sthreshley, Larry PhD; Cloherty, Gavin A. PhD; Berg, Michael G. PhD; Rodgers, Mary A. PhD. (2020) Complete Genome Sequence of CG-0018a-01 Establishes HIV-1 Subtype L. JAIDS Journal of Acquired Immune Deficiency Syndromes: Volume 83 - Issue 3 - p 319-322

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Genomics Revolution

Guest Hosts: Sheree Nobles & Joshua Gregory

Episode 47: Human Papillomavirus (HPV)

Script:

Josh- Hello everyone, and welcome to this episode of Genomics Revolution! We’re your guests hosts

today, Sheree Nobles and Joshua Gregory. Today we’ll be talking about Human Papillomavirus,

or HPV as it’s commonly known. This is a sexually transmitted infection, and to keep on topic with this year’s theme, it’s a virus.

Sheree- There isn’t a scientific name for it, so much as a bunch of scientific names, as there are over 100

different human papillomaviruses. These viruses can cause not only genital warts, but warts

elsewhere, and have even been linked to cervical cancers.

Josh- Five types of HPV were found prior to 1983, but most of those HPVs were present in animals, not

humans. HPV 6 was found by German virologist Harold zur Hausen shortly before HPV 11 was

found by the same man in 1983. Shortly after that, he found HPVs 16 and 18, which together are

present in roughly 70% of cervical cancers.

Sheree- zur Hausen was originally studying cervical cancer, and the only reason he thought to look for

viruses was because the cancer seemed to be “infectious” despite cancer not behaving the way

the should . It was when his friend, a U.S. researcher named Richard Shope, told him about a

virus in rabbits that would cause warts and cervical cancer did he think about the possibility of a

virus causing the disease.

Josh- It’s important to note that most HPVs don’t cause cancer, or any symptoms at all. More often

enough, HPV is present in a human, but does not cause any symptoms before the immune

system removes it. It’s only a few types of the virus that can stay around long enough to induce

cancer, like HPV 16 or 18. This is because the virus affects a cell’s growth cycle while trying to

reproduce, causing the cells to grow rapidly and for warts to form, and sometimes causing

tumors as well.

Sheree- This is why it’s important to understand the virus. Cervical cancer only has a 66% survival rate,

so if it can be avoided, it should be. Because HPV is so closely related to the cancer, it makes

sense that we should try to understand it completely to try and stay safe from deadly diseases.

It’s also important to realize that someone can be carrying the virus without showing any

symptoms, and could possibly transfer it to another person, spreading the virus and its

dangerous symptoms.

Josh- Not only should we understand the virus, but everyone should also get tested regularly to make

sure whether or not they’re carrying HPV. Now the virus itself isn’t too complicated. Its genome is made of double-stranded DNA in a circular formation, with a singular DNA molecule. It is roughly 7916 base pairs in length with 8 ORFs, though this data is subject to slight change between different types of HPV.

Sheree- There were three findings that we thought were really important. One, HPVs are separated into

two unofficial classifications: low and high risk. Most human papillomaviruses never cause any

symptoms, let alone serious diseases, but even still, they can be transferred between people. If

someone has a compromised immune system, an HPV that doesn’t hurt one person could hurt

them. Two, certain HPVs are much more likely to cause cervical cancer.

Josh- HPVs 16 and 18 are some of the more dangerous types of the virus you can get. Together they are

present in approximately 70% of all cervical cancer cases. This is due to their ability to target

retinoblastoma (Rb) protein families and p53 in our cells. This can induce telomerase

production, causing the cell to be unable to repair damaged DNA, a crucial task for cancer cells

to continue to survive. And last but not least, HPV is total is present in around 99.7% of cervical

cancer samples, as found in 1999 by a group of scientists including U.K. Researcher Professor

Julian Peto.

Sheree- These findings are important for several reasons. They tell us that anyone could be carrying HPV

at any time and not know it. This is a common trend among some viruses, much like the corona

virus that is currently causing an uproar. They also tell us that cervical cancer, which only has a 66% survival rate, is so closely linked to HPV that we should take every care to avoid contracting HPV. Lastly, knowing the mechanism of HPV causing cancer brings us a step closer towards finding an actual cure for existing HPV infection, not just prevention, a vaccine, or surgery.

Josh- This has been an episode of Genomics Revolution, and we thank you all for listening to us ramble

about a virus.

Sheree- We’ve had some fun recording this for you, and we hope you’ll take away from this the

importance of understanding viruses and the diseases they can cause.

Josh- This has been Josh,

Sheree- and Sheree,

Josh- and we hope you have a great day, take care!

References:

IARC Working Group on the Evaluation of Carcinogenic Risk to Humans. “Human Papillomavirus (HPV)

Infection.” Human Papillomaviruses., U.S. National Library of Medicine, 1 Jan. 1970, www.ncbi.nlm.nih.gov/books/NBK321770/.

“HPV: the Whole Story, Warts and All.” Cancer Research UK - Science Blog,

scienceblog.cancerresearchuk.org/2014/09/16/hpv-the-whole-story-warts-and-all/.

Burk, Robert D., et al. “Human Papillomavirus Genome Variants.” Virology, Academic Press, 31 Aug.

2013, www.sciencedirect.com/science/article/pii/S0042682213004388.

Liu, Ying, et al. “Whole-Genome Analysis of Human Papillomavirus Types 16, 18, and 58 Isolated from

Cervical Precancer and Cancer Samples in Chinese Women.” Scientific Reports, Nature Publishing Group UK, 21 Mar. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5428204/#!po=25.0000.

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Genomics Revolution

Guest Hosts: Abbey Anderson & Samantha Mansfield

Episode 46: Hepacivirus (Hepatitis C)

Script:

Hello! This is Sammie and Abbey coming at you live from a safe social distance! Today we are here to talk to you about Hepacivirus which is commonly known as Hepatitis C virus. We are going to spend some time talking about when and how the virus was discovered, the reasons that we should care about this virus, the genome of Hepatitis C, and key findings about HepC virus.

Sammie: Every story starts somewhere and the uniqueness of this virus is how it was discovered. This virus was first named in 1989. Doctors knew that this might have had something to do with blood transfusions, but it was called non-A, non-B hepatitis because they could not pinpoint the exact virus down. Later it became known that there were several different genotypes of the hepatitis C virus. This virus is widespread and curable now a days if caught early enough. The spread of this virus occurs by sexual intercourse, blood to blood contact, or improperly sterilized equipment (2). While we now understand how to take precautions against HepC, many people today are still infected by the virus through needle sharing. With the present opioid epidemic, especially in our great state of Ohio, we must understand the vast impacts that drug sharing can cause. This leads into why we should care about the virus that turns into a serious disease.

Abbey: According to the CDC, there are 2.4 million people in the United States alone living with hepatitis C. Worldwide, HepC affects more than 170 million people. This virus is often ignored until it is too late. There are two phases of hepatitis C which occurs when the person is infected but shows no symptoms. Even if there are symptoms, most people never realize until they are diagnosed with liver disease. According to the San Francisco Department of Health, in 15-40% of the persons with acute hepatitis C, the immune system will fight off the infection and there will be no presence of the virus within 6 months and the liver heals completely. In most other people, the immune system cannot clear the virus and after 6 month time window this is then classified as chronic hepatitis C. This disease will cause the liver to become more and more inflamed and scarred over several years. According an article written by the University of Texas Hepatitis C is the leading cause of liver cancer (5).

Sammie: According to a microbiology textbook edited by Dr. Tan Seng-Lair, The hepatitis C virus belongs to the family Flaviviridae. This family genome consists of a positive strand RNA molecule that ranges in size from 9.6 to 12.3 thousand nucleotides.The Hepatitis C virus open reading frame is known to contain 9,024-9,111 nucleotides, depending on the genotype. It is a double stranded RNA molecule. Hepatitis C virus encodes a single polyprotein, and this is processed to generate 10 polypeptides (1). The number of predicted coding proteins is 11.

Abbey: Now it is time to talk about why sequencing this viral genome helped our understanding of Hepatitis C. Remember those genotypes mentioned earlier? Yep, they really come into play here. From sequencing the HepC virus, scientists have discovered that this virus exists in 8 different genotypes, all with different geographical distributions according to a study that was looking at where the highest burden of HepC occurs in Southeast Asia (3). The analysis of Hepatitis C Virus nucleotide sequences has worked as an epidemiological marker, allowing scientists to trace the source of HepC infection within a given population (6). Until 2019, it was believed that HepC only existed in 6 genotypes with more than 50 subtypes. The best part is, not all of these 8 genotypes have been sequenced! In case you are interested in classifying the next Hepatitis C type, the complete genome sequence must differ from other sequences by 30% in genotype or by 15% in subtype (4). From sequencing these genomes, researchers have also been able to determine which genotypes and subtypes are more prevalent in specific regions around the world. They often follow patterns of human migration. For example, subtypes 1a, 1b, 2a, 2b, 2c, and 3a are found worldwide and make up the largest portion of HepC infections (4). Other subtypes, recall that there are over 50, are extremely rare and are found in restricted geographical locations such as West Africa, Central Africa, India, and even Canada.

Sammie: Finally, we should mention the most important use of sequencing the HepC genome...HepC treatments. According to the National Institute of Health, by having a sequenced genome, scientists began to target different genes in order to develop drugs that would combat Hepatitis C. Some drugs target the gene responsible for the activity of HepC polymerase, the enzyme that aids in replication of the virus. Other drugs target genes that encode the virus’s structural proteins.

Abbey: While it may seem that we know a lot about Hepatitis C virus today, we are still a long ways away from a vaccine. Because this virus mutates so rapidly and exists in so many genotypes, making a vaccine is more complicated than it seems. We hope you enjoyed learning a little bit more about Hepatitis C, and remember, make sure you avoid needle sticks! See you next time on Genomics Revolution.

References:

1) Dubuisson, J. (2007). Hepatitis C virus proteins. World Journal of Gastroenterology, 13(17), 2406. doi: 10.3748/wjg.v13.i17.2406

2) Hepatitis C. (n.d.). Retrieved April 5, 2020, from https://www.sfcdcp.org/infectious-diseases-a-to-z/hepatitis-c/

3) Ngoc, C. L., Thanh, T. T. T., Lan, P. T. T., Mai, T. N., Hoa, T. N., My, N. N., … Vizions. (2019, March 19). Differential prevalence and geographic distribution of hepatitis C virus genotypes in acute and chronic hepatitis C patients in Vietnam. Retrieved April 5, 2020, from https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212734#sec005

4) Spitz, N., Barros, J., doO, K., Brandao-Mello, C., & Araujo, N. (2019). The First Complete Genome Sequences of Hepatitis C Virus Subtype 2b from Latin America: Molecular Characterization and Phylogeographic Analysis . Viruses, 11(11). doi: 10.3390/v11111000

5) Underferth, D., & MD Anderson Cancer Center. (2019, May 8). Hepatitis C and liver cancer: What to know. Retrieved April 5, 2020, from https://www.mdanderson.org/publications/focused-on-health/HepatitisC-liver-cancer-What-you-need-to-know.h16Z1591413.html

6) Zein, N. N. (2000). Clinical Significance of Hepatitis C Virus Genotypes. American Society for Microbiology. doi: https://dx.doi.org/10.1128/cmr.13.2.223-235.2000

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Brad jumps in with a trailer for 4 episodes dealing with viruses that are transmitted by sex and transfer of bodily fluids.

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Genomics Revolution

Guest Hosts: Ciara Love & Cara Katzendorn

Episode 45: Hepatitis B Virus

Script:

Hi, my name is Ciara and today we’re going to talk about a virus called Hepatitis B. This virus is a type of species in the orthohepadnavirus genus as well as a member of the hepadnaviridae family. This virus causes a world-wide known disease called Hepatitis B or HBV. HBV causes liver cancer and the vaccine that was invented for Hepatitis B was one of the first anti-cancer vaccine. The virus was discovered in 1965 by Dr. Baruch Blumberg, which he had won a Nobel Prize for. He worked with a microbiologist named Irvine Millman, to help develop a blood test for the virus that blood centers were using in 1971. The first vaccine was originally the virus treated by heat, and then later in 1986 genetically engineered Hepatitis B vaccines were created. We should care about understanding this virus because it affects everyone and some people carry this virus their whole lives. This virus is a partial double dna stranded molecule that usually replicates by reverse transcription and has between 3182-3248 base pairs depending on the genotypes.

The hepatitis B genome includes four open reading frames for viral proteins. These four groups are surface antigens, core protein, polymerase, and protein X whose function is unclear but may be connected to this virus’s influence on the development of liver cancer. Therapies and vaccines continue to advance in the hopes of finding cure for this disease. In 2015, a study published in the Journal of Clinical microbiology demonstrated researchers improving techniques of identifying this virus throughout all 10 different genotypes found in patients. They designed a mix of primers based on sequencing from over 5000 Hbv patients that would help in creating a more analytically sensitive PCR amplification for detection of this virus. This may be used as a universal detection method for all genotypes of this disease.

In 2012 researchers set out to identify the functional effects of HBV viral integration into the human genome, in hopes of indicating how this virus connects with liver cancer. They found that the virus had many possible effects by insertional mutagenesis, viral promoter-driven transcriptional up-regulation, and genomic instability. This study opens possibilities for future studies that will hopefully be able investigate medicines to prevent the development of liver cancer in HBV patients.

Research continues to develop our understanding of this virus and the diseases associated with infection, and a cure for patients in on the horizon.

Resources:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313610/

https://www.ncbi.nlm.nih.gov/pubmed/12480564

https://www.hepb.org/prevention-and-diagnosis/vaccination/history-of-hepatitis-b-vaccine

https://hbvdb.lyon.inserm.fr/HBVdb/HBVdbGenome

http://garfield.library.upenn.edu/classics1979/A1979HW50100001.pdf

https://www.ncbi.nlm.nih.gov/pubmed/26112647

https://jcm.asm.org/content/53/6/1831

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317142/?report=reader#!po=46.4286

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Genomics Revolution

Kiyana Caver & Brittany Weaver

Genetics Podcast Transcript

Episode 44: Poliovirus

Script:

Hello, I am Kiyana & I am Brittany and we will discuss the virus poliovirus which causes polio. Polio can also be known as poliomyelitis. It stems from the Greek meaning of an inflammation of the gray matter. In the early 20thcentury there weren’t many diseases that had parents worried about the health and wellbeing for their children. However, poliomyelitis is something that parents were fearful of their children getting. Poliomyelitis was a disease that was more common to catch during the summer as that is when polio was more likely to come about, in warmer weather. This disease is very contagious and it is caused by a virus that attacks the nervous system, it also can lead to spinal cord and or brain stem paralysis if it is not treated soon. The people that are mostly affected by it are younger children, usually children become affected with the disease before they turn 5 years old (7 & 6). Polio was most likely spread through contact between people by nasal and oral secretions and by coming into contact with contaminated feces. Polio enters through the mouth, multiplies through the digestive tract where it continues to multiply. Polio was discovered in 1908 by Karl Landsteiner and Erwin Popper. Poliovirus was discovered as scientists were proving that it was not a bacterium that had been causing paralysis, but instead it was a virus (5). Despite it being discovered it the early 1900’s, poliovirus did not peak in the United States until around 1952 when almost 60,00 cases had sparked throughout the United States.

Poliovirus is a RNA genome with a protein capsid made up of one single RNA positive sense stand, that is about 7500 nucleotides long. There is one primary RNA molecule in the genome, which has been shown to have 8 unused start codon sites that are in front of the active AUG start codon. The molecule also has 23 RNA hairpin secondary structures (3). Poliovirus has one main cleavage of a precursor polypeptide called NCVP 1 which encodes for proteins VP 1, 2, 3, and 4 (1). Poliovirus originated from a single precursor molecule called NCVPOO [noncapsid viral protein (NCVP] (2). Poliovirus is a well known and studied virus because between the period of the 1940’s-1950’s the virus was at its most epidemic (8). Polio had crippled around 35,000 people every year in the US alone. A study was preformed in 1994-1995 that showed over 1 million people had survived poliovirus but, over 443,000 people reported having been paralyzed, as it affects a persons nervous system. During the epidemic when it was at its highest affect there were reports of about 27,000 people reporting paralyzation and about 6,000 deaths. When poliovirus was sequenced it led to many discoveries that affected the treatment and prevention of polio in the world. Research found that there were 3 serotypes of poliovirus that caused paralysis in diffferent ratios, type 1 was the most paralytic and accounted for about 80% of paralyzation, type 2 had 8% and type 3 had 13%. This finding was important because it allowed for more focus to be pulled onto why one type was more paralytic than others. Another key finding was that of inactivated poliovirus also known as IPV being introduced for polio in 1955 and oral poliovirus also known as OPV in 1961. These were both used to protect immunized patients and it was found that OPV could spread from the vaccinated to close companions when in contact, increasing the immunity in the US, which was seen when the number of annual inciddences fell drastically. Knowing that OPV was a good way of immunization it was studied along with the wild poliovirus sequence to see how many doses of OPV was approximately needed to eradicate wild poliovirus. The US and other countries tested the possibility of eradicating the wild poliovirus and was first succesful in Cuba through the eradication of wild poliovirus. It was found that if a child by the age of 12 months had 3 doses of OPV that percentage of cases of wild poliovirus decreases. The eradication of all indigenous wild poliovirus occurred in 1991 (4).

This is Brad Goodner and I need to step in with an add-on to Brittany’s last sentence. After success in Cuba, the eradication of wild poliovirus in 1991 was from the entire Western Hemisphere through an organized immunization effort using the oral poliovirus vaccine. Since then, efforts have continued worldwide and we are so close to complete eradication of polio that immunization programs have switched from the oral vaccine to the inactivated vaccine to prevent OPV-derived shedding of live virus that might cause rare infections on non-immunized individuals. Hopefully, we are only a few years away from saying goodbye to this small but devasting virus.

This has been our genomic podcast on poliovirus, thank you for listening and we hope you now know more about poliovirus.

References:

1. Rekosh, David. “Gene Order of the Poliovirus Capsid Proteins.” Journal of Virology, vol. 9, no. 3, Mar. 1972, pp. 479–487., doi:10.1128/jvi.9.3.479-487.1972.

2. Nomoto, A., et al. “Complete Nucleotide Sequence of the Attenuated Poliovirus Sabin 1 Strain Genome.” Proceedings of the National Academy of Sciences, vol. 79, no. 19, Jan. 1982, pp. 5793–5797., doi:10.1073/pnas.79.19.5793.

3. Simoes, E A, and P Sarnow. “An RNA Hairpin at the Extreme 5 End of the Poliovirus RNA Genome Modulates Viral Translation in Human Cells.” Journal of Virology, vol. 65, no. 2, Feb. 1991, pp. 913–921., doi:10.1128/jvi.65.2.913-921.1991.

4. Nathanson, N., and O. M. Kew. “From Emergence to Eradication: The Epidemiology of Poliomyelitis Deconstructed.” American Journal of Epidemiology, vol. 172, no. 11, 26 Oct. 2010, pp. 1213–1229., doi:10.1093/aje/kwq320.

5. “NMAH: Polio: The Polio Genome.” NMAH | Polio: The Polio Genome, 1 Feb. 2005, https://amhistory.si.edu/polio/virusvaccine/livingchem.htm

6. “Polio.” Mayo Clinic, Mayo Foundation for Medical Education and Research, 9 Dec. 2017, www.mayoclinic.org/diseases-conditions/polio/symptoms-causes/syc-20376512.

7. “Poliomyelitis.” World Health Organization, World Health Organization, www.who.int/news-room/fact-sheets/detail/poliomyelitis.

8. “Post-Polio Syndrome Fact Sheet.” National Institute of Neurological Disorders and Stroke, U.S. Department of Health and Human Services, www.ninds.nih.gov/disorders/patient-caregiver-education/fact-sheets/post-polio-syndrome-fact-sheet.

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Genomics Revolution
Mit Patel and Andrew Pemberton
Episode 43: Variola Virus (Cause of smallpox)

Script:
     Welcome to the Genomic Revolution Podcast! This is Mit Patel and Andrew Pemberton from the 2020 Hiram College Genetics course hosting this episode on an orthopoxvirus known as the Variola virus. This virus causes the disease that many of our parents or grandparents may have gotten when they were younger but rather for us millennials we are vaccinated for it. The variola virus or VARV causes the disease smallpox. However, there are some mysteries as to how it originated. According to Igor Babkin, an author of The Origin of the Variola, the descriptions of smallpox can be seen in ancient literature books from India which were written in 6th or even 15th century BC. Historical evidence shows that smallpox has been seen in many countries including India, china, egypt, and even parts of europe. The variola virus has been proven to be only transmitted between sensitive people but it does not show any signs of affecting an animal. Therefore as a result, the majority of the sensitive people will have two outcomes: be immune or die which will cause the virus to fade. Many researchers have come to conclude that animal domestication, land farming, and large human settlements about 6000 to 10000 years ago truly caused the emergence of smallpox (Babkin, 2015).

Now you listeners may be wondering why I should care about a disease that we already have a vaccination for? But, truly understanding the history and information on how the resolution was found, gives the idea of how important this virus is. When smallpox was around, it was certainly a devastating disease. On average, out of 10 people about 3 of them would die. And due to this, control efforts started to be implemented. One of the first methods was the use of variolation. Variolation, named after the variola virus, was first used in Asia where a small dried smallpox scab was blown into the nose of an individual. This individual would then contract the disease but a milder form. At the end, the individual would be immune to smallpox. Due to this, only about 1%-2% percent died rather than 30%. By 1700, variolation had spread to India, Africa, and the Ottoman empire. Europeans and Americans tended to variolate by puncturing the skin (CDC, 2016).

On the other hand comes the factual information. So the variola virus is a double stranded DNA with a length of approximately 190 kbp, specifically 186,102 base pairs. A 102 kbp is compromised for a central conserved domain, which encodes for multiple different proteins. The virus encodes for about 200 proteins altogether all having different functions. About 80 of those encoded proteins are located in the terminal regions of the genome, where proteins related to host immune invasion are encoded.

The variola virus belongs to the genus of ​Orthopoxvirus a​ nd is in the family of Poxvirus (Babkin and Babkina). Research done by Mackett and Archard performed DNA sequencing of several organisms from the genus of Orthopoxvirus (Mackett and Archard). Using restriction endonuclease, they analyzed the genome structure of several viruses and discovered that there is a central conserved region in Orthopoxvirus organisms (Mackett and Archard). It was determined that these highly conserved regions are important to vital function of the virus, including DNA replication and repair, transcription and such (Mackett and Archard). The sequenced DNA also showed variation of the terminus end. This variation is how the Orthopoxvirus viruses vary and is how monkeypox virus infects monkeys and why variola virus is specific to humans (Mackett and Archard). Genome sequencing of the variola virus and closely related virus has revealed which regions of the DNA determine function of the Orthopoxvirus including the variola virus.

Another area where the genome sequence of Variola Virus has been used is to create a phylogenetic tree, or a tree that shows evolutionary relationships among different organisms and in our case viruses. In a paper published by Smithson and other authors, their core group members used one of the first sequenced ancient variola virus genomes. With this, they removed sequencing tags and conducted manual gap-spanning reads. This new assembly was used along with other orthopoxvirus genomes like camelpox and taterapox to determine the last common ancestor of the VARV virus. Their analysis of these different genomes including variola virus lead them to conclude that single nucleotide polymorphisms and amino acid changes in the vaccinia virus ortholog associated to the VARV host specificity and virulence. Furthermore, it was found that these traits were introduced prior to the rise of recent pox viruses. An interesting fact from this paper is that when comparing the ancient and modern VARV genome sequences there is measurable drift in Adenine and thymine richness (Smithson, 2017).

A large worry of many is the reemergence of the smallpox virus. In a paper by Theves and others, they exhumed bodies from an elite burial dating back to 1730s to 1740s, due to the burial time being in the winter and lack of trauma, the authors hypothesized that they died because of a pathogen. At first they thought it might have been a bacterial pathogen, but when they could not find any evidence they searched for a virus (Thèves et al.). After some searching they were able to identify some DNA fragments that were from the poxvirus family, since they were humans, it was smallpox (Thèves et al.). The worry of reemergence prompts researchers to sequence all variations of the variola virus, even ancient variations, in order to have the best possible chance to combat the variola virus if it were to mutate and evolve to infect people again.

We would like to thank you all for listening and we would also like to thank Professor Goodner for allowing us to join Genomics Revolution! We hope that you got a great understanding about the variola virus.

References:
Babkin, Igor, and Irina Babkina. "The Origin Of The Variola Virus". Viruses, vol 7, no.3, 2015, pp. 1100-1112. MDPI AG, doi:10.3390/v7031100. Accessed 9 Apr 2020.

“History of Smallpox.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 30 Aug. 2016, www.cdc.gov/smallpox/history/history.html.

Mackett, M, and L.C. Archard. "Conservation And Variation In Orthopoxvirus Genome Structure". General Virology, vol 45, no. 3, 1979, pp. 683-701., Accessed 4 Apr 2020.

Smithson, Chad et al. “Re-Assembly and Analysis of an Ancient Variola Virus Genome.” Viruses vol. 9,9 253. 8 Sep. 2017, doi:10.3390/v9090253

Thèves, C. et al. "The Rediscovery Of Smallpox". Clinical Microbiology And Infection, vol 20, no. 3, 2014, pp. 210-218. Elsevier BV, doi:10.1111/1469-0691.12536.

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Brad jumps in with a short trailer introducing some viruses that we have conquered or nearly conquered.

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Genomics Revolution

Brianna Bays and Melika King

Episode 42

Influenza Type B Virus

Script:

Welcome to Genomics Revolution. This is Brianna Bays and Melika King from the 2020 Hiram College Genetics course hosting this episode over Influenza virus type B commonly known as the flu virus. The common symptoms are fever, muscle aches, headache, lack of energy, sore throat, and nasal congestion (1). Complications can rise due to immune status and underlying medical conditions (1).

In 1892 two physicians Richard Pfeiffer and Shibasaburo Kitasato worked at a lab in Berlin claimed discovery of a new bacterium (2). Since the flu is seasonal, other people had a hard time investigating the correlation between influenza B and the true cause of the illness. Until the next outbreak in 1918 when Peter Olitsky and Frederick Gates provided strong evidence to prove influenza B was the cause (2). Modern techniques used to identify influenza B are reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence assays and rapid molecular assays (1).

All human influenza viruses are enveloped negative-strand RNA viruses with segmented genomes containing seven to eight gene segments (3). Influenza B has eight and it comprises about 14,000 nucleotides (3). This genome has four proteins in the envelope: hemagglutinin(HA), neuraminidase (NA), NB, and BM2 (3). HA and NA are surface proteins and these are considered antigenic sites where the body’s immune system develops antibodies to bind to (3). To test for antigenic changes, scientists can use hemagglutinin inhibition assay essentially seeing how well antibodies bind to it making the influenza virus inactive. The NB protein is believed to be an ion channel (3) and the BM2 protein is a proton channel that is important for the uncoating process (4). Uncoating refers to the deliverying of the viral genome to the host cell for replication. Influenza viruses recognizes N-acetylneuraminic (sialic) acid on the host cell surface (4). N-acetylneuraminic are found on glycoconjugates (5).

Having the knowledge about this genome, scientists then can compare it to other viral genomes of influenza B to see if there are any genetic variations. That allows them to create a more effective vaccine. To look at the genome the CDC uses techniques like ‘the Sanger reaction’ and advanced molecular detection (1). It is important to understand influenza B just like any other virus because it can be prevented from spreading and helps scientists come up with an effective treatment. This virus is commonly seen in humans and seals so we do not see as many genetic changes as we do with influenza A viruses (1). Currently influenza B viruses are classified into two genetic lineages - Yamagata and Victoria (6).

It is important to understand influenza B just like any other virus. Having scientific knowledge about this virus and knowing its genome can help correlate to studies of viruses that may come about in the future and help us gain insight about their mechanisms, based on the genomes' similarities and differences. Knowing the mechanisms also allows scientists to come up with an effective treatment not just for Influenza B but other viruses as well. Lastly, everyone having an understanding of how a virus like influenza B spreads, we all can help prevent or slow down the infection rate. Thank you for listening.

References:

1. Influenza Signs and Symptoms and the Role of Laboratory Diagnostics | CDC. (2020). Cdc.gov. Retrieved 7 April 2020, from https://www.cdc.gov/flu/professionals/diagnosis/labrolesprocedures.htm#symptoms
2. Taubenberger, J. K., Hultin, J. V., & Morens, D. M. (2007). Discovery and characterization of the 1918 pandemic influenza virus in historical context. Antiviral therapy, 12(4 Pt B), 581–591.
3. The A, B, and C of influenza virus. (2020). Virology.ws. Retrieved 7 April 2020, from https://www.virology.ws/2009/09/22/the-a-b-and-c-of-influenza-virus/
4. Imai, M., Watanabe, S., Ninomiya, A., Obuchi, M., & Odagiri, T. (2004). Influenza B virus BM2 protein is a crucial component for incorporation of viral ribonucleoprotein complex into virions during virus assembly. Journal of virology, 78(20), 11007–11015. https://doi.org/10.1128/JVI.78.20.11007-11015.2004

5. Ronald L. Schnaar, in Advances in Carbohydrate Chemistry and Biochemistry, 2019

6. Types of Influenza Viruses. (2019). Centers for Disease Control and Prevention. Retrieved 10 April 2020, from https://www.cdc.gov/flu/about/viruses/types.htm

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Genomics Revolution

Benjamin Blake Erman and Ciza Sadoke

Episode 41: Influenza Virus Type A

Script:

Hello and welcome to Genomics Revolution. Today’s episode is hosted by me, Blake Erman and my partner, Ciza Sadoke, which we’ll hear from later on in the podcast. We are from Brad Goodner’s 2020 Hiram College Genetics course and today we will be focusing on the virus Influenza Type A. This virus causes Influenzae which can give you the symptoms of; a fever, chills, headaches,muscle aches, feeling tired and weak, sneezing, stuffy or runny nose, sore throat and cough (healthdirect, 2018). This disease has been thought to be around in humans for about 6000 years although the first instance of influenza symptoms were recorded around 2400 years ago. Despite this long period of time Influenza wasn’t truly discovered until 1918. This disease was discovered not from the study in humans but through studies of animal diseases by veterinarian J.S. Koen. Koen noticed the close similarities of the disease in pigs that he was working on compared to the then ongoing “Spanish” influenza pandemic of 1918. However, the Spanish influenza pandemic has not been the only influenza type A caused pandemic. According to a review article by Susan Baigent and John McCauley Influenza Type A has caused six pandemics throughout both the 19th and 20th centuries (Baigent, 2003). These pandemics have resulted from the introduction of genes from an animal-derived virus being put into the genetic background of a currently circulating human virus by the process of reassortment (Baigent, 2003). Due to reassortment being a highly developed ability of the disease and the disease causing high mortality throughout time in both humans and animals it is important to understand it to prevent its spread.

It wasn’t until 1933 through the diligent work of researchers Christopher Andrews, Patrick Laidrow, and Wilson Smith that the first human influenza virus was discovered (Kuszewski, 2000). Through their work a vaccine was made and the influenza virus was found to belong to the Orthomyxoviridae family. This family can be divided into two genera, the first includes both influenza A and B viruses while the second the influenza C virus (Kuszewski, 2000). For today, we will be focusing on this first genera, the one including influenza A. The type A virus has many subtypes that are distinguished by the antigenic properties of surface glycoproteins of the virus (Kuszewski, 2000). These glycoproteins are haemagglutinin (HA) and neuraminidase (NA) and they have 15 and nine different types respectively (Kuszewski, 2000). These multiple different types add to influenza type A’s complexity and is why it is a recurring disease. Most of the pandemics previously mentioned were caused by different compositions of this virus such as H1N1 or H2N2. And now to pass it off to my partner.

Hello my name is Ciza Sadoke and I will be finishing the rest of today’s podcast. Picking up where my partner left off, Influenza type A’s genome consists of eight negative sense single-stranded RNA molecules and they can range in size from 890 nucleotides to 2341 nucleotides (McCauley, 1983). These eight RNA molecules total up to about 14,000 nucleotides in length (Virology, 2009) and encode for eleven proteins (Samji, 2009). There are two ways that the influenza type A virus can change, and these changes are continuously happening at random in the genome. One of the ways the influenza virus can change is through Antigenic drift. These are small changes in the genes of the influenza virus that can lead to changes in the surface proteins. This means that they get recognized by the immune system and are capable of triggering an immune response. The genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments (Kim, 2018).

The other way the influenza type A virus can change is through antigenic shift. Antigenic shift occurs when there is an abrupt major change in the influenza A virus. This can result in new HA and NA proteins in the influenza viruses that can infect humans. These shifts can result in different influenza A subtypes in humans. One way shift can happen is when an influenza virus from an animal population gains the ability to infect humans. When a virus is produced that can infect human cells and it has new subtypes of these proteins. The people population will now have to little no protection against it, and therefore the virus will be rapidly spread throughout population. This antigenic shift process is responsible for three major influenza pandemics in the 20th century, including the Spanish flu in 1918, which killed 3% of the worlds entire population at the time (Taubenberger,2006). Also, due to the segmented genome of influenza type A, recombination can occur quite frequently and make a form of the virus that populations have no immunity against (McCauley, 1983). Even current human influenza virus vaccines are only protective against currently circulating strains and close variants of the virus (Baigent, 2003). These vaccines contain inactivated mixture of types A and B strains and this would not have been possible without its sequenced genome. This is all that we have time for today. Thank you for listening to today’s episode of Genomics Revolution. We hope you have enjoyed and have a good day!

References:

Baigent, S. J., & Mccauley, J. W. (2003). Influenza type A in humans, mammals and birds: Determinants of virus virulence, host-range and interspecies transmission. BioEssays, 25(7), 657–671. doi: 10.1002/bies.10303

Influenza A (flu). (May 2018). Retrieved April 5, 2020, from https://www.healthdirect.gov.au/influenza-a-flu

Influenza Type A Viruses. (2017, April 19). Retrieved April 5, 2020, from https://www.cdc.gov/flu/avianflu/influenza-a-virus-subtypes.htm

Kim, H., Webster, R., & Webby, R. (2018, March 01). Influenza Virus: Dealing with a Drifting and Shifting Pathogen. Retrieved April 10, 2020, fromhttps://www.liebertpub.com/doi/abs/10.1089/vim.2017.0141?rfr_dat=cr_pub

Kuszewski, K., & Brydak, L. (2000). The epidemiology and history of influenza. Biomedicine & Pharmacotherapy, 54(4), 188–195. doi: 10.1016/s0753-3322(00)89025-3

McCauley, J. W., & Mahy, B. W. J. (1983). Structure and function of the influenza virus genome. Biochemical Journal, 211(2), 281–294. doi: 10.1042/bj2110281

Noronha, J., Liu, M., Squires, R., Pickett, B., Hale, B., Air, G., . . . Scheuermann, R. (2012, May 15). Influenza Virus Sequence Feature Variant Type Analysis: Evidence of a Role for NS1 in Influenza Virus Host Range Restriction. https://jvi.asm.org/content/86/10/5857

Samji T. (2009). Influenza A: understanding the viral life cycle. The Yale journal of biology and medicine, 82(4), 153–159.

Taubenberger, J., & Morens, D. (2006, January). 1918 Influenza: The mother of all pandemics. Retrieved April 10, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3291398/

“Influenza Virus RNA Genome.” Virology Blog Header Image, 16 Dec. 2009, www.virology.ws/2009/05/01/influenza-virus-rna-genome/.

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Genomics Revolution Podcast

Allison Slutz & Cole Filer
Episode 40: Measles & Morbillivirus

Hi all. Welcome back to another episode of Genomics Revolution. Today you are hosted by Cole Filer and myself, Allison Slutz. We are part of the 2020 Hiram College genetics course and will be talking about the Measles morbillivirus, the virus that causes the measles.

The genus that morbillivirus is apart of, is the family Paramyxoviridae (1). The measles virus genome typically consists of 115,894 nucleotides, and encodes for 8 proteins: 6 structural (N, P, M, F, H, L) and 2 non-structural (V and C) (5). This family has enveloped viruses that have negative non-segmented single-stranded RNA genomes (1). This family also has several members that cause several issues. These are highly infectious in their nature and spread by the respiratory system (1). At the cellular level, infections from morbillivirus are initially caused by the binding of hemagglutinin glycoprotein to cellular receptors and the two receptors found in wild-type morbilliviruses are CD150 also referred to as signaling lymphocyte activation molecule F1, and the poliovirus receptor-like 4 (1).

One of the key findings that emerged from the sequenced virus genome is the number of different strains that exist, and tracking the mutation of the virus, which would allow for better development of vaccines and prevention of the spreading of measles.

Before moving forward, it’s important to understand the history of measles. The first documentation was by a Persian doctor in 9th century AD (3). Later, in 1757, a Scottish physician by the name of Francis Home demonstrated that measles are caused by a blood-borne infectious agent (3). A notable date for America is the first domestically reported case. During the time of the European immigration in 1765, the measles was first reported (3).

We should care about this virus because if we didn’t understand how it was transmitted and how to prevent it, it would kill a lot of people, and a large portion of those would be children. Measles are transmitted by coughing and sneezing, this is because the virus lives in the nose and throat of a person with this illness (2). This virus has the potential to live in the air for two hours and up to 90% of people who do not have immunity and come in contact with an infected person, will contract measles (2).

Just for the sake of reference, there are roughly 200 deaths per 100,000 cases in immunocompetent people, and around 100 cases of encephalitis per 100,000. Additionally, 1 in every 20 children that contract measles end up developing pneumonia as a complication, which happens to be the most common cause of death in young children from measles (4).

As previously stated, measles cause a significant amount of people to die, specifically children. The current vaccine used in the United States is the measles, mumps, rubella, and varicella developed in 1968 by John Enders and colleagues (3). In 2014, there was an increase of death from the measles (1). This could be correlated to some individuals and groups within first world countries that don’t believe that vaccines are necessary. Viruses do not discriminate between social classes and a person’s overall wealth. This is why everyone should protect themselves with vaccines. Vaccines are the best way to prevent the measles. We hope you enjoyed this episode of Genomics Revolution on the measles. Please continue to tune in to hear our peers on other notable viral genomes that have molded our history.

Works Cited:

1. Vries RD, Duprex W, Swart RD. Morbillivirus Infections: An Introduction. 2015;7(2):699–706.

2. Transmission of Measles. 2018 Feb 5 [accessed 2020 Apr 10]. https://www.cdc.gov/measles/transmission.html

3. Measles. 2018 Feb 5 [accessed 2020 Apr 9]. https://www.cdc.gov/measles/about/history.html

4. Measles Data and Statistics. Center for Disease Control. 2019 Apr 16 [accessed 2020 Apr 9]. www.cdc.gov/measles/downloads/measlesdataandstatsslideset.pdf

5. Phan MVT, Schapendonk CME, Munnink BBO, Koopmans MPG, Swart RLD, Cotten M. Complete Genome Sequences of Six Measles Virus Strains. Genome Announcements. 2018 [accessed 2020 Apr 9];6(13). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876482/. doi:10.1128/genomea.00184-18

Cool Audio That Goes with This Episode:

​“I’m a Virus.” Sciencemusicvideos, 2012. www.youtube.com/watch?v=kYf_Sl8W3qY&t=2s

​“D.J. Tools Let’s Get Ready to Rumble Sound Effect.” All Sounds, 2015. www.youtube.com/watch?v=Ozol5AYYg5c

​Paxton, Tom. “I’ve got the measles.” I’ve got a yo-yo., Pax Records, 1997. www.youtube.com/watch?v=iyJ9Tvv8aWw

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Zach Walker & Alainna Conroy

Virus: Varicella zoster virus

Disease: Chickenpox/ Shingles

Welcome to today’s episode of Genomics Revolution. I am Zach Walker and am here with Alainna Conroy and we will be discussing the varicella zoster virus. We will be referring to this as the VZV.

BACKGROUND/ DISCOVERY:

In 1888, Von Bokay first observed that the VZV was related herpes zoster virus or shingles when children contracted VZV from adults who had shingles. Then in 1954, Thomas Weller took cell cultures from VZV lesions to scientifically distinguish VZV from shingles. After this in the 1970s, Japan developed the Oka strain of the varicella vaccine.

WHY WE CARE:

VZV causes chickenpox or shingles. Chicken pox results from initial infection of the VZV and occurs in children. The VZV can remain silenced in individuals and become activated later in life resulting in shingles.

GENOME:

VZV is a member of the varicellovirus genus and belongs to the alpha-herpesvirus family. The VZV genome has at least 70 genes, most of them homologs in herpes simplex virus.

In 1986, the complete sequence of the VZV genome was determined by Davison and Scott. They determined the genome is variable in size, but the sequence they looked at was 124, 884 base pairs and contained 70 genes between 2 strands of DNA. The VZV genome encodes at least 71 unique proteins. The sequence is linear with a single unpaired nucleotide on each end. These will pair together and become circular in cells that are infected with the virus.

FINDINGS FROM SEQUENCING

After sequencing the VZV genome, Davidson and Scott determined that there are a lot fewer repeats among the sequence compared to HSV-1 and the places with repeats are unrelated to HSV-1. The research describe these repeats as accumulated parasitic sequences that possibly happen through recombination, in regions that they do not cause a selective disadvantage. This affects the size of some of the encoded proteins and could have a role in different functions of these genes.

By looking at the sequence and determining the gene layout, Davidson and Scott were able to determine that both the VZV and HSV-1 have similar gene layouts. Many of the genes encode similar proteins with specific properties. Research reports that there are also a limited amount of regions with significant differences. This indicates that the proteins are highly conserved and can be determined that the functions of VZV and HSV-1 are very similar.

In a study done by Argaw et al, they were able to determine distinguishing parts in the nucleotide sequence between the originally sequenced VZV and the VZV Oka vaccine strain. They looked at about 34,000 from the 3’ end, they were able to see numerous changes that they describe as “nonconservative amino acid substitutions in coding sequences for virus gene products” (1154). The biggest change occurred in ORF62 where a glycine was substituted for an arginine leading to a cleave site. The study reports that mutations in ORF62 may factor into attenuation of the Oka vaccine strain. There is also a nucleotide insertions that allow a distinction.

Another finding by Lebrun et al is that ORF9p binds to the adaptor protein complex 1 also known as AP-1. AP-1 is involved in the intra-cellular transport and moving proteins between endosomes and the trans-Golgi network. With this interaction between ORF9p and AP-1 it allows the secondary envelopment of VZV. However, in the same study conducted by Lebrun et al they were able to determine that leucine 231 is conserved among alphahepresviruses and was critical in interaction between ORF9p and AP-1. To help show this, they mutated leucine 231 to alanine in ORF9p and found that by mutating leucine 231 to alanine it strongly impaired the viral growth of VZV.

This ends today’s episode of Genomics Revolution, thank you for your time and we hope you were able to learn something new!

Works Cited:
Argaw, T., Cohen, J. I., Klutch, M., Lekstrom, K., Yoshikawa, T., Asano, Y., Krause, P. R. (2000) Nucleotide sequences that distinguish Oka vaccine from parental Oka and other Varicella-Zoster Virus isolates. The Journal of Infections Diseases, 181, 1153-1157.

Centers for Disease Control and Prevention, “Varicella” wonder.cdc.gov/wonder/prevguid/p0000108/p0000108.asp

Cohen, J. I., The Varicella-Zoster Virus Genome. Curr Top Microbiol Immunol., 342, 1-14.

Davison, A. J., Scott J. E. (1986). The complete DNA sequence of Varicella-Zoster Virus. J. Gen. Virol. 67, 1759-1816.

Lebrun, M., Lambert, J., Riva, L., Thelen, N., Rambout, X., Blondeau, C., Thiry, M., Snoeck, R., Twizere, JC., Dequiedt, F., Andrei, G., Sadzot-Delvaux, C. (2018) Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity. J Virol. 92, 1-22

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Genomics Revolution Podcast

Episode 38: The Viral Age Begins

Host: Brad Goodner, Professor of Biology & Biomedical Humanities at Hiram College

Transcript:

Welcome back. Now that we have a basic definition of a virus – a RNA or DNA genome surrounded by a protein shell called a capsid, let us put that definition into some historical context. When did we humans come to realize that viruses existed? When did we realize that they were different from cellular life forms? The Viral Age began, in terms of human recognition, around 120 years ago. Yet, it was not a human disease that brought us to this realization or even a disease of another animal. It was a disease of tobacco. You heard me right, tobacco.

Tobacco was a major cash crop in the U.S. and around the world in the late 19th century. As you know, it it the leaves that matter. They get dried and cured, then rolled and chopped to make smoking products, chewing tobacco and snuff. Tobacco mosaic disease ruins leaves. It is called mosaic because the leaves of infected tobacco plants have a mottled color. This pattern reflects the path of infection as the virus moves throughout a given leaf. The disease causes leaves to be much less efficient at photosynthesis. Slower growth means fewer leaves to use.

By the late 19th century, the germ theory of disease had gained a lot of support from European researchers such as Robert Koch in Germany and Louis Pasteur in France. Pasteur had put the last nail in the coffin of spontaneous generation, the very old idea that some diseases, food spoilage and rotting meat was due to new life forms arising out of non life. In fact, these issues were due to the presence of specific microbes. Scientists started finding microbial causes for many diseases and other issues such as wine spoiling into vinegar. Koch came up with a set of steps for proving that a particular microbe caused a particular disease – what we now call Koch’s postulates. In 1886, Mayer showed that he could take leaves from a tobacco plant suffering from mosaic disease, crush the leaves and squeeze out some juice, paint that juice onto the leaves of a healthy plant and see the disease occur. Ivanowski went further in 1892 to show that he could pass the juice from infected leaves through a filter that normally catches bacteria and the filtered juice could still cause disease. Mayer and Ivanowski both thought that tobacco mosaic disease was caused by some type of bacterium, maybe just a really small one (Bos, 1999). Martinus Beijerinck thought differently. He repeated Ivanowski’s filtration experiment and went on to show that the causal agent of tobacco mosaic disease could diffuse through a block of agar like a small chemical. Even as small as they are, most bacteria cannot do that and so in 1898 Beijerinck put forward a strikingly different proposal – tobacco mosaic disease is caused by something different, in his words “a contagium vivum fluidum” (Bos, 1999). He could not define it further, but it was clear to him that the pathogen was not a bacterium. Beijerinck’ s “contagium vivum fluidum” turned out to be the first virus ever discovered which now goes by the name Tobacco Mosaic Virus or TMV.

Next, TMV was the first virus to be crystallized by the American Wendell Stanley in 1935 for which he won a partial share of a Nobel Prize in 1946 (Norby, 2008). It is important to note that Stanley thought the particles he crystallized were pure protein. It was actually not pure protein as shown by Bawden and Pirie in England in 1936 – there was RNA in the crystallized sample as well. Many scientists at the time and since then believed that Bawden and Pirie’s work was equal to or more important than that of Stanley, but it was Stanley whose name is on the prize (Norby, 2008).

In 1956, Frankel-Conrat showed that TMV RNA alone could produce full blown disease when applied to the surface of wounded tobacco leaves (Creager, 1999). This data proved that the viral genome codes for all the necessary proteins needed for disease symptoms and to produce infectious viral particles (Scholthof et al., 2011). Just a year later, Andre Lwoff, who would go on to share a Nobel Prize for his work on gene regulation, wrote out a description of a virus that still works today – “…infectious, potentially pathogenic, nucleoproteinic entities possessing only one type of nucleic acid, which are reproduced from their genetic material, are unable to grow and to undergo binary fission ...’’ (Lwoff, 1957).

TMV has continued to be the subject of much basic and applied research (Scholthof, 2004; Zaitlin, 1999). Yet for us, it will always be the start of the Viral Age!

See you next time on Genomics Revolution as we begin to hear from guest hosts of the 2020 Hiram College Genetics course on some other important viruses and their genomes.

For More Information on TMV:

Bos, 1999. Philosophical Transactions of the Royal Society of London, part B 354:675-85. Beijerinck’ s work on tobacco mosaic virus: historical context and legacy.

Creager et al., 1999. The Plant Cell 11:301-8. Tobacco mosaic virus: pioneering research for a century.

Norrby, 2008. Archives of Virology 153:1109-23. Nobel prizes and the emerging virus concept.

Lwoff, 1957. Journal of General Microbiology 17:239–253. The concept of virus.

Scholthof, 2004. Annual Review of Phytopathology 42:13-34. Tobacco mosaic virus: a model system for plant biology.

Scholthof et al., 2011. Molecular Plant Pathology 12:938-54. Top 10 plant viruses in molecular plant pathology.

Zaitlin, 1999. Philosophical Transactions of the Royal Society of London, part B 354:587-91. Elucidation of the genome organization of tobacco mosaic virus.

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Genomics Revolution Podcast

Episode 37: A New Season, A New Normal

Host: Brad Goodner, Professor of Biology & Biomedical Humanities at Hiram College

Transcript:

Welcome to our 2nd season of Genomics Revolution. I am your host, Brad Goodner. Along with this new season of podcasts comes a new normal that all of us are dealing with – the COVID-19 pandemic. I hope that you and your loved ones are not just safe and healthy, but that you are finding new ways to engage with the world. I know here in northeast Ohio where I teach at Hiram College, it is a very different feeling this 3rd week of March than it was just 2 weeks ago. I now teach courses and work with students using a virtual classroom. I am trying to embrace the challenge of making my virtual interactions as meaningful and fun as my face-to-face interactions were in the past. All because of a viral outbreak called COVID-19.

Last week during our spring break, while I was preparing materials to help the students in my Spring 2020 Genetics course write new episodes for this podcast, it struck me that we should embrace our new normal. Why not focus this season of podcasts on viruses. You might wonder - what do viruses have to do with genomics? That calls for a definition – what is a virus?

A virus is a noncellular parasite. Cellular lifeforms on planet Earth all share certain features – they are bounded by a lipid bilayer membrane, they use double-stranded DNA as their genomes, they make their own proteins, and they transform energy and carry out metabolism. Viruses are much much simpler. Among all viruses, there are only two shared components. One, a genome, but there are 4 flavors to choose from – some viruses have double-stranded DNA like cells, some have single-stranded DNA, some have double-stranded RNA, and some have single-stranded RNA. Two, the viral genome is surrounded by a protein-based shell called its capsid. Beyond a genome inside a capsid, viruses can differ greatly from one another. Some are surrounded by membrane derived from a host cell, while others are not. Most viral genomes are composed of one molecule of DNA or RNA, but some viruses have multi-component genomes. Some virus particles are rigid while others are flexible. However, no matter its genome, capsid and other features, every virus has one or more cellular organisms as a host. There is no cellular organism on Earth that does not have at least one virus that can infect it. We humans have lots of viral pathogens to worry about and maybe a few that infect us but do not cause us great harm.

COVID-19 is caused by one such human viral pathogen called a coronavirus. Let us learn more about coronaviruses and other viruses, and in doing so feel more empowered that we can make it through the ongoing pandemic. Along the way, we will see how viruses have in fact impacted humans throughout our history.

Take care of yourself and those around you. Genomics Revolution will help keep you informed and entertained. Stay tuned.

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Welcome to Genomic Revolution. This is Curtis Swearingen from the 2019 Hiram College Genetics course hosting this episode on the genome Saccharomyces cerevisiae S288C. This strain is from the fungal division Ascomycota and the family Saccharomycetaceae. Saccharomyces cerevisiae is an eukaryotic microbe. More specifically, it is a globular-shaped, yellow-green yeast belonging to the Fungi kingdom, which includes multicellular organisms such as mushrooms and molds. Natural strains of the yeast have been found on the surfaces of plants, the gastrointestinal tracts and body surfaces of insects and warm-blooded animals, soils from all regions of the world and even in aquatic environments. Most often it is found in areas where fermentation can occur, such as the on the surface of fruit, storage cellars and on the equipment used during the fermentation process. S. cerevisiae is famously known for its role in food production. It is the critical component in the fermentation process that converts sugar into alcohol, an ingredient shared in beer, wine and distilled beverages. It is also used in the baking process as a leavening agent; yeast releasing gas into their environment results in the spongy-like texture of breads and cakes. Because of its role in fermentation, humans have known about and used S. cerevisiae for a long time. Archaeologists have found evidence of a fermented beverage in a pot in China as early as 7000 BC, and molecular evidence of yeast being used in fermentation was found in a wine jar dating back to 3150 BC. Isolation of the species did not occur until 1938, when Emil Mrak isolated it from rotten figs found in Merced, California. Taking advantage of its unique reproductive cycle, in the 1950s Robert Mortimer performed genetic crosses that used the isolated fig strain and other yeast strains obtained through other researchers. As a result, he created a new strain called S288c, which is the species strain we are talking about, which was then used as a parental strain in order to isolate most of the mutant strains currently used in research. Furthermore, this strain was then used to sequence the S. cerevisiae genome. The entire genome was sequenced 1996, being the first genome to be entirely sequenced. 12.068 Million bp are spread out across 16 chromosomes and codes for about 5885 proteins. In the genome, 5,885 genes code for proteins, 275 code for tRNA, 40 code for snRNA's, and 140 genes code for ribosomal RNA. 4% of the genome is comprised of introns. Out of all the genes that code for proteins, 11% of the protenome is devoted to metabolism, 3% to energy production and storage, 3% to DNA replication, 7% to transcription and 6% to translation. Nearly 430 proteins are involved in intracellular trafficking, and 250 proteins have structural roles.

Because S. cerevisiae is a eukaryote, there are many cool things that scientists have done with it genetic information and genetic tools. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death. They have also been used to make proteins to help combat Alzheimer’s and Parkinson’s diseases in humans. Some cool facts on S. cerevisiae is that a heat tolerant strain of to be used to make ethanol and use that ethanol from the S. cerevisiae rather than from starch based plants because they are limited as a alternative to petroleum. The strain of Saccharomyces can make 42g of ethanol with 97.4g of sugar, which is a 84.36% fermentation efficiency. Hopefully one day we can stray away from the use of petroleum in the near future. Another cool fact is because S. cerevisiae is a eukaryotic cell is has been used to make human insulin for those affected by diabetes. With its help the global insulin market is expected to reach $70.6B by the year 2023. With the help of Saccharomyces cerevisiae, it can allow for easier and cheaper access for people to essential proteins to maintain a healthy life. One last cool fact is even though there are many beneficial things S. cerevisiae can do, with too much exposure to taking as a probiotic can cause sickness in people. There was a case with 57 patients with the infection and 60% were in the ICU and it was detected that 26 patients were given S. cerevisiae as a probiotic and 17 patients died. The use of S. cerevisiae should be reassessed, especially in patients that are ill or immunosuppressed. The chances
are still very low in getting the disease for humans. S. cerevisiae is a cool species with its ability to make human proteins and help make energy efficient products. We have yet to find the full potential of this species and hopefully
in the near future it can help us even more. Thanks for listening.

WORK CITED
1.) Ballesta, Isabella. 2010, September 26. A Microbial Biorealm page on the genus Saccharomyces cerevisiae.
https://microbewiki.kenyon.edu/index.php/Saccharomyces_cerevisiae

2.) Nielsen, Jens. “Production of of biopharmaceutical proteins by yeast: advances through metabolic engineering” Bioengineered vol. 4,4 (2012): 207-11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728191/

3.) Mason, Robert P and Flaviano Girorgini. “Modeling Huntington disease in yeast: perspectives and future directions” Prion vol. 5,4 (2011): 269-76. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012407/

4.) Patricia Muñoz, Emilio Bouza, Manuel Cuenca-Estrella, Jose María Eiros, Maria Jesús Pérez, Mar Sánchez-Somolinos, Cristina Rincón, Javier Hortal, Teresa Peláez, Saccharomyces cerevisiae Fungemia: An Emerging Infectious Disease, Clinical Infectious Diseases, vol. 40,11 (2005) Pg. 1625–1634. https://academic.oup.com/cid/article/40/11/1625/445600

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Welcome back to Genomics Revolution.  Brad Goodner jumping back in to our Survey of Genomes guest-hosted by students from the 2019 Hiram College Genetics course.  You have probably been wondering “hey, when are they going to talk about the human genome?”  We will get to it in time, but I am big believer that we humans need to see the biological world through a non-human dominated lens.  Life on Earth, contrary to what our eyes tell us, is actually dominated by microbes – in cell number, in biomass, and in diversity.  That is even true for the major branch of the tree of life where we humans sit – the eukaryotes.

As you probably know, we humans are in the evolutionary family commonly known as the great apes.  We are in the order Primates, the class Mammalia, the phylum Chordata meaning we have a notochord during our development (different from a spinal cord) and the kingdom Metazoa better known as the animals.  Yet, did you know that there are microscopic animals?  Some of the most numerous animals on Earth are tiny roundworms, mites, and tardigrades (commonly called water bears).

We humans are also in the domain Eucarya or Eucaryota.  All eukaryotes – animals, plants, algae, fungi and amoebas to name just a few, share a common ancestor whose cells had, among other things, a double membrane-bound nucleus, a double-membrane-bound organelle called a mitochondria, some other membranous organelles such as endoplasmic reticulum (ER) and Golgi apparatus, well-developed actin- and tubulin-based cytoskeleton systems and a special way to separate their chromosomes called mitosis.  Yes, mitosis is only seen in eukaryotes.  You don’t see these features in the domains Bacteria and Archaea.  Yes, the available data suggests that Bacteria invented the ancestral forms of actin and tubulin, but these cytoskeleton systems in Bacteria are nowhere near as complex as we see in eukaryotes especially in terms of molecular motors and regulatory proteins.  Yes, Archaea invented new ways of organizing and compacting DNA, but eukaryotes have taken DNA organization and compaction to multiple levels of complexity that are critically linked to regulating gene expression.  To finish this line of thought, there is growing evidence that the earliest proto-eukaryote was a cell from the Archaea lineage that engulfed a member of the Bacteria lineage, specifically an organism from the alpha-Proteobacteria.  Instead of only one cell winning, the engulfer and the engulfed ended up as a co-dependent “cell of cells” as aptly described by Carl Woese and his collaborators.  The engulfed alpha-Proteobacteria cell evolved into what we now call the mitochondria – keeping a small subset of genes (in our case as mammals, keeping about 15 or so genes), losing most of its now non-essential genes, and having some other genes move into what would become the nucleus.  Other genes in current eukaryotic nuclear DNA appear to be of Archaea origin.

Now eukaryotes are found in virtually all habitats, large and small, on Earth.  We usually think of the blue whale, the human, the redwood, the daisy, the kelps of the brown algae and the button mushroom.  These macrobes, as we might call them, are visible to the naked eye because each organism is composed of millions to trillions of cells working with and/or signaling each other.  In episode 12, Kaitlyn Morse discussed the genome of the most well-understood macrobe, the fruit fly Drosophila melanogaster.  Each fruit fly is made up of 5 million or so cells.  However, for every macrobe we can easily see, there are billions, if not trillions, of single-celled eukaryotes or multicellular eukaryotes with just a few hundred to a few thousand cells in the body.  In episode 14, Brett Bentkowski introduced us to the unicellular fission yeast, Schizosaccharomyces pombe.  In the next couple of episodes we will hear about some more important yet tiny eukaryotes.  So I hope you will stay tuned, listen and learn.  Until then, take care.

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Hey guys welcome to this guest podcast of Genomics Revolution hosted by yours truly Hunter Jenkins of the 2019 spring Genetics class of Hiram College.  In this episode, we will be zeroing in on the specific strain Fusobacterium nucleatum ATCC 25586 which I will now refer to as F. Nucleatum.  This strain is a subspecies of Fusobacterium nucleatum named nucleatum which can be found in the human oral cavity, (cough) your mouth.  This organism is from a Genus of Fusobacterium, and family Fusobacteriaceae which I believe previously resided under the family of Bacteroidaceae.  F. nucleatum is closely related to Fusobacterium necrophorum which is commonly found in the intestines of most animals especially mammals.  This alone shows that Fusobacterium can range in many ways to survive and show wildly unique characteristics to those in its family. 

F. nucleatum has been originally studied back in the early 1900’s however not our specific strain.  With newer technology circa 2002, The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics pipeline.  The genome is contained on a singular circular chromosome which holds 2.17 million base pairs encoding 2,046 proteins.  On its own F. nucleatum is generally harmless to humans, however its ability to help form dental plaque and coaggregate with other bacteria can make it worrisome and lead to common disease such as gingivitis.  F. nucleatum’s ability to coaggregate is due in large part to their outer membrane proteins.  These outer membrane proteins were newly found as they previously would get stuck at the top of analytical gels.  Due to this we previously only knew of one porin that could be created.  However we now know that there are eleven ORFs encoding outer membrane proteins.  We have also found several proteins that are closely related to the outer membrane protein family (omp)A-F.  Given that the outer membrane proteins are typically correlated with pathogenesis we can hope to make vaccinations through closer evaluation and research of F. nucleatum (Kapatral et al).  According to Rodrigues et al., F. nucleatum is proven to be more prevalent in the oral cavities of those whom have gingivitis and chronic periodontitis.  However their study also shows that it is in high accordance with Aggregatibacter actinomycetemcomitans and that they work as a symbiotic relationship increasing their populations.  In an experiment Bolstad et al attempted to create a model of the adherence created by F. nucleatum outer membrane proteins, specifically the 40kDa proteins.  By ligating the ORF of each protein into vectors, they were capable of comparing it to the know structure of F. nucleatum strain Fev1 by looking at conserved sequences and variability of polypeptides. Their results show sixteen highly conserved polypeptide sequences which traverse the outer membrane and are highly variable amongst the strains ATCC 25586, Fev1, and ATCC 10953.

That’s all I have for you guys today I hope you enjoyed your time and make sure you have proper dental hygiene to prevent the build up of Fusobacterium nucleatum. 

Erratum of podcast: 
       1) I said early 19th century instead of early 1900’s.
       2) PCR does not tell about protein structure.  My mistake.
       3) If a mature protein sequence goes through a membrane one or more times, the correct term is that it traverses the membrane.

Works Cited:
Bolstad, Anne Isine, H. B. Jensen, and J. Tommassen. "Sequence variability of the 40-kDa outer membrane proteins of Fusobacterium nucleatum strains and a model for the topology of the proteins." Molecular and General Genetics MGG 244.1 (1994): 104-110.

Kapatral, Vinayak, et al. "Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586." Journal of bacteriology 184.7 (2002): 2005-2018.

Rodrigues, Viviane Aparecida Arenas, et al. "Qualitative, quantitative and genotypic evaluation of Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum isolated from individuals with different periodontal clinical conditions." Anaerobe 52 (2018): 50-58.

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Welcome to Genomics Revolution. This is Hannah Mann from the 2019 Hiram College Genetics course hosting this episode on Buchnera aphidicola sub species Acyrthosiphon Pisum, or Buchnera sp. APS. Named after Paul Buchner, a pioneer in the field of symbiotic microbiology of sap sucking insects.

Buchnera sp. APS is a type of microbe found in the gut of pea aphids (Acyrthosiphon Pisum). The endosymbiotic relationship between these two organisms goes back over 160 million years and is tied to the creation of 5 essential amino acids that two organisms share. The pea aphids maintain this relationship by passing the bacteria through their eggs using a cell called a bacteriocyte (1). The bacteriocyte is really where the study of Buchnera aphidicola begins. In 1858, Thomas Huxley was describing an organ-like structure called a mycetoma – a collection of bacteriocyte cells in an organ like structure (2). Huxley thought that this mycetoma only contained granules of yolk material used for the aphid eggs, but in 1910 Pierantoni and Sule disproved this idea and showed that the granules were Buchnera microorganisms (2).

Paul Buchner entered the scene in the early 20th century alongside Pierantoni and Sule and furthered the understanding of the aphid/ Buchnera symbiosis. Using electron microscopy, Buchner discovered that the bacteriocytes housed the Buchnera cells in its cytoplasm and that there are 3 membranes that separate the Buchnera from the bacteriocyte – an inner and an outer Gram-Negative membrane and a 3rd that envelopes the Buchnera cell known as the symbiosomal membrane that is created by the aphid (2). In addition to structural elements, Buchner theorized the nutrient relationship of the symbionts – meaning he believed that the aphids and the bacteria supplied each other with amino acids that the other couldn’t make on their own. These theories were later confirmed by a series of diet assays, aposymbiotic aphids - meaning living apart from - and whole genome sequencing from the 1960s to the 1990s (2).

The genome of Buchnera sp. APS was sequenced using whole genome random shotgun sequencing – a method which uses a library of the bacteriocytes containing the Buchnera that have been sheared into random fragments, blunted, and subjected to multiple rounds of sequencing and fragmentation to obtain a reading (1). The result of the sequencing revealed that genome consisted of one circular chromosome of 640,861 bp size, and two circular plasmids – the pLeu plasmid of 7,786 bp size with 7 ORFs containing the leu ABCD operon and the pTrp plasmid that contains at least 2 tandem repeats the trpEG operon that generates 12-16 copies of Tryptophan for the host (1). The Buchnera genome codes for 574 protein genes and 36 RNA genes, of these genes 55 genes are used to code nutrients for the host (1).Interestingly enough, while the Buchnera contain so many nutrient based genes, it lacks many genes needed to synthesize structural lipids for its membranes – these elements are provided to the bacteria by the host and further cement the endosymbiotic relationship.

All this information is well and good, but what exactly are we trying to learn from these gut buddies? Why is this important to us? By diving deeper into the Buchnera and pea aphid relationship, we have the potential to learn how co-adaptive evolution occurs and how to separate endosymbionts in ways that could affect our work in gene transfers and protein synthesis. Recent studies in weevils found that endosymbionts would leave the bacteriocytes when antimicrobial genes responsible for symbiont growth and transmission were silenced (3). Aside from the mechanisms, we can learn a bit more about Eukaryotes by studying these endosymbiotic relationships. Think back to introductory biology and the endosymbiotic theory – it is believed that the organelles of our cells and other eukaryotes evolved from bacterial cells that formed similar endosymbiotic relationships to that of the aphid and the Buchnera (4). By studying the aphid and Buchnera, we can see how two species adapt to each other and supply essential products of life.

Now, these are just my thoughts on why I think these discoveries are important, but think about how much further we could push our knowledge of the human body if we understood our organelles or bacterial relationships better – like in areas of mitochondrial mutations or protein mutations – if we can figure out how other organisms share and separately synthesize essential proteins, perhaps we can apply similar systems in health treatment to compensate for these mutations. If we keep furthering our base knowledge, who knows what we could learn or tools we can develop? After all, CRISPr came from yogurt bacteria and now it is being used to modify the human genome, perhaps diabetes treatments could come from the mechanisms of the endosymbionts that would allow us to synthesize insulin outside of the pancreas? While pea aphids and their gut buddies seem insignificant in the scope of human knowledge, what lies in the stomach could be the key to a world of new possibilities.

Thank you for listening to the Genomics Revolution, this is Hannah Mann, signing off.

References
(1) Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y., & Ishikawa, H. (2000). Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature, 407(6800), 81-86. doi:10.1038/35024074
(2) Shigenobu, S., & Wilson, A. C. (2011). Genomic revelations of a mutualism: the pea aphid and its obligate bacterial symbiont. Cellular and molecular life sciences : CMLS, 68(8), 1297-309.
(3) Login, F. H., Balmand, S., Vallier, A., Vincent-Monegat, C., Vigneron, A., Weiss-Gayet, M., . . . Heddi, A. (2011). Antimicrobial Peptides Keep Insect Endosymbionts Under Control. Science, 334(6054), 362-365. doi:10.1126/science.1209728
(4) Tamames, J., Gil, R., Latorre, A., Peretó, J., Silva, F. J., & Moya, A. (2007). The frontier between cell and organelle: Genome analysis of Candidatus Carsonella ruddii. BMC Evolutionary Biology, 7(1), 181. doi:10.1186/1471-2148-7-181

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Hello all, this is Tae’lor Jones from the 2019 Hiram College Genetics course, and today on this episode I will reveal some interesting information on the genome Thermotoga maritima MSB8, which I will refer to as T. maritima from here on out. So, sit back and fasten your seat belts because you’re going to learn all the incredible wonders of this amazing organism just within 3-5 minutes! T. maritima, a non- spore- forming, or in other words non-pathogenic, rod -shaped bacterium belonging to the order Thermotogales, was originally isolated from a geothermal heated marine sediment near Vulcano, Italy, and has an optimum growth temperature of 80ºC (176ºF). Though it is capable of growing in waters of 55-90ºC (131-194ºF) this is the only bacterium known to grow at such a high temperature; the only other organism known to live in environments this extreme are members of the domain, Archaea. This similarity suggests that a lateral gene transfer may have occurred between thermophilic Eubacteria and Archaea within ancient times. To further explain T. maritima’s unique evolutionary relationship to other microbial species the genome was sequenced from the type strain T. maritima MSB8, which was utilized, and when using the whole-genome random sequencing method to identify this lateral relationship you would not believe what was discovered. 

Before pondering on the amazing discovery lets discuss some general features of the genome T. maritima. While being a single circular chromosome consisting of 1,860,725 base pairs and encoding for 1,877 proteins within its genome it has several heat and cold shock proteins that are most likely involved in metabolic regulation and response to environmental temperature changes (2). Of the eubacteria sequenced to date, T. maritima has the highest percentage (24%) of genes that are most similar to archaeal genes. Eighty-one archaeal like genes are clustered in 15 regions of the T. maritima genome that ranges in size from 4 to 20 kilobases. Due to conservation of gene order between T. maritima and Archaea in many of the clustered regions unraveled the amazing discovery of the lateral relationship between Eubacteria and Archaea (2). Genome analysis also discovered numerous pathways involved in the degradation of sugars and plant polysaccharides suggesting that the environment in which T. maritima is found is rich in organic material, with the predominant mechanism of transport being ATP (2). While making all these tremendous findings highly important in stabilizing the fluidity of the membrane at such high temperatures. 

While listening to this episode I know you’ll may be wondering how this organism serves any relevance within the world and why one should be so intrigued about understanding its magical works. Well, your curiosities are about to be unfolded. As an anaerobic fermentative chemoorganotrophic organism, simply referring to a requirement of an organic source of carbon and metabolic energy (2). T maritima catabolizes sugars and polymers and produces carbon dioxide and hydrogen gas as by-products of fermentation, for review the meaning of fermentation is defined as a metabolic process that produces chemical changes in organic substrates through the action of enzymes. This organism is also capable of metabolizing cellulose as well as xylan: a polysaccharide found in plant cell walls, yielding H2 (hydrogen gas) that could potentially be utilized as an alternative energy source to fossil fuels (2). Additionally, this species of bacteria can reduce Fe (III) to produce energy using anaerobic respiration (2). Collectively, these attributes indicate that T. maritima has become resourceful and capable of metabolizing a host of substances in order to carry out its life processes. 

Since the initial findings of this organism’s genome many experiments have been conducted with the utilization of T. maritimas genome sequence. For instance, Swapnil et al. grew this hyperthermophilic bacterium on a variety of carbohydrates to determine the influence of carbon and energy source on differential gene expression (3). With the wide-ranging collection of such networks in T. maritima allowed one to suggest the capabilities of this organism being able to adapt to a variety of growth environments containing carbohydrate growth substrates (3). Shannon et al. discovered that beyond the information obtained for T. maritima, expression-based strategies can be used for improving genome annotation in other microorganisms, especially those for which genetic systems are unavailable (1). All of which was empathized after a comprehensive analysis of genome wide expression patterns during growth of the hyperthermophilic bacterium T. maritima on 14 monosaccharides and polysaccharide substrates that were undertaken with the goal of proposing carbohydrate specificities for transport systems and transcriptional regulators (1). 
 Who would’ve known that this rod-shaped organism within the outer membrane of the cell would demonstrate such complexity? While being the only known organism to live in such extreme environments to do so. Thanks for listening. 

References:
(1) Conners, Shannon B.; Montero, Clemente I.; Comfort, Donald A.; Shockley, Keith R.; Johnson, Matthew R.; Chhabra, Swapnil R.; Kelly, Robert M. (2005). “An Expression Driven Approach to the Prediction of Carbohydrate Transport and Utilization Regulons in the Hyperthermophilic Bacterium Thermotoga maritima” Journal of Bacteriology. 187(21): 7267-7282.
(2) Karen E. Nelson; Rebecca A. Clayton; Steven R. Gill; Michelle L. Gwinn; Robert J. Dodson; et al (1999). “Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima” Nature. 399 (6734), 323-329.
(3) Swapnil et al (2003). Carbohydrate-induced differential gene expression patterns in the hyperthermophilic bacterium Thermotoga maritima. The Journal of Biological Chemistry. 278:7540-7552

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Welcome to Genomics Revolution! My name is Nicole Ryman and I will be hosting this episode to discuss the genome of a bacterium with a Guinness World Record! Deinococcus radiodurans  claimed the record for “Most radiation-resistant lifeform” by being capable of withstanding 1.5 million rads of gamma radiation, which is about 3,000 times the lethal amount to kill a human. The bacterium is capable of not only surviving, but also reproducing in environments that would be lethal for any other organism. Deinococcus radiodurans R1 was the first strain of deinobacteria to be discovered by Arthur W. Anderson in 1956 after observing red bacteria in spoiled canned meat after being exposed to radiation (White, et al. 1999). 

The secret to this bacteria’s profound resistance to extreme conditions? It’s multi-genomic structure! The D. radiodurans R1 genome is composed of two chromosomes, ranging in size between 2.65 Mbp and 412 kbp, one megaplasmid of 177 kbp, and one small plasmid of 46 kbp (Hau et al. 2017). Overall, the genome encodes about 3,195 predicted genes. The bacterium carries between four and ten copies of its genome, rather than the usual single copy. The additional genomes seem to allow the bacterium to recover at least one complete copy of its genome after exposure to extreme conditions such as radiation, oxidation, desiccation, or UV light without dying or undergoing induced mutation (Eggington et al. 2004). 

A combination of factors has positioned D. radiodurans as a promising candidate for the study of mechanisms of DNA damage and repair, as well as developing beneficial operations for toxic waste cleanup and stabilization of radioactive waste sites. The D. radiodurans sequencing project was funded by the US Department of Energy (DOE), which is interested in using the bacterium in environmental cleanup. DOE is responsible for multiple radioactive waste sites around the country, some of which are contaminated with heavy metals and toxic chemicals. Researchers have produced new strains of the bacterium to detoxify toxic waste sites which contain radioactive material (Brim et. al 2000). 

Within the United States, radioactive wastes have contaminated millions of cubic yards of soil and trillions of gallons of ground water. Researchers have engineered multiple strains of D. radiodurans to convert heavy metals, organic and inorganic chemicals found in radioactive waste sites into a much less toxic form. These genetically engineered strains may not be able to completely dispose of the radiation, but they may accelerate the cleanup process and save money (Brim et al., 2006). Meanwhile, scientists are also pursuing more “out of this world” opportunities of D. radiodurans—outer space. The organism could be used in simulations to help scientists predict where to search for life on Mars and other planets. In addition, researchers suggest further potential uses of D. radiodurans in space. The bacterium could pose a use for space travel sewage treatment processes or in environmental engineering to make the surface more suitable for human colonization (Leuko et al. 2017).

Supposedly, there is no environment on Earth that exposes life to more than a fraction of the amount of radiation this bacterium can withstand. Since this bacterium is often isolated from extreme environments where severe conditions of temperature, pH, salts, or toxic compounds are present, genome analyzation offers the opportunity to understand how cells are able to exist and restore damaged chromosomal DNA in harsh conditions. Clearly this bacterium’s remarkable capabilities of cleaning up toxic waste, exploring extreme environments, and engineering new processes, should be continually studied for society’s benefit. Understanding the substantial endurance of D. radiodurans of extreme physical, chemical, and biological conditions opens up a field of indefinite possibilities… maybe anything is possible after all. Thanks for listening. 

References:

Brim, H., S. McFarlan, J. Fredrickson, K. Minton, M. Zhai, L. Wackett, and M. Daly. 2000. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments. Nature biotechnology 18: 85-90. 

Brim, H., et al. 2006. Deinococcus radiodurans engineered for complete toluene degradation facilitates Cr(VI) reduction. Microbiology 152: 2469-2477. 

Eggington, J., N. Haruta, E. Wood, and M. Cox. 2004. The single-stranded DNA-binding protein of Deinococcus radiodurans. BMC Microbiology 4: 1–12. 

Hau, X., Y. Hua. 2016. Improved complete genome sequence of the extremely radioresistant bacterium Deinococcus radiodurans R1 obtained using PacBio single-molecule sequencing. American Society for Microbiology 4: 1-2. 

Leuko, S., et al. 2017. On the stability of Deinoxanthin exposed to Mars conditions during a long-term space mission and implications for biomarker detection on other planets. Frontiers in microbiology 8: 1-11. 

White, O., et al. 1999. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286: 1571-1577.

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Welcome to Genomics Revolution. This is Kerington Vickers from the 2019 Hiram College Genetics course hosting this episode on the genome of Chlorobium tepidum TLS. I will call this C. tepidum from here on out. This strain of thermophilic green sulfur bacteria was isolated from acidic high sulfide hot springs on North Island, New Zealand (3). 

This organism is part of the Chlorobiaceae family which are also called green sulfur bacteria. Green sulfur bacteria use sulfide ions as electron donors for photosynthesis. They are known to be found in depths of up to 145 m in the Black Sea, with low light availability. The genome of C. tepidum has been completely sequenced. The single circular chromosome in this organism is approximately 2.15 Mb in size. There are a total of 2,337 genes (of these genes, there are 2,245 protein coding genes and 56 tRNA and rRNA genes). This organism can be found growing in dense mats over hot springs as well as other warm muds and bodies of water that contains sufficient hydrogen sulfide.
So far we know that C. tepidum were found in hot springs in New Zealand, the whole genome is sequenced and contains 2,227 genes and we learned about green sulfur bacteria... what else is next? I bet you are wondering the growing conditions are for C. Tepidum.

It is the only known thermophilic member of its family which means it thrives at relatively high temperatures. It has been recorded to grow optimally at 48 degrees Celsius (this is 118.4 degrees Fahrenheit). If that was us we would not be able to survive in this type of environment for long periods of time. They grow best in pH levels between 6.0 and 4.5. They described the bacteria as gram negative and rod-shaped. It is able to harvest light through special photsynthetic systems known as chlorosomes.

These unique features make studies of Chlorobia important for understanding the evolution and mechanisms of photosynthesis and energy metabolism. So what makes this organism survive in these environments? Why should we care about this organism?

C. tepidum is a valuable model for the green sulfur bacteria because it is easily cultivated and naturally transformable. According to Wahlund et al., C. tepidum while under photautotraphic conditions can be cultured in about two hours which makes this faster than any other anoxygenic phototroph. This makes it the ideal tool for research in basic studies of photosynthesis and autotrophy in green bacteria.

Unlike Chromatium and Thiothrix who are two other sulfur-producing bacteria, C. tepidum deposits the elemental sulfur outside the cell. When comparing genomes, many of the genes are highly conserved among the photosythetic species. Their function is not clear but they are suspected to play an important role in photosynthesis or photobiology. C. tepidum has been shown to have strong similarities to many Archael species between their metabolic processes.
C. tepidum contains duplications of genes involved in biosyntheic pathways for photosynthesis and the metabolism of sulfur and nitrogen. Unlike regular photosynthesis that synthesizes foods from carbon dioxide and water and generates oxygen as a byproduct, while Chlorobia performs anoxygenic photosynthesis. This is helpful for humans unlike how Chlorobium tepidum does it because we cannot use their byproducts of elemental sulfur.

It is important to know that currently C. tepidum does not cause any diseases. Mutants have been found and studied such as bchK in C. tepidum and it lacked BChl c., this means the mutant grew slower. BChlc is important because all photoautotrophic organisms rely on chlorophyll (Chl) or bacteriochlorophyll (BChl)- based photosynthesis. BChl c in C. Tepidum is a mixture of four homologous that carry different modifications at the C8 and C12 positions and had been demonstrated that these side chains are derived from methylation reactions involving SAM. There is so much to learn from this genome and I hope this sparks your interest in it to further your knowledge, I know it has for me. Thanks for taking the time to listen to this guest speaker podcast.

References:
(1) Eisen et al., 2002.  Proceedings of the National Academy of Sciences USA 99:9509-9514.  The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic green-sulfur bacterium.
(2) Frigaard et al., 2003.  Photosynthesis Research 78: 93-117.  Chlorobium tepidum: insights into the structure, physiology, and metabolism of a green sulfur bacterium derived from the complete genome sequence.
(3) Frigaard et al., 2002.  Journal of Bacteriology 184:3368-3376.  Chlorobium tepidum mutant lacking bacteriochlorophyll c made by inactivation of the bchK gene, encoding bacteriochlorophyll c synthase.
(4) Wahlund et al., 1995.  Journal of Bacteriology. 173:2583-2588.  Genetic transfer by conjugation in the thermophilic sulfur bacterium Chlorobium tepidum.
(5) Wahlund et al., 1991.  Archives of Microbiology 156:81-90.  A thermophilic green sulfur bacterium from New Zealand hot springs, Chlorobium tepidum sp. Nov.

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Welcome back to Genomics Revolution.  This is Brad Goodner.  I freely admit that I am a sports nut.  I love watching humans compete against themselves and each other under stressful conditions.  I also grew up intrigued by extreme athleticism in non-human animals – the world’s fastest mammal or longest-distance migrant.  I don’t think I am alone in being intrigued by survival and even more so by high performance under conditions that seem way beyond the norm.  I bring this up today because when it comes down to it, microbes are the ultimate extreme athletes (1).

Many of the microbial genomes we have considered so far come from organisms that grow best under conditions that we humans consider very comfortable – plenty of oxygen as an electron acceptor for aerobic respiration, plenty of reduced organic compounds as food (think carbs and fats), temperatures between 20 and 45oC, a neutral pH around 7, and at most a touch of salt in the water.  For example, Agrobacterium tumefaciens strain C58, an organism I have worked on for over 25 years and spoke on in episode 7, would happily grow in a flask strapped to my hip containing a watery extract of the same food that I eat.  That said, many other microbes grow best under one or more conditions that we would consider extreme.  Let us consider four extreme conditions.

First and foremost, we humans are totally dependent on oxygen and we struggle to imagine life without it.  Antoine von Leeuwenhoek published a short report in 1680 on two extracts of ground pepper, one in an open tube and the other in a tube that was heat-sealed (2).  Leeuwenhoek was surprised that the sealed tube not only contained microbes, what he called “animacules”, but that the most abundant microbe in the sealed tube was not seen in the open tube.  He did not know how to explain the differences he saw, but his report certainly documents what we now know is anaerobic microbial life a century before the discovery of oxygen and its importance for aerobic life. Louis Pasteur, one of the founders of experimental microbiology, was the first to truly understand and write about anaerobic microbes in 1861 as he was studying a fermentation that produces butyric acid as a byproduct (3).  Such a fermentation leads to rancid butter.  Pasteur figured out that the microbes producting butyric acid grew in the absence of oxygen, but even more surprising that these same microbes could be killed by bubbling oxygen through the solution.  He wrote the first description of what we call obligate anaerobes.  The Bifidobacterium strains discussed in episode 17 do well in our colons because of the absence of oxygen at the end of our GI tracts.  The pathogen Clostridium perfringens in episode 24 and the archaeaon Methanococcus jannaschii in episode 28 are also obligate anaerobes.  Other anaerobes don’t use oxygen but are not killed by it.  They are called aerotolerant anaerobes.  Going one step further, some anaerobes grow fine without oxygen but will use oxygen for aerobic respiration when possible.  The pathogens E. coli O157:H7 from episode 8 and Vibrio cholerae strain El Tor 16961 from episode 9 act this way and are called facultative anaerobes.  Finally, some microbes use oxygen but only at very low concentrations.  These microaerophiles, as we call them, include the pathogen Campylobacter jejuni discussed in episode 16.  We humans cannot do any of these no-oxygen or very low-oxygen alternative growth strategies for more than a few minutes without severe consequences.

Second, I grew up in north central Texas where summer days reached 100 oF (37.8 oC) and more than occasionally 110oF (43.3oC).  Even in the shade, the heat would just wear you out.  We humans do best at slightly cooler temperatures and so do many microbes known as mesophiles whose optimum growth temperature is between 20 and 45oC.  Organisms whose optimum growth temperatures are between 45 and 80oC are called thermophiles including the archaeaon Thermoplasma acidophilum from episode 10.  Aeropyrum pernix strain K1 from episode 27 takes it up another notch as a hyperthermophile whose optimal growth temperature is greater than 80oC.  For reference, 80oC is twice as hot as your steaming hot tub on the back deck.  Yowsa! 

Third, if you are like me you have jumped when you have accidentally gotten some lemon juice in your eye or in a cut.  Imagine if you were bathed in lemon juice all the time.  That mouth-puckering citrus liquid has a pH between 3 and 2, meaning it is 10,000 to 100,000 times more acidic than water.  How can anything live in such an environment?  Ask Thermoplasma acidophilum who we already know is a thermophile, but it is also an acidophile that thrives at pH 2 and can still grow at pH as low as 0.5.  On the other end of the pH spectrum are alkaliphiles who prefer a very basic lifestyle at pH >9.

Fourth and finally, have you every soaked your aching feet in some water containing Epsom salt?  Great for a short while, but what living in a very salty solution all the time.  Our bodies spend a lot of energy and regulatory power to maintain internal cellular salt concentrations in a very narrow range.  So do most other organisms across all 3 domains of life.  Organisms in salt water have to work that much harder to osmotically regulate.  Seawater has an average salinity of 3.5% (35 grams of salts per liter), but there are microbes who love living in the Great Salt Lake or the Dead Sea at up to 27% and 33.7% salinity, respectively.  Halobacterium strain NRC-1 from episode 26 is one such extreme halophilic member of the Archaea that is part of a lineage that evolved a novel salt-dependent growth strategy that involves accumulating high internal levels of KCl instead of NaCl.

In the next several episodes, we will hear about many more extremophiles who live in amazing places right here on Earth.  Our guides will be more of my students from the 2019 Hiram College Genetics course.  Stay tuned.

For more information:
(1) Merino et al., 2019.  Living at the Extremes: Extremophiles and the Limits of Life in a Planetary Context.  Frontiers in Microbiology 10:780.

(2) Gest, 2004.  The discovery of microorganisms by Robert Hooke and Antoni van Leeuwenhoek, Fellows of the Royal Society,  The Royal Society 58:12.

(3) Pasteur, 1861. Animal infusoria living in the absence of free oxygen, and the fermentations they bring about [translated from French].  Competes Rendus de l’Academie des Sciences 52:344-7.

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Brad Goodner here. It is great to be back with you after some more student-hosted episodes of Genomics Revolution. Did the title of today’s episode bring a chuckle or a smile to your face? It is one of many memorable lines from one of my favorite movies – The Princess Bride. Vizzini, the big-brained Sicilian kidnapper says “Inconceivable” every time the so-called “Man in Black” makes it past a huge obstacle. Vizzini’s hired swordsman, the Spaniard Inigo Montoya finally turns to Vizzini and says “you keep using that word. I don’t think it means what you think it means.”

Inigo Montoya’s phrase could also apply to a word all biologists know – prokaryote. It is in my opinion, as I tell my students early and often, “the dreaded P-word” that has outlived its usefulness and should never be used again.

Prokaryote literally means “before kernel” or “without kernel” where kernel refers to a membrane-bound nucleus within a cell. Prokaryote is literally the antonym of eukaryote which means “true kernel” or having a membrane-bound nucleus within a cell. The prokaryote-eukaryote distinction played a major role in how biologists, and especially microbiologists like myself, looked at organisms in the mid- to late-20th century as biology became more cellular-focused. The terms are attributed back to Edouard Chatton, a French biologist who studied protozoa. As described in a great 2005 review by Jan Sapp (1), Chatton first used the terms prokaryote and eukaryote in a 1925 paper where he was trying to phylogenetically place a particular protozoan species. Building on work that goes back to Haeckel and others in the late 19th century, Chatton made a case that his protozoan species of interest shared essential features seen in other eukaryotes: a membrane-bound nucleus, a flagella or cilia with associated basal bodies, and exhibited mitosis. On the other hand, bacteria, spirochaetes, and blue-green algae, what we now call Cyanobacteria, lacked these structures and were lumped under the term prokaryote. Chatton didn’t care about relationships within prokaryotes, but he did care deeply that his beloved protozoans were eukaryotes just like fungi, animals and plants.

So why should we care about this term prokaryote so much? It turns out that Chatton’s terms gave two famous microbiologists, Roger Stanier and C.B. van Niel, a way out of a problem that had vexed them for over two decades. Stanier and van Niel wanted a robust way to infer evolutionary relationships amongst bacteria like E. coli, Pseudomonas aeruginosa, Treponema pallidum, and Mycobacterium tuberculosis that we have heard about in past Genomics Revolution episodes. They had tried using cell shape and biochemical tests with no success. In their 1962 paper entitled “The Concept of a Bacterium” (2) Stanier and van Niel had reached a point of utter frustration, but Chatton’s terms at least gave them a way of saying what these bacteria WERE NOT. They WERE NOT EUKARYOTES. They were prokaryotes.

Whether or not Stanier and van Niel believed that all prokaryotes share a more recent common ancestor with each other than any of them shared with eukaryotes is not totally clear, it was this very hypothesis that most microbiologists accepted just on the grounds of cell complexity. Robert Whittaker in his 1969 Science article (3) appeared to agree with this hypothesis with his five kingdom model for classifying cellular life on Earth with all prokaryotes in the kingdom Monera. Yet around this same time, data started emerging that held the real answer. Emile Zuckerkandl and Linus Pauling in 1965 talked of DNA, RNA and protein sequences as information macromolecules of history because mutations were markers laid down over evolutionary time (4). It kept getting easier and cheaper to sequence these linear polymers and in 1977, Carl Woese and George Fox had enough data for the right macromolecule, the 16S/18S rRNA found in all ribosomes in all cellular lifeforms, to answer the question (5). Do all eukaryotes share a more recent common ancestor with each other than with prokaryotes? YES. Do all prokaryotes share a more recent common ancestor with each other than with eukaryotes. NO. The prokaryotes split into two groups that appeared to be equidistant from each other as they were from eukaryotes. Woese and Fox called these two groups Eubacteria and Archaebacteria and suggested that these groups were two of three “primary kingdoms” for cellular life on Earth. With more data in later papers, the names were shortened to Bacteria and Archaea to sit alongside of Eucarya not as “primary kingdoms” but rather as the three domains of life, the highest most encompassing evolutionary groupings. Even the New York Times took notice and put it on the front page – “Third Domain of Life Found.”

As a freshman at Texas A&M University in the early 1980’s, I got the chance to hear George Fox give a series of visiting lectures on evolution. I don’t think I really grasped at that time how much Woese and Fox had turned the microbial world upside down. As more and more data accumulated, it became increasingly clear that the Archaea were a truly distinct group. At first, we thought they were limited to a few weird extreme habitats, but now we know they are everywhere including inside our own GI tracts.

We heard about one Archaea genome several episodes ago, that of Thermoplasma acidophilum. In the next few episodes we will hear about a few more genomes from this 3rd domain of life. Stay tuned to expand your horizons, but please let the dreaded P-word Rest In Peace. It doesn’t mean what you think it means.

References:
(1)Sap, J., 2005. Microbiology & Molecular Biology Reviews 69:292-305. The prokaryote-eukaryote dichotomy: meanings and mythology.

(2)Stainer, R. & C.B. van Niel, 1962. Archives Microbiology 42:17-35. The concept of a bacterium.

(3)Whittaker, R.H., 1969. Science 163:150-163. New concepts of kingdoms of organisms.

(4)Zuckerkandl, E. & L. Pauling, 1965. Journal Theoretical Biology 8:357-366. Molecules as documents of evolutionary history.

(5)Woese, C.R. & G.R. Fox, 1977. Proceedings of National Academy of Sciences USA 74:5088-5090. Phylogenetic structure of the prokaryotic domain: the primary kingdoms.

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Hey, Brad Goodner here.  It has been several episodes since I last got the chance to talk with you.  I hope you are enjoying the Survey of Genomes episodes guest hosted by students from my 2019 Hiram College Genetics course.  There are more of those episodes to come, but I wanted to jump in and provide some context.  So far, 4 out of the 7 student-hosted episodes have dealt with human pathogens.  Why sequence their genomes?  Don’t we just want to kill pathogens in order to cure infectious disease?

No doubt about it, healthcare practitioners not only want to diagnose infectious disease but also to treat it in such a way that the pathogen is no longer hurting the patient.  Over the past 70+ years, treatment for most non-viral infectious diseases has included the use of antimicrobial compounds like penicillin.  Each antimicrobial compound has a specific target, usually an enzyme or other larger macromolecule whose action is essential to the life of the pathogen.  In a perfect world, the antimicrobial compound will kill or inhibit the growth of the pathogen without impacting the patient.  However, the closer the evolutionary relationship of the pathogen to its host, the harder it is to find a pathogen-specific target.  For example, it is much harder to kill a worm infection than to kill a bacterial infection.  Sequencing the genome of a particular pathogen can show us how similar pathogen target macromolecules are to those in potential patients.  If there is sufficient difference, we have more options for how to stop the pathogen.  A genome sequence also illuminates the mechanisms by which the pathogen may resist particular antimicrobial compounds and a genome sequence may allow us to find potential new antimicrobial targets that are unique to a particular pathogen.

While antimicrobials have greatly improved human health, they are not foolproof.  The evolution of resistance in many pathogens has made it much harder to control certain infections.  However, if we understand how pathogens cause disease then maybe we can stop them from hurting us without having to kill them.  This relatively new idea is called the anti-infective strategy and it may allow us to control infections without the worry of selection for resistance in the pathogen.  A genome sequence of a pathogen compared to a closely related nonpathogen can help us better understand how that particular pathogen interacts with a potential host and how it might take advantage of the host and cause disease symptoms.  This knowledge may give us new options for blocking attachment of pathogen cells to our cells or for blocking one of its mechanisms for manipulating our cells for its own advantage.

Finally, better understanding how particular pathogens take advantage of a host and cause disease symptoms can provide us with pathogen-based tools that might have a future positive medical use under the right conditions.  For example, you have probably seen TV commercials for BoTox as a treatment for chronic migraines or to decrease frown lines or crow’s feet on one’s face.  BoTox is a medical treatment involving a known bacterial toxin called botulinum toxin A.  This toxin targets neuromuscular junctions and causes muscle paralysis in the relaxed state, also called flaccid paralysis.  Too much of this toxin through botulism food poisoning and muscles all over the body stop working.  This can lead to a risk of death due to impairment of breathing or other critical muscle-related functions.  However, very tiny amounts of this toxin when applied to very specific muscles in the face or scalp can relax those muscles for long periods of time providing relief from problems due to uncontrollable muscle contractions.  There are several other microbial toxins that have been turned into medicines or research tools.

There is an old saying that you should keep your friends close but your enemies even closer.  That can also apply to a pathogen through the use of its genome sequence.  Thanks for listening. Now lets get back to our survey of genomes and see how other life forms on Earth make a living.  See you next time.

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Welcome to Genomics Revolution. This is Sam Hitchcock from the 2019 Hiram College Genetics course and I will be hosting this episode on the genome of Ralstonia solanacearum GMI1000. R. solanacearum is a beta-proteobacterium, which is a class of proteobacteria that occupy diverse environments as pathogens living within hosts. R. solanacearum is no different, this pathogen is soil borne and infects roots. Different strains of R. solanacearum are able to attack 200 different hosts. The first strain of R. solanacearum to have its genome sequenced was Ralstonia solanacearum GMI1000, this took place in 2002 (3). The complete genome was sequenced in order to better understand pathogenicity and host response.

The genome of Ralstonia solanacearum GMI1000 is 5,810,922 base pairs long. 3,716,413 of which lie in a circular chromosome. The other 2,094,509 base pairs are in a circular plasmid. It was found that both of these replicons have a mosaic structure, which provides evidence for acquisition of genes through horizontal gene transfer (2). The genome was also found to encode 5,129 predicted proteins. It was found that many of the encoded proteins were equivalent to known pathogenetic proteins in other organisms. This further supported the idea that Ralstonia solanacearum GMI1000 is a pathogen. Studying the pathogenicity of this organism was also one of the main goals for sequencing this organism (2). 

Ralstonia solanacearum GMI1000 strain most notably targets tobacco plants and tomato plants, when it attacks these plant it causes them to wilt. The genome sequence of this strain was used to study what caused this wilting. It was found that a double inactivation of both the avrA gene and the popP1 gene allowed GMI1000 to wilt the tobacco plants. Both avrA and popP1 genes are type III secretion system effectors (1). One interesting fact that the genome of GMI1000 showed was that there were over 40 possible type III secreted effector proteins identified. Then by comparing with other genomes it was found that bacterial plant pathogens and animal pathogens have their own distinct arrays of specialized type III dependent factors. Meaning that the effector proteins in this organism could not be found in an animal pathogen (2).

The sequencing of Ralstonia solanacearum GMI1000 allowed scientists the opportunity to study plant pathogens and host response at a genomic level. It also showed how this strain was able to cause wilt in many host plants. Then through later genome sequencing of different R. solanacearum strains researchers found that almost all genes encoding type III secretion system effectors are conserved. However, the promoters differ between differing strains. In the future these differences can be looked at to understand why certain strains attack certain plants (3).
Thank You for watching.

References:
 (1) Poueymiro et al., 2009. International Society for Molecular Plant-Microbe Interactions (5):538-50. Two type III secretion system effectors from Ralstonia solanacearum GMI1000 determine host-range specificity on tobacco.
(2) Salanoubat et al., 2002. Nature 415, 497-502. Genome sequence of the plant pathogen Ralstonia solanacearum.
(3) Ying et al., 2017. Frontiers in Microbiology 8:974. Genome Sequencing of Ralstonia solanacearum CQPS-1, a Phylotype I Strain Collected from a Highland Area with Continuous Cropping of Tobacco. 

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Hello all, today I will be talking about Halobacterium NRC-1, or ATCC 700922. Halobacterium is a 
genus of Halobacteriaceae. The Halobacterium species is actually not a bacteria, however, it belongs to 
the archea domain. Halobacterium was originally studied in the 1960s, brine shrimp, who are filter 
feeders, would consume the Halobacterium, in turn, flamingos would eat the brine shrimp, giving them 
the pigment that they are. The genus Halobacterium ("salt" or "ocean bacterium") consists of several 
species of the Archaea with an aerobic metabolism which requires an environment with a high 
concentration of salt; many of their proteins will not function in low-salt environments. These organisms 
can be found in the Great Salt Lake, the Dead Sea, and other waters with high salt concentrations.
Halobacterium is used for postgenomic analysis. Halobacterium is also used as biomediation 
agents, they have been used for soil remediation . Some strains of Halobacterium are being explored for 
medical applications for their radiation-resistance mechanisms. Bacterioruberin is a carotenoid pigment 
found in Halobacterium which decreases the bacteria’s sensitivity to UV radiation and other types of 
radiation. It has been shown in studies that the absence of bacterioruberin increases the sensitivity of 
the bacterium to oxidative DNA-damaging agents. Halobacterium also exhibits high intracellular 
concentrations of potassium chloride which has also been shown to confer radiation resistance. 
Halobacterium are also being explored for the pharmaceutical applications of bioactive compounds they 
produce, including anticancer agents, antimicrobial biosurfactancts, and antimicrobial metabolites. They 
possess genes for DNA replication as well.

The genome is 2,571,010 base pairs long, there are 3 circular strands, the large chromosome is 
2,014,239 base pairs long while the other two mini chromosomes are 191,346 and 365,425 base pair 
long respectively. The minichromosomes are A-T rich, they have a GC content of 58% and 59% compared 
to the large chromosome which possesses a GC content of 68%.

References
Balakrishnan, Arjun, et al. “Halobacterial Nano Vesicles Displaying Murine Bactericidal Permeability-
Increasing Protein Rescue Mice from Lethal Endotoxic Shock.” Nature News, Nature Publishing Group, 
20 Sept. 2016, www.nature.com/articles/srep33679.

Evans, Jessica J., et al. “Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium 
Salinarum in Survival of DNA Damage.” MDPI, Multidisciplinary Digital Publishing Institute, 20 Apr. 2018, 
www.mdpi.com/2073-4425/9/4/223.

Halobacterium Sp. NRC-1.” Microbewiki, microbewiki.kenyon.edu/index.php/Halobacterium_sp._NRC-1.

Kennedy, Sean P., et al. “Understanding the Adaptation of Halobacterium Species NRC-1 to Its Extreme 
Environment through Computational Analysis of Its Genome Sequence.” Genome Research, Cold Spring 
Harbor Lab, 1 Jan. 1970, genome.cshlp.org/content/11/10/1641.full.

Ng, Wailap Victor, et al. “Genome Sequence of Halobacterium Species NRC-1.” PNAS, National Academy 
of Sciences, 24 Oct. 2000, www.pnas.org/content/97/22/12176.

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Welcome to Genomics Revolution. This is guest host Stephanie Cipa from the 2019 Hiram College Genetics course. I’m going to be talking about two strains of bacteria today; Bifidobacterium longum NCC2705 and Bifidobacterium longum ssp. longum 35624, previously known as Bifidobacterium infantis 35624. From here out I will be referring to them as B. longum 2705 and B. longum 35624. Lets dive right into it! 

B. longum has one circular chromosome made up of about 2,256,646 base pairs. In B. longum 2705 this creates up to 1,725 proteins. To really understand this bacteria I’m going back to the start of all our human lives, infancy. Babies are cute, squishy, and full of B. longum. In 1899, French pediatrician, Henry Tissier, observed a y-shaped microorganism in the stool of an infant with diarrhea. This microorganism turned out to be B. longum 35624, a strain strongly present in the gastrointestinal tract of babies. These bacteria are mostly anaerobic, gram positive microorganisms that are very helpful to us. Bacterium in the Bifidobacteriaceae family live in the guts of humans and help with digestion. While heavily present in babies, the population of B. longum diminishes over time due to the addition of many other gut microbes that we gain as we mature. B. longum is believed to be acquired by breastfeeding as the mother passes on helpful bacteria to the baby or during birth when the baby passes through the vagina, another location where this bacteria is known to live. These bacteria help by breaking down “nondigestable” plant polymers and oligosaccharides. The result of their fermentation is lactic and acetic acid, two products that help maintain intestinal pH as well. They can also limit the growth of harmful bacteria like E. coli. Understanding bifidobacterium helps us understand how it works with other bacteria in our guts to digest food and how it helps maintain homeostasis.

Many positive results have come from the discovery of B. longum. In 1907, Elie Metchnikoff suggested that this bacteria could be beneficial as a probiotic. It was one of the first to be considered for this role. Probiotics are supplements of live bacteria believed to be helpful for digestion and gut health. B. longum is believed to help bad bacteria pass through the gut easier and ease constipation and diarrhea. The most popular probiotic for Bifidobacterium is B. longum 35624, the strain from infants. It is marketed under the name “Align” and claims to be a purified strain of bifidobacteria meant for every day consumption. Of course there are hundreds of other products out there if this brand doesn’t “align” with your tastes!

Sequencing this genome in 2002 revealed to researches just how well B. longum have adapted to live in the human gut. They found multiple sequences coding for large proteins specialized for catabolism on a variety of oligosaccharides. A few of these proteins appeared to be specialized for “nondigestible” plant polymers. Because of the range of catabolic capabilities, B. longum appear to be well suited for life in the gastrointestinal tract. It also helps them compete with other bacteria present in the gut and may account for why we contain this bacteria from infancy to death.  Furthering these findings in 2007, another team of researchers discovered the sequences for 19 permeases in B. longum 2705 all meant for different carbohydrates. Amongst the genes were permeases for lactose, maltose, fructooligosaccharides, and more.  Again this just shows how well this bacteria has adapted to our personal gut jacuzzi of bacteria and digesting food. 

One more remarkable discovery that stemmed from genome exploration was the link between human breast milk and substrates for enzymes B. longum creates in infant guts. Essentially, researchers analyzed human breast milk and enzymes secreted by B. longum and found that 5 of the most abundant oligosaccharides in the milk were the preferred substrates for those enzymes. The researchers believed this to be an example of coevolution. This means that as humans and B. longum evolved, they effected each other’s evolution. This is fascinating to me because it has taken millions of years for this relationship between human and bacteria to perfect itself. They mutually benefit from this relationship because the bacteria digests abundant milk molecules, making it a strong competitor to survive in the gut, and the infant benefits by being able to digest the breast milk.

All three of these discoveries highlight just how extensively B. longum has evolved and adapted itself to fit into the highly specific environment that exists in the human gastrointestinal tract. Not only does it live there, but it also digests molecules and improves the health of the gut as well. These discoveries help scientists understand how and why certain relationships exist in the world. Not many people would consider the importance of a tiny bacteria for the overall health of a human. But, as B. longum shows us, sometimes it’s the tiny guys who make all the difference and the next time you’re using the restroom and it all goes according to plan, make sure you thank your gut bacteria for all the help in making it possible.

References:
“Bifidobacterium longum NCC2705.” Bacmap Genome Atlas, http://bacmap.wishartlab.com/organisms/125.
“B. Longum- A common probiotic strain.” Humarian, 14 Feb. 2017, https://humarian.com/b-longum-common-probiotic-strain/.
Parche, Stephan, et al. "Sugar transport systems of Bifidobacterium longum NCC2705." Journal of molecular microbiology and biotechnology 12.1-2 (2007): 9-19.
“Proteome-Bifidobacterium longum (strain NCC 2705).” Uniprot, https://www.uniprot.org/proteomes/UP000000439.
Schell, Mark A., et al. "The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract." Proceedings of the National Academy of Sciences 99.22 (2002): 14422-14427.
Sela, D. A., et al. "The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome." Proceedings of the National Academy of Sciences 105.48 (2008): 18964-18969.
“The role of Bifidobacterium longum in a healthy human gut community.” Microbe Wiki, https://microbewiki.kenyon.edu/index.php/The_role_of_Bifidobacterium_longum_in_a_healthy_human_gut_community.

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Hi my name is Brayla Stokes and today will be talking about Aeropyrum pernix K1 and this genome is a species of Archaea.  It was the first crenarcheote which is just a category of Archaea and first aerobic member of Archaea with a complete genome sequence.  This organism helped us link the relationship between the three domains of life.  In 1999 Aeropyrum pernix was discovered while heating and vent marine water sediments in Japan.  It is circular in shape and its diameter is 1 micrometer.  Its genome is 1,669,696 base pairs in size.  We see a growth response starting at 70 degrees Celsius and it continues to function in temperatures up until 100 degrees.  Aeropyrum pernix can be qualified as a hyper-thermophile.

Now the reason the that we should care about this genome is one because it has opened up doors for us to answer question since it was the first of it’s kind to have a complete genome sequence. While doing my research I came across some facts that made me even more curious about the  genome and how it works.  It lacks coding genes for histones which are found in eukaryotes and euryarchaeotes but rather it has genes coding for small basic DNA binding proteins.  These can be functionally compared to histones proteins found in eukaryotes.

Aeropyrum pernix lacks homologs of the prokaryotic cell division genes ftsZ and MinD.  The problems with that is that those two genes are found in all bacteria and archaea that we know of today.  The two genes are also associated with cell division in different species.  So what makes this genome so different that is doesn’t need key factors but can still function as if it has them?  It was found that this genome doesn’t have as many eukaryotic genes compare to other genes.  The reason that I think this is important is because that we are discovering different genome everyday and I don’t think Aeropyrum pernix is the only genome out there that is able to do such a thing.  Therefore it could potentially solve scientific mysteries. 

Lastly the discovery of this genome help solve some problems dealing with the relevance of lateral gene transfer. Which is the sharing of genes between bacteria and archaea.  This help us link how certain species are related and how species have evolved over time.  It is important for us to know what use to work and figuring out why it was no longer need for survival.  Thank for listening to my podcast.  I hope you have retained some of important information that I have presented today. 

References:
David M, F. and W. Ford, D. (2019). Genomics: Lessons from the Aeropyrum pernix genome.  Current Biology. Available at: https://www.cell.com/current-biology/fulltext/S0960-9822(00)80074-3

Kawarabayasi, Y, et al. “Complete Genome Sequence of an Aerobic Hyper-Thermophilic Crenarchaeon, Aeropyrum Pernix K1.” DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes, U.S. National Library of Medicine, 30 Apr. 1999, www.ncbi.nlm.nih.gov/pubmed/10382966.

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Welcome back to Genomics Revolution, this is Kiara Jeffrey from the 2019 Hiram College Genetics course hosting this episode on the genome of Pseudomonas aeruginosa strain PAO1, which I will call P. aeruginosa from now on. This rod-shaped, gram-negative bacterium belongs to the Bacteria class gamma-Proteobacteria and the family Pseudomonadaceae (2). P. aeruginosa was first discovered in 1882 when a French bacteriologist and chemist, Carle Gessard, noticed a blue and green color develop on the bandages of two of his patients’ wounds. Through an experiment, he found that this odd color development was the result of the bacterium’s water-soluble pigments, which turn blue and green when subjected to UV light (3). However, the specific PAO1 strain was not identified until 1954, after it was isolated from a patient’s wound in Melbourne, Australia (2).

If you noticed, both extractions came from wounds, which is quite common for P. aeruginosa and also a major reason why it’s studied. This is because this bacterium is considered an opportunistic pathogen. As a result, it commonly causes infections in individuals with severe burns, UTIs in patients with catheters, and pneumonia in respirator patients (6). However, it is most infamous for its potentially lethal effect in cystic fibrosis patients. The lungs of these patients are similar to other environments that P. aeruginosa inhabits, like coastal marine habitats, soil and marshes, in that they contain moisture. This allows the pathogen to grow large colonies in the lungs of cystic fibrosis patients, which could lead to death (6). In addition to its ability to live in a diverse set of conditions, P.aeruginosa is also resistant to a wide variety of antibiotics and has even become resistant to new ones over the course of treatment (3). Given all of this, scientists decided to sequence its genome, which has since served as a reference for Pseudomonas genetics (2).

P.aeruginosa’s genome consists of 6,264,403 base pairs in a single, circular chromosome and, in total, encodes 5,570 proteins (6). Looking into this bacterium’s large genome has given scientists insight in regards to its abilities to resist antibiotics, to act as a pathogen, and to adapt.

Sequencing the genome gave scientists a better— yet still incomplete— understanding of how P. aeruginosa is resistant to many antibiotics. The pathogen is able to accomplish such a feat in part due to a combination of naturally encoded and imported resistance mechanisms and mutations. It was found that naturally, the pathogen’s genome codes for enzymes, like AmpC cephalosporinase, that break down antibiotics in such a way that they can no longer cause harm (1). P. aeruginosa also decreases the amount of non-specific porin proteins coded for and introduces more specific channels for importing essential nutrients, which serves as a way to limit antimicrobial substances from entering. There are also genes that code for components of outer membrane multi-drug efflux pumps that recognize specific antibiotics and pump them out of the cell. This gives its membrane a relatively low permeability to antibiotics (4). Lastly, it was found that mutations and changes in chromosomes can result in the over-expression of these resistance genes, greatly increasing the bacterium’s ability to be such a resilient organism (3). 

Another fascinating story scientists were able to piece together from sequencing its genome was some of the mechanisms by which this pathogen affects its host. In addition to the resistance genes previously mentioned, some of the 5,570 ORFs also account for a number of virulence factors including pili, flagella, quorum sensing proteins, exotoxin A and Type III secretion systems. The proteases mentioned can function in a variety of ways, which can range from breaking down proteins from the organism that it’s infecting and even killing off competing bacteria within the same vicinity. The Type III secretion systems produce toxins that can kill off some of its host’s cells (5). It’s no wonder these infections are so difficult to treat!

However, even more impressive is P. aeruginosa’s ability to grow in such a wide variety of environments. It can also live off of a wide array of organic carbon sources, but let’s take a look at additional encoded tools. Some of these include quorum sensing genes— which serve to pick up environmental signals and activate/deactivate other genes accordingly, motility switches— which control whether the pathogen remains stationary or moves, biofilm formation, and the regulation of the antibiotic resistance and virulence genes previously mentioned (4). Research has shown that deactivating motility and lowering virulency can actually help P. aeruginosa survive, as it helps to protect them from unfavorable environments. One may think the ability to move from less than optimal conditions would be more advantageous, but it turns out, switching off this gene allows the pathogen to form a colonized, biofilm structure, which— through a safety-in-numbers kind of logic— helps prevent damage from the host’s variety of immune responses and other competing species (4). Amazing.

Thanks for listening.

References:
(1) Hare, N.J. et al., 2012. J. Proteome Res 11:2. Proteomics of Pseudomonas aeruginosa Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin.
(2) Klockgether, J. et al., 2010. J. Bacteriol 192:4. Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains.
(3) Lister, P.D.; Wolter, D.J.; and Hanson N.D., 2009. Clin Microbial Rev 22:4. Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Mechanisms.
(4) Moradali, M.F.; Ghods, S.; and Rehm, B.H.A., 2017. Frontiers in Cellular and Infection Microbiology 7:39. Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence.
(5) Sood, U. et al., 2019. Front. Microbial. 10:53. Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.
 (6) Stover, C.K. et al., 2000. Nature 406. Complete Genome Sequence of Pseudomonas aeruginosa PAO1, an Opportunistic Pathogen.

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Welcome to Genetics Revolution. This is Ashley Redman from the 2019 Hiram College Genetics course hosting this episode of clostridium perfringens. Clostridium perfingens, CP for short, is a gram positive anaerobic, spore-forming, bacterium known to be the most widely distributed pathogen in nature. It is commonly found in soil, sewage, raw meat, and in the intestines of animals and humans as a member of the normal flora. The clostridium perfringens strains are classified into five groups, types A-E on the basis of their production of the four major toxins known as the alpha, beta, epsilon, and iota-toxins. Clostridium perfringens causes several human diseases, including food poisoning,  and gas gangrene. Clostridium perfringens (C. perfringens) is one of the most common causes of food poisoning in the United States. According to some estimates, this type of bacteria causes nearly a million illnesses each year.

During World War II, it was estimated that hundreds of thousands of soldiers died of gas gangrene as a result of battlefield injuries, and clostridium perfringens was widely recognized as being the most important casual organism of the disease. After the cells or spores entered into the body through injury, the organism grows rapidly in the host tissue, producing various toxins and enzymes that case massive destruction of the host tissues. The infection often lead to systematic toxemia, shock, and death unless promoted antibiotic and surgical treatment was given.

The  complete genome of c. Perfringes contains a 3,031,430-bp sequence that comprises 2,660 protein coding regions. The genome includes a 3.53 Mb chromosome, respectively, the genome  includes  five circular plasmids. C. perfringens chromosome sequences identified to be around  ~247 kb.

One key finding from the genome sequence, was that the genome contains typical anaerobic fermentation enzymes leading to gas production but no enzymes for the tricarboxylic acid cycle or respiratory chain. Various saccharolytic enzymes were found, but many enzymes for amino acid biosynthesis were lacking in the genome. Twenty genes were newly identified as putative virulence factors of C. perfrin- gens, and we found a total of five hyaluronidase genes that will also contribute to virulence. The genome analysis also proved an efficient method for finding four members of the two-component regulon. 

Another study used the complete genome of Clostridium perfringens to identify   numerous toxins produced, which are responsible for severe diseases in man and animals.  For example, delta toxin was characterized to be cytotoxic for cells expressing the ganglioside GM2 in their membrane. The study reported the genetic characterization of Delta toxin. Delta toxin consists of 318 amino acids, its 28 N- terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), alpha toxin and leukotoxins.

Lastly, another study investigated the virulence associated genome content and the genetic relationship among clostridium perfingens isolated from healthy and necrotic enteritis infected chickens and turkeys, applying genome sequencing. The pathogens associated with necrotic enteritis in chickens has not been examined in diseased turkeys. Strains expressing the NetB toxin are the main cause of necrotic enteritis in chickens and has a remarkable imact on animal welfare and the production economy in the international poultry industry. Whole genome sequencing of clostridium perfingens stain A was used to determine that the pathogenesis of necrotic enteritis in turkeys appears to be different fron that of broiler chickens. 

It is important to understand the mechanism of this bacterium because it is the most common cause of food borne illness. By using genome sequencing, researchers have been able to understand the mechanism of clostridium perfingens and genetically study how it infects humans and animals. This knowledge can then be applied to create ways to make sure humans do not contract this food borne illness. Another benefit from the geneome sequencing and understanding clostridium perfingens, it can keep soldiers healthier on the battle field, which strengthens a military force. If soilders contract gangrene we can create an effective treatment for the potently deadly illness. 
Who knew such a small organism could cause gangrene and be the primary cause of food borne illness. Thanks for listening. 

References:
Ronco, Troels, et al. “Genome Analysis of Clostridium Perfringens Isolates from Healthy and Necrotic Enteritis Infected Chickens and Turkeys.” BMC Research Notes, vol. 10, July 2017, pp. 1–6. EBSCOhost, doi:10.1186/s13104-017-2594-9.

Awad, Milena M., et al. “Functional Analysis of an FeoB Mutant in Clostridium Perfringens Strain 13.” Anaerobe, vol. 41, Oct. 2016, pp. 10–17. EBSCOhost, doi:10.1016/j.anaerobe.2016.05.005.

Manich, Maria, et al. “Clostridium Perfringens Delta Toxin Is Sequence Related to Beta Toxin, NetB, and Staphylococcus Pore-Forming Toxins, but Shows Functional Differences.” PLoS ONE, vol. 3, no. 11, Nov. 2008, pp. 1–13. EBSCOhost, doi:10.1371/journal.pone.0003764.

Mehdizadeh Gohari, Iman, et al. “Plasmid Characterization and Chromosome Analysis of Two NetF+ Clostridium Perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis.” PLoS ONE, vol. 11, no. 2, Feb. 2016, pp. 1–20. EBSCOhost, doi:10.1371/journal.pone.0148344. 

Shimizu, Tohru, et al. “Complete Genome Sequence of Clostridium Perfringens, an Anaerobic Flesh-Eater.” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 2, Jan. 2002, p. 996. EBSCOhost, doi:10.1073/pnas.022493799.  

FoodSafety.gov. “Clostridium Perfringens.” FoodSafety.gov, U.S. Department of Health and Human Services, 27 Oct. 2009, www.foodsafety.gov/poisoning/causes/bacteriaviruses/cperfringens/index.html. 

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Welcome to Genomics Revolution. This is Ka Shing Allan So from the 2019 Hiram College Genetics course hosting this episode on the genome of Campylobacter jejuni, or C. jejuni.

C. jejuni falls under the Campylobacteraceae family, and the Campylobacter genus. Campylobacter jejuni was discovered by Theodor Escherich. In 1886, Escherich was studying stool specimens and large intestinal mucous correlated to diarrhea in kittens and in neonates. He published his result in “The Intestinal Bacteria of the Infant and Their Relation to the Physiology of Digestion”. Escherich would grow cultures from infant stool samples and he successfully identified 19 different bacteria including Campylobacter jejuni.

C. jejuni is one of the highest causes of food poisoning in the United States. In 2011, the CDC predicted over 845,000 C. jejuni-related illness each year.

One particular strain of C. jejuni, strain NCTC11168, has a circular chromosome of only 1.6 million bp and two additional plasmids of 35 thousand bp each. Due to how small the C. jejuni genome is, it was genetically modified into CjCas9 for more efficient CRISPR-Cas9. Up to 1,654 proteins are predicted to be encoded by the C. jejuni genome.

Something very interesting about the C. jejuni genome is how few repeated sequences it has with only four repeated sequences consisting of three copies of the ribosomal RNA operon which are 6 thousand bp in size and three duplicates of the open reading frame. Repeated sequences are important as they could suggest highly preserved sequence, for example, the ribosomal RNA operon seen here. Repeated sequences are also important as tandem aid in mutations. A lack of repeated sequence suggests either slow mutation, or mutation through a different method.

Which brings us to another interesting fact about C. jejuni, its inability to perform DNA repair. C. jejuni does mutate, in fact some genomic regions show as much as three times more variation when compared with another organism like E. coli under similar conditions. This rapid phase of variation suggest that C. jejuni was unable to repair it’s own DNA. DNA sequencing found that the direct repair genes ada and phr, among other repair genes, were no where to be found in the C. jejuni sequence. C. jejuni mutates, simply by not repairing their DNA!!!

Another interesting fact about the C. jejuni genome is how few operons or cluster of genes there are. Some sets of proteins are still encoded by the same operons, for example the ribosomal protein operons. However, up to 617 families of genes are scatter through out the genome. For example, the Tyrosine, Aspartic Acid, and Glutamine synthesis genes can be found scattered randomly. While it seems to make it difficult to study, on the contrary, it allows us to focus on how important the genes that are grouped together must be. For example, the lipopolysaccharide gene clusters must have be involved in the synthesis of surface structures.

Isn’t C. jejuni interesting. Thank you for listening.

Bacon, D. J., Alm, R. A., Burr, D. H., Hu, L., Kopecko, D. J., Ewing, C. P., . . . Guerry, P. (2000). Involvement of a Plasmid in Virulence of Campylobacter jejuni 81-176. Infection and Immunity, 68(8), 4384-4390.
Kim, E., Koo, T., Park, S. W., Kim, D., Kim, K., Cho, H., . . . Kim, J. (2017). In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nature Communications, 8, 14500.
Shulman, S. T., Friedmann, H. C., & Sims, R. H. (2007). Theodor Escherich: The First Pediatric Infectious Diseases Physician? Clinical Infectious Diseases, 45(8), 1025-1029.
Parkhill, J., Wren, B. W., Mungall, K., Ketley, J. M., Churcher, C., Basham, D., . . . Barrell, B. G. (2000). The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature, 403(6770), 665-668.

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Welcome to Genomics Revolution. This is Jake Lininger from the 2019 Hiram College Genetics course. Today we will look at Rickettsia prowazekii or R. prowazekii for short.

During WWI, epidemic typhus infected around 30 million humans (1). R. prowazekii had been discovered previously as a causal agent for typhus, or typhus fever, but how it infects humans was yet to be discovered. In 1928, Dr. Charles Nicolle received a Nobel prize for discovering the vector for this infection; human body louse. Humans that develop typhus are infested with lice that carry R. prowazekii until skin is broken and the bacteria is able to invade the body. R. prowazekii is transmitted via the feces of lice. The bacteria remain infective for months, or in the case of Brill Zinsser Disease, R. prowazekii remains viable for years and may infect when the host’s defenses are down. Without treatment, fatalities were around 30%. Today, antibiotics are used to treat the infection. While infection is rarer today than in the 1900s, lice of the flying squirrel look to be the leading carrier of R. prowazekii in North America.

In 1998, R. prowazekii was the first alpha-proteobacterial, gram-negative genome to be sequenced. The strain used was the Madrid E strain of R. prowazekii, named after a patient who died in 1941 of epidemic typhus. The single circular chromosome of R. Prowazekii has 1,111,523 base pairs. The genome consists of 834 protein-coding genes, averaging in length of 1,005 base pairs. This represents about 75% of the genome leaving the rest to non-coding DNA (1). The large amount of non-coding DNA along with other factors of the genome point to evolutionary ties to mitochondria via horizontal gene transfer.

Since the sequencing of R. prowazekii in 1998, other Rickettsia species have been sequenced (2). Rickettsia conorii, known for causing Mediterranean spotted fever, exhibits 804 of the 834 genes from R. prowazekii. Data suggests a divergence of the genus while they exhibited a near perfect collinearity. Another study discussed R. prowazekii, R. conorii, and R. typhi in having similar sequences revealing the presence of four genes with potential membranolytic activities (3). The same paper also hypothesized and proved that expression in Salmonella of the R. prowazekii gene could lead to host cell infection.

Rickettsia prowazekii is classified as a bioterrorism agent due to its small size, low infectious dose, and high morbidity and mortality. Antibiotic resistant strains of R. prowazekii could be engineered to inflict a lot of damage (3). Regular bathing and laundering can prevent the spread of human lice, reducing the probability of R. prowazekii infection. And, for as cute as having a pet flying squirrel sounds, it is probably best to avoid contact with those little guys. Thanks for tuning in.

References:
1) Andersson, S. G., Zomorodipour, A., Andersson, J. O., Sicheritz-Pontén, T., Alsmark, U. C. M., Podowski, R. M., ... & Kurland, C. G. (1998). The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature, 396(6707), 133.
2) Ogata, H., Audic, S., Renesto-Audiffren, P., Fournier, P. E., Barbe, V., Samson, D., ... & Raoult, D. (2001). Mechanisms of evolution in Rickettsia conorii and R. prowazekii. Science, 293(5537), 2093-2098.
3) Whitworth, T., Popov, V. L., Yu, X. J., Walker, D. H., & Bouyer, D. H. (2005). Expression of the Rickettsia prowazekii pld or tlyC gene in Salmonella enterica serovar Typhimurium mediates phagosomal escape. Infection and immunity, 73(10), 6668-6673.

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Welcome to Genomics Revolution. I’m Taylor Yamamoto from the 2019 Hiram College Genetics course hosting this episode on the genome of the bacteria Escherichia Coli O157:H7. I will be calling it E. Coli from now on. This strain of E. Coli is the most harmful strain to humans because it produces a toxin, called Shiga toxin, that causes bloody diarrhea and hemolytic-uremic syndrome, which is when red blood cells get damaged, and then cause a blockage in the kidneys. This can lead to life-threatening kidney failure. Comparatively, nonpathogenic E. Coli often inhabit the human gut without any adverse affects. E. Coli O157:H7 is normally spread fecal-orally, and has caused major gastrointestinal illness outbreaks in both North America and Asia. In this podcast, we will be focusing on the genomic sequence of the E. Coli O157:H7 strainthat caused an outbreak in Sakai City, Osaka, Japan in 1996.

The complete sequence of the chromosome is 5,498,450 base pairs in length. Additionally, the strain also has a large virulence plasmid that is 92,721 base pairs, and a cryptic plasmid that is 3,306 base pairs. In total, the genome is 5,594,477 base pairs long. The Sakai strain is 859,000 base pairs longer than the nonpathogenic strain of E. Coli, but its not like its just tacked onto the end of the sequence. There is a lot of the sequence that is conserved between the two strains, which probably represents the chromosome backbone that most E. Coli strains share, but there are also regions that are unique to the Sakai strain.

In order to cause an infection, bacteria first need to stick to the tissues they are hoping to infect. This is done via fimbriae, which act kind of like Velcro and help the bacteria stick to its target tissue. On the Sakai chromosome, there were fourteen regions that were identified in association with the production of this fimbriae. Five were conserved in the nonpathogenic strain, five were partially conserved in the nonpathogenic strain, and four were unique to the Sakai strain. One of the genes found in this region was actually found to be similar to a gene that codes for fimbriaein Salmonella.

The genomic sequencing of the Sakai strain can be used in the future identification and study of this harmful E. Coli strain. For example, it was used by Gadri et al when their lab was investigating the effects the presence of other bacteria can have on the proliferation rates of various strains of E. Coli O157:H7.

Because there have been multiple gastrointestinal illness outbreaks worldwide, Manning et al (3) have developed a system to identify SNPs in various strains of E. Coli and how they correspond to level of severity of the infection.

So, while this is definitely an international issue, you listening to this podcast is the first step in furthering education, and coming to a better solution. So thanks for listening.

References:

(1) Marouani-Gadri, N., Augier, G., and Carpentier, B. (2009). Characterization of bacterial strains isolated from a beef-processing plant following cleaning and disinfection – Influence of isolated strains on biofilm formation by Sakai and EDL 933 E. Coli O157:H7. Retrieved from https://www.sciencedirect.com/science/article/pii/S0168160509002499?via%3Dihub.

(2) Hayashi, T., Makino, K., Ohnishi, M., Kurokawa, K., Ishii, K., Yokoyama, K., Han, C.G., Ohtsubo, E., Nakayama, K., Murata, T., Tanaka, M., Tobe, T., Iida, T., Takami, H., Honda, T., Sasakawa, C., Ogasawara, N., Yasunaga, T., Kuhara, S., Shiba, T., Hattori, M., and Shinagawa, H. (2001). Complete Genome Sequence of EnterohemorrhagicEschelichia coli O157:H7 and Genomic Comparison with a Laboratory Strain K-12. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/11258796.

(3) Manning, S., Motiwala, A., Springman, A., Qi, W., Lacher, D., Ouellette, L., Mladonicky, J., Somsel, P., Rudrik, J., Dietrich, S., Zhang, W., Swaminathan, B., Alland, D., and Whittam, T. (2007). Variation in virulence among Clades of Escherichia Coli O157:H7 associated with disease outbreaks. Retrieved from https://www.pnas.org/content/105/12/4868.

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Hello everyone, My name is Tim Stucky and today I’ll be taking you through the genetics of Caulobacter crescentus. Caulobacter was first proposed in 1935 by Henrici and Johnson, two researchers who found the bacteria in microscope slides with samples from a freshwater lake. It is considered a stalked organism, meaning it has a long tubelike extension in its mature form. This organism fit into the Caulobacteriales which was a newly found order for bacteria at the time of its discovery. It is a gram-negative bacteria that lives in nutrient poor areas of fresh water bodies. There are two main strains of C. Crescentus, C15 which is the natural freshwater strain and NA1000 which is the experimental strain isolated in the 1970’s. Caulobacter is a very important tool for studying the cell cycle because of its odd development. There are two distinguishable developmental stages that it will go through which makes it easy to look at how its genome is used throughout its life. It gives rise to daughter swarmer cells that are mobile and have flagella. Soon they begin to secrete polysaccharide adhesins known as holdfast which makes it bound to the surface it is on. Once it is bound to a surface, it sheds its flagellum and grows a stalk, or a long tubular extension. In the stalk phase, it is mature and ready to replicate.

Caulobacter is tightly regulated in how it can only replicate its DNA one time while in the stalk phase. It will replicate its single chromosome and divide into two more swarmer cells. In the C. crescentus 4,016,942 base pair genome, there are 3767 genes averaging 969 base pairs in size. There are 3763 proteins thought to be produced by the genome with 1012 being hypothetical and 2751 having matches to known proteins. This genome has the largest ratio of signal transduction proteins made to any other bacteria that has been fully sequenced. This has allowed researchers to test and gain a much deeper understanding of these pathways and how they are used in regulating the cycles of development. Researchers have also found that there were 13 undiscovered RNA Polymerase sigma factors made in C. crescentus. Sigma factors help regulate changes in gene expression by coupling with outside stimuli. This has significance in the organism’s development and has allowed deeper research into gene expression. The last point of importance is that it initiates replication in different ways depending on the media it grows on. This has allowed researchers to study in depth, how cells adapt and change their replication control based on the nutrients provided to them.

This bacteria has been vital in cell cycle research and has provided insight and discovery since its discovery in the 1930’s. Thanks for joining me and I hope you enjoyed learning about Caulobacter crescentus and its contributions to genetics.

References

Gorbatyuk, B., & Marczynski, G. T. (2004, December 09). Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus. Retrieved from https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2958.2004.04459.x

Nierman, W. C., Feldblyum, T. V., Laub, M. T., Paulsen, I. T., Nelson, K. E., Eisen, J., . . . Fraser, C. M. (2001, March 27). Complete genome sequence of Caulobacter crescentus. Retrieved from https://www.pnas.org/content/98/7/4136.short

POINDEXTER, J. S. (1964, September). BIOLOGICAL PROPERTIES AND CLASSIFICATION OF THE CAULOBACTER GROUP. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC441226/?page=2

Toh, E., Kurtz, H. D., & Brun, Y. V. (2008, November 01). Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps. Retrieved from https://jb.asm.org/content/190/21/7219.short

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Welcome to Genomics Revolution. This is Anna Pallante and this episode will focus on the genome of Mycobacterium tuberculosis H37Rv. Mycobacterium tuberculosis is a pathogenic bacteria from the family Mycobacteriaceae that was discovered in 1882 by Robert Koch (1). Koch was able to isolate the bacteria from tissue samples from animals that were suffering from tuberculosis. However, he had to develop and use new staining and culturing techniques in order to do so. When the tissue samples were stained with methylene blue, M. Tuberculosis turned bright blue while the surrounding tissue and other bacteria were all turned brown (1). All infected samples showed the bright blue rod shaped bacteria that Koch originally named tubercle bacillus. In order to prove that this bacteria was truly responsible for tuberculosis, Koch grew the isolated bacteria on a solid medium and then inoculated guinea pigs. Soon after, the guinea pigs developed tuberculosis (1).

When Mycobacterium tuberculosis was first discovered, it was estimated that tuberculosis killed 1/7 of all humans (1). Much research was done to discover the cause of this disease as well as prevention and curative measures. While many antibiotics have been found to treat tuberculosis, it is still one of the top ten causes of death in the world, killing approximately 1.6 million people every year (2). Multi drug resistant tuberculosis has become a major health crisis and studies continue to be done in order to find genetic reasons for resistance and new medications that can fight resistant strains.

M. tuberculosis H37Rv was sequenced in 1998. The genome contains one circular chromosome with approximately 4.41 Mbp with a G + C content of 65.6%. The genome encodes about 3,924 proteins (3). 

Mycobacterium tuberculosis is incredibly adept at lipid metabolism and production. The genome encodes around 250 distinct enzymes that are involved in lipid metabolism (3). This is more than almost any other bacteria. For example, E.coli only has about 50 enzymes for lipid metabolism. This genome also encodes enzymes that allow M.tuberculosis to be proficient lipid producers. The genome encodes biosynthetic enzymes similar to other bacteria, plants, and animals which allows M. Tuberculosis to contain an example of almost every lipid biosynthetic system (3).

M. Tuberculosis is an incredibly successful pathogen, and its virulence comes mainly from the ESX-1 secretion system (4). This is a type VII secretion system which allows transport through the heavily lipidated cell wall through specialized membrane proteins (5). The ESX-1 system is comprised of a cluster of proteins that allows for transport and give the bacteria its virulence. While the system is not fully understood, study of the genome has revealed that the EspL protein is responsible for a great amount of ESX regulation (4). When the espL gene was inactivated through a mutation, M. Tuberculosis was no longer able to secrete ESX-1 substrates and the amounts of various Esp proteins involved in the system were altered (4).

Multi drug resistant and extensively drug resistant tuberculosis are a major health threat due to the increased mortality rates because the antibiotics used to treat them are ineffective. The genomes of antibiotic resistant strains of M. Tuberculosis are being studied and compared to the drug sensitive H37Rv strain in order to find the genetic reason for drug resistance. The pncA gene has been found to be a major factor in the resistance to pyrazinamide, which is a first line treatment of tuberculosis (6). A study revealed that 70% of multidrug resistant and 96% of extensively drug resistant M. Tuberculosis strains had mutations in the pncA gene (6). A total of 69 polymorphism in the pncA gene were identified in the study. Fifty two of these were single nucleotide polymorphisms, and 67 were nonsynonymous mutations (6). These findings and others provide the basis for determining the cause of drug resistance and offer a platform to build off of in the future. Thank you for listening.

References

(1)Koch, 1882. The Germ Theory of Disease 15: 109-115. The etiology of tuberculosis.

(2)World Health Organization 2018. Online: https://www.who.int/news-room/fact-sheets/detail/tuberculosis. Tuberculosis.

(3)Cole et al., 1998. Nature 393: 537-544. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.

(4)Sala et al., 2018. PloS Pathogens 14(12): e1007491. EspL is essential for virulence and stabilizes EspE, EspF, and EspH levels in Mycobacterium tuberculosis.

(5)Green and Mecsas, 2016. Microbiology Spectrum 4(1): 10.1128. Bacterial Secretion Systems – An overview.

(6)Allana et al., 2017. Emerging Infectious Diseases 23(3): 491-495. pncA Gene Mutations Associated with Pyrazinamide Resistance in Drug-Resistant Tuberculosis, South Africa and Georgia.

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My name is Daijah Sek, and on today’s episode I will be discussing Treponema pallidum which many of you may already be familiar with. In 1905, scientists Schaudinn and Hoffman identified this spiral-shaped bacterium as a causative agent of syphilis. However, we now suspect with more recent research done that this may have originated and been present as far back as in the Columbus era in the 1400s in Europe. It’s subspecies can cause yaws and bejel in restricted regions of the world. Though they are more constrained to tropical climates.

As said by Fraser et al., Treponema pallidum has been a difficult organism to study experimentally because it is completely dependent on a mammalian host for sustained growth and viability. If the exact conditions are not met, the organism cannot be studied. The genomic sequence of T. pallidum brings about a great deal of useful information that would be unlikely to find by any other approach. A more complete understanding of the biochemistry of this organism derived from genome analysis may provide a foundation for the development of a culture medium for T. pallidum, which opens up the possibility of future genetic studies.

As we know, syphilis is a relatively common STI/STD that is still around today. While we have an effective treatment of penicillin or other appropriate antibiotics should a patient be allergic, it is still useful to study this organism and to care about it because of its presence today and because there is still much more to learn about what can be done for people who have syphilis in more serious forms such as latent and tertiary where fatality is on the table.
In 1998, Fraser et al. sequenced the T. pallidum genome and determined that it hast 1,138,006 base pairs. This organism contains a single circular chromosome containing 1,041 predicted coding sequences or open reading frames.
In 1999, Centurion-Lara et al. studied Treponema pallidum’s major sheath protein trp K which is a target of opsonic antibody and the protective immune response. It is predicted to have a cleavable signal peptide and is located in the outer membrane of the bacterium. With their research done on rabbits, they hypothesized that this gene family is important to pathogenesis and immunity of syphilis infection. They identify that treponema pallidum is able to cause a multistage disease and establishes life-long infections and that it is important to understand that through protective immunity.

In 2000, only two years after the T. pallidum genome was sequenced, Subramanian et al did a study on comparative genome analyses of Treponema pallidum organism with Borrelia burgdorferi which is another pathogenic spirochete that is known to be the causing agent of Lyme disease. This team was able to compare the genomes and gain insight to the organism’s evolutionary trends and adaptive strategies. This can better help scientists to understand the organism in order to more effectively treat as well as even prevent the diseases. They specifically were able to identify metabolic and signaling pathways that are specifically in pathogens and great targets for therapeutic intervention. Fun fact: this study used the tool BLAST that we have all become very familiar with. The team was also able to identify new protein families in these spirochetes which with further research, they believe can be a resourceful way to study pathogen-host interactions.

References:
Fraser, Claire M., et al. “Complete Genome Sequence of Treponema Pallidum, the Syphilis Spirochete.” Science, American Association for the Advancement of Science, 17 July 1998, science.sciencemag.org/content/281/5375/375.full.

Centurion-Lara, A et al. “Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response” Journal of experimental medicine vol. 189,4 (1999): 647-56.

George M. Weinstock, John M. Hardham, Michael P. McLeod, Erica J. Sodergren, Steven J. Norris, The genome of Treponema pallidum: new light on the agent of syphilis, FEMS Microbiology Reviews, Volume 22, Issue 4, October 1998, Pages 323–332, https://doi.org/10.1111/j.1574-6976.1998.tb00373.x

Subramanian, G et al. “Comparative genome analysis of the pathogenic spirochetes Borrelia burgdorferi and Treponema pallidum” Infection and immunity vol. 68,3 (2000): 1633-48.

https://www.cdc.gov/std/syphilis/treatment.htm

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Hello my name is Nikkia Schady and today I will be talking to you about the organism Thermoplasma Acidophilum. Thermoplasma Acidophilum is a thermoacidophilic archaeon. It is classified as being in the archaea domain due to its membrane lipid composition, evolutionary ribosomal RNA and conserved proteins. (2) Thermoplasma Acidophilum is also in the subgroup Euryarchaeota and it’s strain is Thermoplasma acidophilum DSM 1728. (3) From here on out I will just call it Thermoplasma as a short name.
• This organism was first discovered when it was isolated from self-heated coal refuse piles. (1). It was first isolated by a man named Darland along with his colleagues in the late 1960’s at a Fair Tuck mine in southwestern Indiana in a pH environment of 1.96. They believed it represented a prokaryotic organism but noticed that unlike other bacterial cells it lacked a rigid cell wall and was only separated by a double membrane.
• The size of the genome was first discovered by Ruepp et. al. using a ‘shotgun primer walking’ method that used PCR amplifications and primer-walking strategies to fill in the missing gaps. Ruepp and his coworkers discovered that the genome of Thermoplasma was 1,564,905 base pairs (3).
• Thermoplasma is one of the smallest free-living organisms and Ruepp and his coworkers also found that the genome consisted of a single circular chromosome of the 1.56Mbp. There were no plasmids detected by biochemical or DNA sequencing methods (3). They also found that the Thermoplasma genome contained 1,509 ORF’s and 1/3 had homologs present in all 3 domains of life (3).
• The Thermoplasma is a typical archaeon with a fairly large protein complement of bacterial origin. By conservative search methods they were able to match 620 domains of 537 ORF’s (35.6%) to proteins of known structure. They discovered that Thermoplasma had 1,478 protein genes and 45 RNA genes. These proteins they found included degradation pathway proteins, putative proteins, chaperones, respiratory chain proteins, extracellular proteins and more. (3)
• While doing my research there were some findings I came across that I thought were interesting and worth noting, and I will discuss 3 of them.
• Ruepp et al found their evidence to indicate that there has been much lateral gene transfer between Thermoplasma and Sulfolobus Solfactaricus (a distinct crenarchaeon inhabiting the same environment). At least 252 ORF’s (17%), including protein degradation pathways and various transport proteins resemble Sulfolobus proteins most closely. (3) This first piece of evidence is interesting because it leads to an idea that organisms who live together can swap genes at higher frequencies.
• Thermoplasma can respire anaerobically using sulfur, but no sulfur-respiratory genes similar to those of other archaeon were found. Instead bacteria-like sulfur reducing proteins were identified and appear to be responsible for sulfur metabolism (2). Not all of the species that were found to share similarities with Thermoplasma were completely annotated at the time this article was published so it would be interesting to see the similarities between this organism and others if those sequences were up to date now to give more insight into it’s genome.
• The third piece of evidence was that Thermoplasma inhibits a hot and highly acidic environment, sometimes as low as pH 0.5 in which few organisms are viable. It has adapted to scavenging nutrients from the decomposition of organisms killed by the extreme acidity and requires yeast, bacterial or meat extract when grown in culture. (3). This information is important and useful because it could give insight into how other organisms similar to Thermoplasma can live in such conditions while missing some of the cell structure that other thermophilics have. The key point is that the proteins in this organism could tell us a lot about how it functions in these environments.
• Thermoplasma Acidophilum is an organism that could give us insight into how organisms in the same environment can transfer DNA to each other and serve as a lab tool to study how different organisms similar to this can live in extreme habitats which makes it worth understanding. That’s all I have for today, thanks for listening!
References:
1. Darland et. al., 1970. American Association for the Advancement of Science 170: 1416-1418.
A Thermophilic, Acidophilic Mycoplasma Isolated from a Coal Refuse Pile.
2. DeLong, E. 2000. Genome Biology. Extreme genome.
3. Ruepp et. al., 2000. Nature Research 407: 508-513. The genome sequence of their
thermoacidophilic scavenger Thermoplasma acidophilum.

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Welcome to Genomics Revolution. I am Brett Bentkowski, from the 2019 Hiram College Genetics Course and it is my pleasure to host this episode on Schizosaccharomyces pombe, the so called fission yeast. Fission yeast was first reported in 1893, by Paul Lindner, who isolated if from East African millet beer. It gets its species name pombe from the Swahili word for beer: pombe. It then went on to be used by Urs Leupold for genetic study, and then by Murdoch Mitchison for studying the cell cycle, both around the 1950s. S. pombe’s common name, fission yeast, comes from how the cell divides: it grows at the cell tips and then divides by medial fission, so that two identical and equal daughter cells are created. This physical consistency makes it easy to see why it was a good model organism, and why it was an excellent choice to be the sixth genome sequenced.

Specifically, we’re working with the 972h- strain here, though there are approximately 160 natural strains. The sequence of fission yeast was reported in Nature in 2002, making it the sixth eukaryotic genome to ever be sequenced. It was preceded by some of our other friends in the genetics world, including Saccharomyces cerevisiae (another useful yeast species) and Drosophila melanogaster (the fruit fly). The Wellcome Trust Sanger Institute and thirteen other laboratories made up the S. pombe European Sequencing Consortium, or EUPOM, which sequenced the genome with a 100 kb sequence generated by the Cold Spring Harbor Laboratory. Sequencing was carried out by integrating two pre-existing restriction maps. These maps both contained chimaeric clones, gaps, and inserted elements, so they were problematic. In order to sequence the genome completely, DNA fragments were cloned into M13 bacteriophage (for analysis in E. coli) or cloned into pUC18 plasmids to analyze in other organisms. Random subclones were then sequenced, and Phrap or Gap4 softwares were used for contiguous assembly, using overlapping segments to generate a map of the whole genome. All of the sequences were collected centrally and checked for error by looking for frameshifts in coding regions. There were less than 1/180000 bp errors, and all of those have been resolved except for four. The sequence predicts a maximum of 4824 protein encoding genes spread over three chromosomes, for a total of 13.8 mega-base pairs (1). This organism has a high level of consistency within itself and its daughters, and the fact that it divides into two identical and equal parts makes it an excellent model organism to study the cell cycle.

​In humans, the Cytochrome P450 enzymes are integral in phase I metabolism of drugs. A 2013 study by Neunzig et al. expounds on a previous finding that the S. pombe genome can be used to synthesize these enzymes by recombinantly expressing the human gene in the yeast. They transformed P450 enzymes into yeast and measured 7 different human enzyme activity with coexpression of human oxidoreductases, the homologous oxidoreductase in yeast, and one oxidoreductasefrom a plant, bishop’s weed. The coexpression from the yeast oxidoreductase was found to be equally helpful for two P450 enzymes and more helpful for one other P450 enzyme (2). This shows that the S. pombe genome has biosynthetic components that are useful in humans, paving the way for metabolic treatments and therapies using other organism’s genomes.

​Another discovery from S. pombe is how the body uses zinc. Zinc is a co-factor for over 300 enzymes, but in higher eukaryotes there is no known indicator for zinc deficiency, since the human genome is so large and complex. Fission yeast has a gene called alcohol dehydrogenase 4, which is regulated by zinc. A study done at the Ohio State University finds that this gene is transcriptionally regulated by zinc presence or deficiency. The gene has zinc responsive elements that are regulated at the transcriptional level (3). This discovery puts us on a path to understanding human gene regulation, since now geneticists know what a sequence that is regulated by zinc looks like in a species with many human analogues.

​We can even use fission yeast to determine how a cell responds to damage. Another study worked with RAD9, which controls a human cell-cycle checkpoint protein was used in fission yeast to figure out how DNA damage can induce apoptosis. Fission yeast and humans have very similar RAD9 structures and mechanisms, and this homology allows us to use yeast as a model organism for humans. It turns out that RAD9 can be blocked by Bcl-2 family proteins and may have a role in regulating apoptosis after damage in addition to already known checkpoint control function (4). This is just one more example of how the S. pombe genome can contribute to human medicine and how we understand the body.

​The story of S. pombe has come a long way from being a beer producer to being one of the first eukaryotes sequenced to being a key player in medical research and human innovation. From apoptotic regulation, to transcriptional regulation, to metabolism, Schizosaccharomyces pombe is living up to its status as a great model organism.

1. Wood, V., Gwilliam, R., & Rajandream, M.-A. (2002). The genome sequence of ​​​Schizosaccharomycespombe. Nature, 415(6874), 871–880. ​​​​​https://doi.org/10.1038/nature724

2. Neunzig, I., Hehn, A., Bourgaud, F., Bureik, M., Widjaja, M., Peters, F. T., & Maurer, H. H. ​(2013). Coexpression of CPR from Various Origins Enhances Biotransformation Activity​of Human CYPs in S. pombe [electronic resource]. Applied Biochemistry and ​​​Biotechnology, 170(7), 1751–1766. https://doi.org/http://dx.doi.org/10.1007/s12010-013-​0303-2

3. Jenkins, B. (2012). Mapping zinc-responsive elements in schizosaccharomyces pombe. ​​[Columbus] : Ohio State University, 2012. Retrieved from ​​​​​​http://ezproxy.hiram.edu/login?url=http://search.ebscohost.com​/login.aspx?direct=true&db=cat01905a&AN=ohiolink.b32360643&site=eds-live

4. Komatsu, K., Miyashita, T., Hang, H., Hopkins, K. M., Zheng, W., Cuddeback, S., … Wang, ​H.-G. (2000). Human homologue of S. pombe Rad9 interacts with BCL-2/BCL-xLand ​promotes apoptosis. Nature Cell Biology, 2(1), 1. https://doi.org/10.1038/71316

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Welcome to Genomics Revolution. My name is Alexis Polcawich from the 2019 Hiram College Genetics course hosting this episode on the genome called Yersinia Pestis. This is a gram negative, nonmotile, rod-shaped coccobacillus bacteria that does not form spores and is a facultative anaerobe. This bacterium was discovered in 1894 by Alexandre Yersin in Hong Kong. Yersin was able to isolate the bacterium in culture and identified it via microscope. Jean-Paul Simond was Yersin’s inspiration to do so as in 1898 he discovered that bacteria was transferred from rodents by flea bites, and Yersin thought it would be beneficial to learn more about such bacteria. You can probably guess where this is going… By doing so, he discovered that Yersinia pestis was responsible for causing the plague in humans. This podcast is going to discuss the Yersinia pestis KIMstrain which is responsible for the both the bubonic and pneumonic plagues. The KIM strain is believed to be the first strain discovered as the Yersinia pestis bacteria was found while researchers were looking into the underlying causes of the bubonic plague.

Yersinia pestis is claimed to be a potential weapon of bioterrorism because it can effectively evade it’s host’s immune system and otherwise go unnoticed until it is too late and the symptoms become too severe for the body to fight them off. This tiny bacterium is responsible for drastically high mortalities throughout the course of history, some people today still become infected by it. However, it is now treatable with present day antibiotics and does not always result in death unless the case is very severe.

The genome of Yersinia pestis contains one circular chromosome of 4,600,755 bp with an average G+C content of 47.64%. This genome encompasses 4,198 open reading framesand contains three different plasmids called pPCP1, pMT1, and pCD1.

One key finding was in a study done in 2011 that looked at developing a possible vaccine for the plaque by constructing a mutant strain of Yersinia pestis that expressed lpxL from the chromosome which resulted in almost all of the lipid A in the cell being hexa-acylated. This means that The lpxL gene is not a gene that normal wild type Yersinia pestis carry. The absence of this protein leads to the production of tetra-acylated lipid A which does not bind to the host receptor, therefore failing to simulate the inflammatory response normally induced by lipid A. By making these hexa-acylated, this allows the inflammation process to happen and the body can start to effectively fight off the bacteria.

A second key finding is now that the genome of this bacterium is sequenced, researches can now trace potentialoutbreaks and analyze the evolution of the bacteria. The differences in certain regions such as the number of inverted/rearranged segments in the different strains of Yersinia pestis in different regions of the world shows how quickly certain strains are mutating compared to others. This can help researches and epidemiologists predict what to expect from a certain strain over a particular period of time and keep up with effective medications/treatments.

A third key finding is that researches can use the sequenced genome and compare it to other sequenced bacterium genomes to identify close relatives of Yersinia pestis. This is very important because if there happens to be a certain bacteria that is very similar to Yersinia pestis and there is already an effective medication found to treat that bacteria, then maybe that medication can be used to treat Yersinia pestis as well. In the study done in 2002 on the genome sequence of Yersinia pestisKIM, the genome was continuously compared to E. Coli K-12. The origin, terminus, and most recent genes encoding DNA replication proteins are very similar to those of E. Coli. Being able to relate Yersinia pestis to a bacteria that is already well known provides researchers with the means to answer a lot of previously unanswerable questions and can even inspire further experiments to test new hypotheses evolving from current knowledge of similar bacterial strains.

Who knew such a tiny organism could be so deadly that it is responsible for billions of deaths throughout history. Without geneticists and epidemiologists (even though they weren’t called such back in the 18-1900’s) the human population could have been completely wiped out by now.

​

References

(1) Auerbach et al., 2007. Plos one 2(8): e770. Yersinia pestis Evolution on a Small Timescale: Comparison of Whole Genome Sequences from North America. https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0000770&type=printable

(2) Butler et al., 2014. Clinical Microbiology and Infection 20: 202-09. Plague history: Yersin’s discovery of the causative bacterium in 1894 enabled, in the subsequent century, scientific progress in understanding the disease and the development of treatments and vaccines. https://www.sciencedirect.com/science/article/pii/S1198743X14608582?via%3Dihub

(3) Losada et al., 2011. Plos One 6(4): e19054. Genome Sequencing and Analysis of Yersinia pestis KIM D27, and Avirulent Strain Exempt from Select Agent Regulation. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084740/pdf/pone.0019054.pdf

(4) Deng et al., 2002. American Society for Microbiology184: 4601-11. Genome Sequence of Yersinia pestis KIM.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC135232/

(5) Sun et al., 2011. Vaccine 29(16): 2986-98. A live attenuated strain of Yersinia pestis KIM as a vaccine against the plague. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073832/

(6) Ziets et al., 2004. International Journal of Hygiene and Environmental Health 207: 165-178. The history of the plague and the research on the causative agent Yersinia pestis.https://www.sciencedirect.com/science/article/pii/S1438463904702771

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Hello, and welcome back to genomics revolution. I am Kaitlyn Morse a guest scientist here today to talk about the organism Drosophila melanogaster, or what we know as the fruit fly. We may think of this insect as a pest but, it has been one of the most useful organisms in a biology/ genetics lab to date. You may be asking how is that possible? A small fly? Let’s discuss why Drosophila has become so important.

The discovery of Drosophila occurs in 1830 but, the first time Drosophila became a popular organism to study was back in 1910 in Thomas Hunt Morgan’s lab. He noticed that a male fly had white-eyes instead of the-wild-type eyes which were red. This lead to years of studying this organism and finding that eye type was found in the sex chromosome and was being passed on. The study of Morgan’s lab Lead to a deeper interest in Drosophila (5).
In the year of 1999, the entire genome of Drosophila was made available to researchers all over the world. They had concluded that the genome is approximately 180 Mb in size (6). The number of proteins found in drosophila was 30,493. The work of these scientists also solidified there are only four chromosomes in Drosophila (2). The first is a sex chromosome ( X chromosome) and the remaining three are autosome. Chromosome two and three are separated into left and right arms ( 2L, 2R, 3L, and 3R) (3). The fourth is a very small autosome. The first physical maps of the chromosomes was actually created in 1910 when the first lab found that the color of eyes was found in the sex chromosome (1). This was Thomas Hunt Morgan’s lab who discovered this as well. We can thank Morgan for the wide historical importance of Drosophila and the large group of scientists that were studying this organism for years after he did.

Why is Drosophila considered a model organism? Why is it so important to biology/ genetics ? First, after working with Drosophila for years and sequencing the genome they realized Drosophila is inexpensive, has a short lifespan, and has a reasonably small genome. The fly only takes 9-10 days to go from a fertilized egg to an adult. This makes it easy for scientists to create many new generations quickly and also study their genome because it isn’t too large in size. Next, Drosophila is know for having Transposons in their genome. As some of us may know, a transposons is a chromosomal segment that can move anywhere in the genome. This movement can cause a mutation, alter genome size, or duplicate the genetic material found in the cell. After the transposons in Drosophila were discovered, they determined they were helping maintain telomere lengths in Drosophila. The transposons are now being used biologically. Laboratories are now using Transposons for mutagenesis ( or causing genetic mutations) and transformations (4). But in my opinion the most important part of the research that has been done on this organism is how their genome is like the human genome. 75% of the genes responsible for human disease are highly conserved in this species. This allows for the study of human disease and for scientists to understand the disease outside of a human patient. In the study “The systematic analysis of Human Disease- Associated Gene Sequences in Drosophila melanogaster 548 drosophila genes were related to the human disease genes. 153 of these are associated with a known mutant allele and 56 others are marked by a P-element insertion in or near the gene (which is a transposons that is more likely to move here) . Knowing humans and drosophila had highly conserved genes allowed for this study to be made possible. The scientists used drosophila to study human metabolic disease. The genes and pathways are the same in the insect as humans which allows for manipulation of these pathways. Scientists have now been able to use this manipulation to cause a fly to be diabetic or even show an obese phenotype. Using Drosophila to study diseases has now led scientists to believe they can create therapeutic ways to treat these diseases by looking at how it affects drosophila first (7).

This organism can lead to many different therapeutic remedies and better understandings of diseases. Using Drosophila to better understand how things work in humans can lead to faster treatments for people. While many people just believe that the fruit fly is just a pest around their house, hopefully now you may have a better understanding how useful this organism is. It is important to continue to study this organism in order to continue to make strides in genetics to help lead to better medical treatment . I hope this podcast was both informational and interesting. Thanks for listening to Genomics Revolution. Kaitlyn Morse Signing off.

References:
1. Fruit flies in the laboratory. (2018, April 04). Retrieved from
https://www.yourgenome.org/stories/fruit-flies-in-the-laboratory
2. Drosophila melanogaster (ID 47). (2015). Retrieved from https://www.ncbi.nlm.nih.gov/genome/?term=drosophila melanogaster.
3. Hales, K. G., Korey, C. A., Larracuente, A. M., & Roberts, D. M. (2015, November). Genetics on the Fly: A Primer on the Drosophila Model System. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649653/#!po=12.3762
4. Cheng, L., Baonza, A., & Grifoni, D. (2018, August 30). Drosophila Models of Human Disease. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136514/
5. Thomas Hunt Morgan and Sex Linkage. (n.d.). Retrieved from
https://www.nature.com/scitable/topicpage/thomas-hunt-morgan-and-sex-linkage-452
6. Adams, M. D. (2004, February 20). The Genome Sequence of Drosophila melanogaster. Retrieved from
http://science.sciencemag.org/content/287/5461/2185?casa_token=oSaTWt9OxqIAAAAA:Pzd5 plb0P9n9IEF1oU8EZkw17VL9n3_JY3XQT9DO7-Vb6DKhILded0YuqOv-H11U7-0lcBPmpg1kXw
7. Reiter, L. T. (2001). A Systematic Analysis of Human Disease-Associated Gene Sequences In Drosophila melanogaster. Retrieved from https://genome.cshlp.org/content/11/6/1114.full.pdf

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Welcome to Genomics Revolution. My name is Kritika Bhau and I am a student at Hiram College and currently taking the course called Genetics. I will be hosting the episode on the organism, Methanococcus jannaschii. It is also known as methanocaladococcus jannaschii. I will be calling the organism M. jannaschii throughout the podcast. M. jannaschii is important because it was also the first archaeon to have its complete genome sequenced. This sequence help identity many genes unique to domain Archaea. But, before we get into the genome analysis of this organism, let us talk more about M. Jannaschii to get some background information as to how it was discovered in the first place. M. jannaschii was first isolated in 1982, in the East Pacific Rise near the western coast of Mexico where it was found from a sample sediment taken from 2600-meter-deep “white smoker” chimney with high temperatures and pressure (1). But, this organisms survives in these conditions. This nature of M. Jannaschii is what intrigued scientists.

When M. Jannaschii’s genome was sequenced, the genomic structure was identified which helped us give more information about the organism’s ability to function in such extreme habitat. M. jannaschii has three chromosomes: large circular chromosome, large-circular extra chromosome and small circular extra chromosome. The large circular chromosome is 1.66 megabase pairs long with total 1729 protein-coding regions. The large circular extra chromosome is 58 kilo base pairs long and has 45 protein coding regions. Finally, the small circular extra chromosome is 16.5 kilo base pairs with 12 protein codings regions (1).

M. jannaschii belongs to a group called methanogens. This means that M. Jannaschii is a methane producer. M. jannaschii is an autotrophic single cell organism which is specialized in undergoing fueling reactions to produce methane as the end product. M. jannaschii is strictly anaerobic and uses only carbon dioxide and hydrogen as primary energy sources. They are very important because they reduce organic products to methane. Looking at the genome, we can understand the metabolic process, enzymes and proteins involved in M. jannaschii and use it to understand the evolution of domain Archaea. While analyzing the genome, it was seen that M. jannaschii genome contains all ribosomal proteins that are common to eukaryotes and bacteria. Moreover, all the subunits found in M. jannaschii show greater similarity to their eukaryotic counterparts than to bacterially subunits meaning that eukaryotes share a common evolutionary trajectory independent to bacteria (1).

To learn more about how M. jannaschii functions, Tumbula and Whitman conducted in vivo regulation studies to learn that in M. jannaschii “genes of a common pathways are seldom clustered in operons” (2). They do not know why this is, but they hope to rationalize their findings as more ORF are confirmed. Additionally, Zhu and et all. Helped identify 963 unique proteins with shotgun proteomic method used in its methanogenesis pathway. Surprisingly, the proteins that were found represent over half of the whole genome of M. Jannaschii. 95% of those proteins are necessary for amino acid biosynthesis, cellular processes, metabolism and transcription (3). This is important as with such we can see how effective this organism uses its small genome to survive in such harsh conditions.

Before, there were two domains based on structure of cells - eukaryotes which have cells with a nucleus like plants and animals. The other domain was prokaryotes which had no nucleus such as bacteria. But M. jannaschii help create this new third domain: Archaea which is made of single-celled organisms without a nucleus. With more genetic research using the genome sequence of M. Jannaschii and similar organisms, we hope to find more interesting mechanisms that help us understand the domain Archaea and its evolutionary history.

References:
(1) Bult, Carol J., et al. (1996). “Complete Genome Sequence of the Methanogenic Archaeon, Methanococcus Jannaschii.” Science, vol. 273, no. 5278, pp. 1058–1073. JSTOR, www.jstor.org/stable/2899534.

(2) Tumbula, D L, and W B Whitman. (1999, July). “Genetics of Methanococcus: Possibilities for Functional Genomics in Archaea.” Molecular Microbiology, U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/pubmed/10411718

(3) Zhu, W., Reich, C. I., Olsen, G. J., Giometti, C. S., & Yates, J. R. (2004). Shotgun proteomics of Methanococcus jannaschii and insights into methanogenesis. Journal of Proteome Research, 3(3), 538-548. http://pubs.acs.org/doi/abs/10.1021/pr034109s.

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Welcome to Genomics Revolution. This is Danielle Vincent from the 2019 Hiram College Genetics course, and I will be your host for this episode on the genome of Vibrio cholerae El Tor N16961. Now, just to make things a bit easier, I’ll go ahead and refer to this organism as V. cholerae for the remainder of this episode. V. cholerae is a gram-negative, gamma-Proteobacterium that you may already recognize as it’s the bacterium that causes the disease Cholera (1). In 1854, during the third pandemic of Cholera, an anaethesiologist named John Snow theorized that this, at the time, unknown, disease was spreading through contaminated water. Snow mapped out 13local, public wells in London and took water samples, and he claimed to see “white, flocculent particles” in some of these samples (3). Around the same time, this organism was seen in Florence by the anatomist Filippo Pacini as he was performing autopsies on individuals that had died during this pandemic. He saw what he referred to as “vibrions”, or little elements, found in the intestinal mucosa and feces, and he believed that this was the cause of the disease (3). V. cholerae was not definitively found to be the cause of Cholera until, in 1884, German scientist, Robert Knoch, successfully isolated the bacterium in pure culture. Robert Knoch also noted that this organism was “a little bent, like a comma” (3).

​Cholera has been known to be an epidemic in Southern Asia for over 1,000 years and has also been the cause of seven pandemics, the first starting in 1817 (1). Cholera is known to be an infection of the small intestines. People who have contracted the disease will experience severe, watery diarrhea and vomiting and can die from dehydration, even just after a few hours (3). Without treatment, this disease is very contagious and lethal. Cholera probably isn’t a disease many of us have to worry about day-to-day. However, individuals in developing countries with unsafe drinking water, poor sanitation, and limited healthcare, can easily fall victim to the disease (3). Over the years, many strains of V. cholerae with varying gene content have been found to cause Cholera in various countries, and the study of this organism is very important as it allows for advances in creating more efficient treatments.

​The originally sequenced El Toro N16961 strain of V. cholerae was found to have a genome size of 4,033,460 base pairs (1). It contains two circular chromosomes that encode for a total of 3,885 proteins, 2,770 of these proteins are found in chromosome 1 while 1,115 proteins are located in chromosome 2 (1).

​Sequencing the genome for this bacterium has played a large role in figuring out the mechanisms in which V. cholerae functions and causes disease. Cholera toxin (CT) and the toxin-coregulated pilus (TCP) are the two main virulence factors.Cholera toxin is an ADP-ribosylating toxin that, when secreted, causes an increase in cAMP levels in the intestinal epithelial cells (2). ToxRS for example, is a transcriptional regulator that aids in the activation of the toxT promoter, and this leads to the activation of various virulence genes (2). Many studies will now use this type of knowledge to find differences between the strains of V. cholerae, as certain conditions, like temperature and pH, can cause decreases in virulence factors (2). One study looked at the expression of cholera toxin in the presence of bile acids and found that El Tor biotype strains, N16961 included, has a much lower production of cholera toxin when exposed to bile acid than say a classical strain of V. cholerae (2).

​As stated before, there are many different strains of V. cholerae, and the genome has become very crucial in differentiating the various strain types. The cholera toxin we just discussed is encoded by two genes, ctxA and ctxB, which are located on the CTXphi prophage (5). Now, epidemic or pandemic V. cholerae can broken down in to serogroups, O1 and O139 (5). The O1 serogroup contains two biotypes, classical and El Tor, like the strain we’ve been discussing. It was found that both of these biotypes have conserved the sequence for the ctxAgene while the sequence for the ctxB gene is slightly different between the two (5). There is a two base change seen at base positions 115 and 203. In the classical strains, cytosines are seen at these postitions while El Tor biotypes express thymines (5). These types of differences have been reliable markers in identifying V. cholerae biotypes and has helped convey the spread of strains in certain geographic locations.

​Severely ill cholera patients will often be treated with antibiotics, but, unfortunately, some strains of V. cholerae have become antibiotic resistant. It seems as though strain resistance patterns evolve overtime, and, from 2007-2010, strains from Democratic Republic of the Congo were seen to be resistant to tetracyclines and ampicillin (4). Later on, from 2011-2012, many of these strains had become resistant to most antibiotics with the exception of cyclines and fluoroquinolones (4). Muchof this data has come from various genomic analyses and whole-genome sequencing. It’s quite apparent that this type of research is vital in understanding and controlling the effects of V. cholerae.

Thanks for listening.

References:

(1) Heidelberg, J.F. et al., 2000. Nature 406:477-84. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae.

(2) Hung, D.T. & Mekalanos, J.J., 2005. PNAS 102(8):3028-33. Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner.

(3) Lippi, D. & Gotuzzo, E., 2014. Clinical Microbiology and Infection 20:191-95. The greatest steps towards the discovery of Vibrio cholerae.

(4) Miwanda, B. et al., 2015. Emerging Infectious Diseases 21(5):847-51. Antimicrobial drug resistance of Vibrio cholerae, Democratic Republic of the Congo.

(5) Son, M.S. et al., 2011. Journal of Clinical Microbiology 49(11):3739-3749. Characterization of Vibrio cholerae O1 EL Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes.

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Welcome to Genomics Revolution. This is Brad Goodner from the 2019 Hiram College Genetics course hosting this episode on the genomes of two strains of the genus Xenorhabdus - Xenorhabdus nematophila ATCC 19061 & Xenorhabdus bovienii SS-2004. I will call them X. nematophila and X. bovienii from here on out. These strains are in the Bacteria division gamma-Proteobacteria and within the family Enterobacteriaceae, meaning they are closely related to well known human gut symbionts and pathogens such as Escherichia coli and Salmonella enterica. However, Xenorhabdus strains are not found in the guts of humans, rather they are gut symbionts of particular soil nematodes of the genus Steinernema. The bacteria are needed for the maturation of the nematodes. Not only that, the bacteria and nematode host work together to kill certain insect larvae and use them as nutrient sources. Wow, that is a complex interaction and that is why a group of labs from the U.S. and Europe, including my lab at Hiram College came together to sequence these two genomes.

One of the biggest problems we had with these genomes is that they have lots of repetitive sequences in them, including transposable elements. This makes it very hard to assemble sequencing reads into the full genome. Think about it – if the same 400 bp sequence is present 10 times in the genome then how do know which fragments go together? This is rarely an issue for a genome in Bacteria or Archaea, but it gave us a lot of headaches for the two Xenorhabdus genomes. To get around this problem, we worked with a company in Madison, WI, called OpGen Technologies to use a genome-wide restriction mapping technique called optical mapping (1). You very very gently lyse bacterial cells on an electron microscopy grid, cut the DNA as it lays on the grid with a particular restriction enzyme and figure out the length and order of the fragments. This strategy was critical to getting these genomes done.

The 2 Xenorhabdus genomes each have a single circular chromosome, ranging in size from 4.26-4.43 Mbp. X. nematophila has an additional plasmid of 155 kbp. The two genomes encode between 4250 and 4350 proteins. The Xenorhabdus genomes were compared to two previously sequenced genomes from a closely related genus, Photorhabdus, that lives in the guts of nematodes of the genus Heterorhabditis (2). The two Bacterial genera inhabit different parts of the nematode gut and the two nematode-bacteria symbioses attack different insect larval hosts. Comparison of the genomes across the two genera showed that there were many orthologous genes in common, some of which probably deal with basic metabolism but also some that deal with living inside a nematode. That said, these shared orthologs only account for about half of any one genome. On the other side of the coin, the two Xenorhabdus strains had many genes in common not found in the Photorhabdus strains and vice versa. This means that these two closely related genera of Bacteria do some really different things. For example, each genus appears to make some unique insect-killing peptides and antimicrobial molecules. Moreover, we also found that the two genera solved the same ecological problems, such as dealing with oxidative stress, using different strategies. These two genera diverged from a common ancestor that might have lived inside nematodes but they have converged on solving certain ecological problems from different angles.

Since the publication of these genomes, several labs have tested hypotheses using wildtype and mutant strains in both in vitro and in vivid experiments. For example, Singh et al. (3) used mutants of X. nematophila unable to make different antimicrobial compounds to figure out how this bacterium kills off competitor Bacterial genera inside a dead insect larva. Murfin et al. (4) figured out that X. boveinii helps its host nematode outcompete related nonhost nematodes.

Bacteria helping a roundworm kill an caterpillar so that both can benefit even in the presence of competing bacteria or other nematodes. Who would have guessed that. Thanks for listening.

References:
(1) Latreille et al., 2007. BMC Genomics 8:321. Optical mapping as a routine tool for bacterial genome sequence finishing.
(2) Chasten et al., 2011. PloS One 6:e27909. The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes.
(3) Singh et al., 2014. Applied & Environmental Microbiology 81:754-64. Role of secondary metabolites in establishment of the mutualistic partnership between Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.
(4) Murfin et al., 2018. Environmental Microbiology doi: 10.1111/1462-2920.14278 [Epub]. Symbiont-mediated competition: Xenorhabdus bovienii confer an advantage to their nematode host Steinernema affine by killing competitor Steinernema feltiae.

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This is Brad Goodner. Welcome back to Genomics Revolution. In our first 6 episodes, we have introduced the terms genome and genomics, talked about how the field of genomics got its start, and looked at the steps of a genome project using the first ever sequenced cellular genome as an example.

Now we will tour through a survey of some sequenced genomes. All three domains of life will be represented, but the Bacteria and Archaea will get the lion’s share. For each genome, we will learn why scientists are interested in the organism, some basic data about the genome, its genes and encoded proteins, a few surprises from the genome sequence, and an example of how scientists took the next step past having the genome sequence. Each genome will be presented by a different student in the 2019 Hiram College Genetics course and they will put their own unique spins on their assigned subjects.

To get us started, I will talk about my favorite genome, #50 in terms of getting published according to my counting. This is the genome of Agrobacterium tumefaciens strain C58, an organism I have worked with on and off since 1983. Agrobacterium, or Agro for short, is a genus from the Bacteria division alpha-Proteobacteria found in soils all over the world and is best known because some strains are plant pathogens. These pathogenic strains contain a plasmid that allows them to do something no other bacterial pathogens can do – transfer a piece of their own DNA into their eukaryotic host cell where the expression of genes on the transferred DNA causes the cells of the plant host to act very very differently. The “transformed” plant cells grow out of control because they make their own growth-stimulating hormones and they produce and secrete some strange compounds that Agro can use as C and N sources. It turns out that Agro has been genetically engineering plants on its own for a long time before any humans thought about the possibility.

I came back to work on Agro in 1996 when I read a paper by Allardet-Servent and coworkers (1) who showed that in strain C58 there were two DNA molecules greater than 1 Mbp that contained rRNA genes. The presence of rRNA genes is usually indicative of a chromosome, but this would mean that strain C58 has 2 chromosomes and there was no previous evidence of this. A group of 8 undergraduates at University of Richmond worked with me to generate and map a large collection of transposon insertions in essential genes of Agro C58. Our paper (2), published in 1999 proved that there 2 chromosomes in strain C58. The larger chromosome is a circle of roughly 3 Mbp,but the smaller 2.1 Mbp chromosome is a linear DNA molecule. Agro‘ s closest relatives in the genus Rhizobium only show 1 circular chromosome of roughly 3.6 Mbp, so we wondered where did the smaller linear chromosome come from? We imagined 3 possibilities. One, the smaller chromosome originated from a breakage event in the original circular chromosome. Two, the smaller chromosome came in from the outside such as a viral infection. Three, some combination of the first two hypotheses. It was this question that drove my lab to start sequencing the Agro C58 genome in 1999.

We started with $3000 to build a genomic library and start sequencing library clones, but we knew the full cost would be closer to a a half million dollars. In the fall of 1999, we presented some of our initial findings at a small research conference that focuses on the biology of Agrobacterium. At the end of the conference, a gentleman approached me. “My name is Steve Slater”, he said, “and I work for a small company called Cereon Genomics. We need to talk but not here. I will call you tomorrow.” On the flight back home, I told my wife Asha that I thought Steve Slater was going to tell me that his group had already sequenced the C58 genome. However, the next day, Steve told me that Cereon Genomics was just beginning to sequence the genome and that they wanted to collaborate with my research team because of our genome map of transposon insertions. Steve rightfully saw the value of using our map to orient and join up the sequenced pieces of the genome. We reached an agreement between Cereon Genomics, its parent company Monsanto Corporation, and my research lab. The agreement required all partners to agree as to when and how to publish the finished work. If any one partner didn’t want to publish, the collaboration would stop.

It was a fun but odd collaboration. My students got to work with a lot more sequence information, but we had to use a dial-in modem connection on one computer to access the company database. This restriction slowed us down but we made consistent progress and were basically finished with sequencing and assembling the genome sequence by the end of 2000. Around that time, we became aware of another collaboration between an academic lab at University of Washington and DuPont Corporation that was also sequencing the Agro C58 genome. It was a race but luckily in the end both collaborations agreed to publish back-to-back articles (3,4) in the journal SCIENCE that came out in December of 2001.

So what we did we learn from the Agro C58 genome sequence? First and foremost, the 2.1 Mbp linear chromosome was evolutionarily derived from a plasmid! The origin of replication on this chromosome is clearly a member of the repABC plasmid family, very similar to those found on two large plasmids in strain C58. Second, the linearity of the second chromosome is due to hairpin loops on each end where the top and bottom strands are connected through a stem-loop structure. In a later paper, we obtained the full sequence of the hairpin loops and showed that the linear chromosome is found only in one subset of Agrobacterium and Rhizobium strains called biovar 1 (5). During replication, the two “old” strands are still connected at their ends. Once the hairpin loops are replicated, an enzyme called protelomerase recognizes the double-stranded hairpin sequences and makes staggered cuts to allow the two new daughter ds DNA molecules to separate and reform hairpin loops on each end. We don’t know yet the evolutionary origin of the hairpin loops and the gene encoding protelomerase. My personal hypothesis is that they came in as part of a linear bacteriophage. Third, comparison of the two chromosomes of Agro C58 with the sequenced single chromosome of Sinorhizobium strain 1021 showed clear evidence that several large chunks of the ancestral circular chromosome moved to the plasmid that became the second chromosome. I had several Hiram College students continue studying this phenomenon and this became part of another follow-up publication (6).

There were a lot more insights gleaned from the Agro C58 genome sequence and we continue to link genes to functions using functional genomics experiments such as the Mariner-type transposon mutagenesis screen going in the 2019 Hiram College Genetics course. However, I will leave those details for another time.

The Agro C58 genome shows us how complex genomes can arise in the Bacteria domain and how genomes can rearrange over time. Now let us see what we can learn from other genomes. Stay tuned to Genomics Revolution.

For More Information on Agrobacterium strain C58 & its genome:
(1) Allardet-Servent et al., 1993. Journal of Bacteriology 175:7869-75.
(2) Goodner et al., 1999. Journal of Bacteriology 181:5160-6.
(3) Goodner et al., 2001. Science 294:2323-8.
(4) Wood et al., 2001. Science 294:2317-23.
(5) Slater et al., 2013. Applied & Environmental Microbiology 79:1414-7.
(6) Slater et al., 2009. Journal of Bacteriology 191:2501-11.

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Welcome back to Genomics Revolution. I am Brad Goodner. Last time we were together, we walked through the strategy used by Craig Venter’s team at TIGR, The Institute for Genomics Research, to sequence the first genome of a cellular organism, Haemophilus influenzae strain Rd.

Today, we will finish up our analysis of the July 1995 SCIENCE article by focusing on the biological implications of knowing the complete sequence of an organismal genome. The H. influenzae strain Rd genome is a single circular chromosome of 1,830,137 base pairs. Previous to this work, the sequence of 122 protein-coding genes and their surrounding noncoding regions had been deposited in GenBank, the world’s foremost database of gene data. The authors of the genome paper, Robert Fleischmann and 39 coworkers, used a published computer algorithm and the previously known coding and noncoding sequences from H. influenzae to construct a model of how the coding sequences differed from the noncoding sequences. This may sound odd, but it turns out that the parts of any given genome that code for proteins, regardless of the specific proteins involved, share key characteristics such as certain dinucleotides, trinucleotides, tetranucleotides, etc.  that are more or less abundant than predicted by the single nucleotide base composition of the genome. These characteristics are unique to each species. Once the computer algorithm had been “trained” to distinguish coding from noncoding regions, Fleischmann and coworkers put the entire genome sequence through the algorithm to predict putative protein-coding genes. For the H. influenzae Rd genome, the algorithm predicted 1743 protein-coding genes or about 1 protein-coding gene per every 1000 base pairs. This rough estimate has held up remarkably well since then across the entire Bacteria and Archaea domains, but it is much smaller that that seen in the Eucarya domain.

Of the 1743 predicted protein-coding genes, 1354 of them had 30% or greater protein sequence identity to genes previously sequenced in other organisms. Evolution keeps what works! However, that does not mean that we know what all of these proteins actually do. 1007 of these genes were similar to known genes that encode proteins of known function. 347 of them were similar to genes encoding “hypothetical” or “conserved hypothetical” proteins. That leaves 389 protein-coding genes with no similarity to previously sequenced genes. Some of these genes turned out to be shared with other organisms but just hadn’t been sequenced yet. However, some of them appear to be unique to the genus Haemophilus. This point appears to be true for all sequenced genomes. Evolution is also eternally creative.

Fleischmann and coauthors found many other interesting biological features from their analysis of the H. influenzae Rd genome. Remember that the Rd strain is a nonpathogenic relative of known pathogenic strains. The Rd genome shows evidence of its pathogenic heritage as some virulence genes and regulatory sequences remain, but it also shows several losses of key virulence genes.

Every genome sequenced since this 1995 breakthrough answers some longstanding questions, illuminates some previously unknown biological capacities, and brings up even more questions and hypotheses for future work.

In future episodes, we will learn more about both well-known organisms and recently discovered ones through their genomes. See you next time.

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Welcome back. I am Brad Goodner, Professor of Biology at Hiram College. We have reached the point of the first genome sequence from a cellular organism, published in July of 1995 in the journal SCIENCE.

As we discussed earlier in Episode 4, Craig Venter and his colleagues at TIGR, The Institute for Genomics Research, came up with a probability-based approach, a shotgun approach, to sequencing a genome. Break it up into pieces and sequence enough pieces to cover the genome at least 5 times to hopefully obtain 99% of the genome sequence.

The 1995 SCIENCE article by Robert Fleischmann and 39 coauthors, including Craig Venter, focused on the genome of Haemophilus influenzae strain Rd. This strain is a nonpathogenic sister of strains that can cause inner ear infections, respiratory infections and even bacterial meningitis. Many of you have been vaccinated against several pathogenic strains of Haemophilus influenzae. The genome of H. influenzae strain Rd is 1.83 million base pairs present as a single circular chromosome. This genome was chosen for its small size and because its G+C content of 38% was very close to that of humans.

Fleischmann and coworkers grew up a culture of the bacterial strain and isolated DNA. They then randomly sheared the DNA into fragments using sonication and separated the fragments using gel electrophoresis. DNA of two size ranges were purified from the gel – 1500 to 2000 bp and 15,000 to 20,000 bp. The purified DNA fragments were then treated with DNA polymerases and exonucleases to generate blunt ends with phosphorylation 5’ ends. The blunt-ended fragments were ligated into a plasmid vector to make two libraries – small insert and large insert.

From the small insert library, the researchers sequenced both ends of over 7000 plasmid clones and one end of over 9000 more clones. The average size of the sequence reads were around 450 bases. Overall, this resulted with over 11.6 million bases of sequence, just over 6X the size of the genome. The shotgun approach is action!

Now the work was turned over to computer algorithms that looked for overlaps between the sequence reads that met a set sequence identity criterion. In this way, the initial sequence reads were assembled into 140 larger fragments called contigs. The researchers estimated that the remaining gaps between the contigs averaged about 100 bases in size. Some of the gaps were due to the randomness of the shotgun cloning methods while other gaps were due to the fact that certain genome fragments were somehow lethal to the E. coli host cells carrying the library plasmid clones.

To close the gaps required human ingenuity. For example, the researchers used the ends of each contig to see if any of them encoded parts of the same protein. If so, they designed PCR primers from each potential adjoining end and using those primers with H. influenzae genomic DNA as the PCR template. In addition, the researchers also used the contig ends as hybridization probes on Southern blots of DNA from the large insert library clones. If the ends of two different contigs hybridized to the same large insert library clone, then the same PCR strategy could be used as well as the two ends of the large insert were sequenced. Using these strategies and a few others, the researchers were able to close all of the gaps.

In this way, Fleischmann and coworkers figured out the first complete genome sequence of a cellular organism. The shotgun strategy was proven a success and became the model for virtually all subsequent genome projects. The cost of this project turned out to 48 cents per finished base pair or just under $900K. Since then, the cost of genome projects has dropped precipitiously to the point that today the same size genome could be sequenced for about $500.

The genome era truly came alive with this publication, but there are biological implications beyond the technological ones. We will deal with the biological implications next time.

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Once the goal of obtaining a human genome sequence had been set by research scientists and several government agencies around the world, the big question was how to organize the effort. Any genome of a cellular organism, but especially the human genome, is a massive amount of information. How do you gather the information and how do you piece it all back together at the end? There was no technology available in the late 1980’s and early 1990’s, and there is still none to this day, that allows one to jump onto a giant DNA strand and determine its sequence. You have to break the genome into lots of pieces, figure out the sequence of all the pieces and put all the sequences back together in the right order so that the virtual genome equals the real physical genome.

Two approaches ended up in a race with each other to sequence the human genome. The larger group was a public consortium of government-funded labs around the world, but mainly in the U.S., the U.K., and Japan. This effort was first led by James Watson of Watson & Crick fame, then by Francis Collins who saw it through to completion. The public effort focused on separating the human genome into individual chromosomes and sub-chromosome pieces to organize the sequencing and simultaneously developing really fine-scale physical maps of each chromosome to help assemble the sequence reads back in the right order. Now there was quite a bit of mapping information known for the human genome already, but much more detail was needed for this mapped-based strategy.

The second, smaller effort was a private affair led by the for-profit company Celera Genomics and several big corporate donors. Celera Genomics and its sister non-profit research organization called The Institute for Genomics Research, TIGR for short, were founded by Craig Venter, a very successful biochemist turned entrepreneur who had once worked at NIH. Venter and his team felt that they had a better strategy – faster, cheaper, and more applicable to any genome of interest. Why wait to develop fine-scale physical maps of a genome? Why not just break the genome into random pieces and sequence them, but here is the rub. You don’t know which random pieces you are sequencing until you have sequenced them. How many random pieces do you have to sequence in order to get virtually all of them? In other words, how hard do you have to work to achieve your goal?

This is actually a problem we have all dealt with on more than one occasion since we were little kids. Think about a really big bag of M&Ms of your favorite flavor. You know that there are seven colors represented in the bag. If you randomly pour out seven M&Ms into your hand, the probability that each color should be represented once is not one. There is an element of random chance in terms of which M&Ms fall out of the bag or in the case of a genome, which DNA fragments you randomly sequence. Craig Venter and his colleagues knew this was true with their so-called shotgun strategy to genome sequencing. In fact, they made use of a statistical distribution, called the Poisson distribution, that simulates such random events. The Poisson distribution can be used to understand random events through the an equation that allows us to calculate the probability of a particular outcome. For example, if the average number of any particular M&M color in your sample is one, what is this probability that a any given M&M color was not seen at all? Using m to represent the average and x to represent the number of interest, the Poisson distribution equation is:

Px,m = (mx . e-m)/x! For the M&M question, P0,1 = (10 . e-1)/0! = (1 . 0.37)/1 = 0.37

This means that there is a 37% probability that if we pour only 7 M&Ms out of the bag that a given color will not be represented. That is not good enough, whether our goal is getting one of each color of M&M or of getting every piece of a genome represented. We can use the Poisson distribution equation to determine how hard we would need to look. We can try different values for m, the average number of times we have seen a particular M&M color or genome fragment, in order to determine the probability of getting no hits. As we have already seen, the probability of getting no hits for a particular M&M color or genome fragment given an average number of hits of 1 is 0.37. For an average number of hits of 2, the probability of getting no hits for a particular genome fragment is 0.14. For an average of hits of 3, the probability of no hits for a particular genome fragment is 0.05. Now we are getting somewhere. If we sequence enough genome fragments to represent 3 times the number needed to cover the entire genome, we should have 95% of it done. If we go up to an average of hits of 5, the probability of no hits for a particular genome fragment is 0.01. Now we have 99% of it done. No need to map a genome first. Just sequence enough pieces to cover the genome 5 times or more.

We will see the shotgun strategy in action in our next episode – the first genome sequenced from a cellular organism. See you then.

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Welcome back. I am Brad Goodner. While genetics as a scientific discipline did not need DNA sequences to get started, it certainly progressed at a much faster clip once one could see what genes actually looked like and determine how genes change due to different mutations. We discussed the Maxam-Gilbert chemical and Sanger enzymatic methods for sequencing DNA strands in our last episode. Their impact was so immediate that Gilbert and Sanger shared ½ of a Nobel Prize just a few short years later. Amazingly, it was Sanger’s second!  The other ½ of that Nobel went to Paul Berg who led efforts in the early 1970’s to develop the methods that we now call recombinant DNA technology or DNA cloning. These techniques involved cutting DNA sequences at specific sites using bacterial enzymes called restriction endonucleases. Restriction is a medical term for cutting and endonucleases are enzymes that cut nucleic acids, in this case double-stranded DNA, within the molecule as opposed to at a free end. For example, the restriction endonuclease BamHI always cuts the DNA sequence 5’-GGATCC-3’. Notice that the complementary strand of DNA is also 5’-GGATCC-3’ just running right to left instead of left to right. Such sequences within a double-stranded DNA molecule are called palindromic sequences. By cutting DNA molecules with different restriction endonucleases and figuring out the sizes of the resulting DNA fragments, scientists could come up with physical maps of a DNA molecule. While they didn’t know from this data alone the complete sequence of the DNA molecule, it was a starting point based on some of the sequence information. By analogy, it was like knowing the layout of streets in a town without knowing every house on every street. In addition to enzymes that cut DNA, recombinant DNA cloning also involved enzymes that could sew DNA fragments back together. The medical term for sewing back up is ligation and these enzymes are called DNA ligases.

This growing physical mapping information about DNA molecules and the initial efforts to sequence fairly small pieces of DNA strands were building on a much older history of genetic maps in different model genetic organisms such as fruit flies, baker’s/brewer’s yeast, and maize. By following the inheritance of different mutations through crosses, geneticists could start to arrange mutations and the genes they were in along linear maps of chromosomes. Through these efforts, they figured out that the number of genetic maps in a given organism usually equalled the number of different types of chromosomes in that organism. In bacteria such as E. coli that lack sexual reproduction, geneticists came up with modifications to their genetic mapping strategies. In most bacteria, the genetic map formed a circle which later matched the true circular nature of the chromosome. These genetic maps were cruder than the physical maps in terms of scale – putting towns in spatial reference to each other rather than individual streets and houses, but genetic maps had a real advantage. They were linked to traits, measurable phenotypes seen in an organism.

All of these tools, old and new, were in the hands of geneticists and other scientists interested in DNA by the year 1980. Over the next 15 years, advances in recombinant DNA technology and DNA sequencing along with sociological changes in the way scientists and governments approached scientific challenges brought forth the Human Genome Project. Here are some of those changes.

Scientists came up with ways to use restriction endonucleases to physically map the human genome. By 1995, the physical map had over 15,000 markers on it. The genetic map of the human genome had 400 mapped traits by 1987.

Scientists came up with ways to handle and clone into plasmids bigger and bigger chunks of DNA – moving from a few thousand base pairs up to over 100 thousand base pairs.
Kary Mullis and colleagues at Cetus Corporation develop a strategy for using DNA Polymerase to replicate user-defined short stretches of DNA over and over and over again to amplify the amount of the user-defined sequence. Their strategy, called Polymerase Chain Reaction, made it easy to obtain workable amounts of specific DNA sequences from a tiny amount of starting material.

Scientists, both at universities and connected to business interests, made Sanger replication-based DNA sequencing into an automated technology. DNA sequencing became more of a standardized service that universities and research institutions provided to their researchers than an individual lab art form.

Big name scientists wrote opinion pieces in the top scientific journals making a case for an all-out effort to sequence the human genome. Discussions about such an effort took place at several research conferences.

In the United States, the National Institutes of Health, NIH for short, and the Department of Energy, DoE for short, independently started plans for sequencing the human genome. NIH makes sense given its mandate to promote human health, but DoE had two good reasons as well. Its governmental charge is to safeguard and promote energy supplies in the US of all types. One energy supply, nuclear power, has clear safety concerns when it comes to exposure to nuclear radiation and subsequent DNA damage. DoE wanted to better understand the impact of radiation on the human genome. DoE had a longer-term energy interest as well – bioenergy in the form of organic carbon polymers stored in algae, crops, and trees. Not as scientifically sexy a topic as the human genome, but very important in its own right. In the end, NIH took the lead but both government agencies were heavily involved in the Human Genome Project. Jim Watson of Watson and Crick fame was picked to head up the new effort. He stayed in the job for 5 years and was replaced by Frances Collins who led the US government-based efforts until it reached its original goal of a complete human genome sequence.

Likewise, government-based scientific agencies in Europe and in Japan made similar decisions to be part of the Human Genome Project.

Early on, several groups of scientists, government agencies and business-based efforts realized that the smaller genomes of other model organisms would be good starting points to better experimental strategies, sequencing technologies, and data analysis tools. There were lots of basic biology interest in these smaller genomes as well.  By the time the first drafts of a human genome sequence were published in 2001, there were already over 50 completed genome sequences of other cellular organisms – many Bacteria, a handful of Archaea, a fungus, a plant, a nematode, and a fruit fly.

Next time, we will consider the two initially competing approaches that were taken to sequence the human genome and why one of those approaches became the standard for all subsequent genomes and metagenomes. Talk to you again soon.

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I am Brad Goodner. Welcome back to Genomics Revolution. To fully understand the impact of having an organism’s complete DNA sequence, its genome, we need to put it into the proper context set by the previous 150 years. Genetics as an experimental science got its start in the middle of the 19th century with Mendel’s inheritance trials on pea plant phenotypes and with Meischer’s biochemical isolation of nuclein, what we now call DNA. Mendel’s ideas on the rules of inheritance in sexually reproducing eukaryotes was generalized into the concept of a gene as a definable unit of genetic information controlling a particular phenotype in the early 20th century, before DNA was confirmed as the genetic material. The work of Beadle and Tatum cemented this concept into “one gene encodes one protein which catalyzes one particular biochemical reaction, typically one step in a biochemical pathway.”

For most of the 20th century, scientists studied one gene at a time. Their typical approach Involved isolating mutants – individual organisms with one or more mutations in a gene of interest that had a noticeable impact on a particular organismal phenotype. Mutations are nothing more than changes in a DNA sequence, but we didn’t have ways to determine a DNA sequence until the 1960’s. Scientists figured out that changes in a DNA sequence can potentially change the sequence of amino acid residues in a protein encoded by that DNA sequence.

By the time I was in high school in the late 1970’s, two groups had worked out methods that allowed labs all over the world to sequence DNA routinely. One method, the Maxam-Gilbert chemical method, started with a DNA strand labeled at one end with a radioactive phosphorus in the 5’ phosphate group. Four tubes containing large amounts of the labeled DNA strand are each exposed to different chemical conditions that lead to breaks in a DNA strand at specific nucleotide residues. In one tube, breaks occurred at purine nucleotide residues. Remember that A and G are the bases in purine nucleotides. In another tube, breaks occurred only at G residues. In a 3rd tube, breaks occurred at pyrimidine, C or T, nucleotide residues. In a 4th tube, breaks occurred only at C residues. Imagine a DNA strand 24 nucleotide residues long with A, C, G, and T residues alternating. ACGTACGTACGT… and so on.
In the first tube, breaks will be induced at A or G purines. Some of the DNA strands will be broken at position 1, others at position 3, others at position 5, others at position 7, and so on. In the second tube where breaks only occur at G residues, some of the strands will be broken at position 3, others at 7, and so on at a 4 base interval. In the 3rd tube, breaks will occur at C or T pyrimidines. Some DNA strands will be broken at position 2, others at position 4, and so on. In the 4th tube, breaks will occur at C residues – some at position 4, others at position 8, and so on at a 4 base interval. If we run the contents of each tube through a jello-like sieving matrix that separates DNA molecules on the basis of size, the smallest DNA fragments will run fastest. Remember our starting DNA strand, a 24-mer ACGTACGTACGT… The fragment breaking at position 1 will run the fastest and will only show up in the tube 1 lane. The fragment breaking at position 2 will be next but it will show up in both the tube 3 lane and the tube 4 lane. You could usually read 100-200 bases of sequence from one gel run. Several reactions to carry out, lots of radioactivity involved that no one wanted to be exposed to. 

In 1979, Sutcliffe published the complete DNA sequence of one of the earliest recombinant DNA molecules, the cloning plasmid pBR322. Plasmids are nonessential extra DNA molecules, usually circles, found in Bacteria, Archaea, and some Eucarya. The plasmid pBR322 is a man-made recombinant molecule, built from several natural DNA pieces. Sutcliffe sequenced pBR322 using the Maxam-Gilbert chemical method. It’s 4362 base pair long sequence was one of the first DNA sequences I worked with when I started graduate school in 1983. The next year, I went to my first research conference where I heard Richard Barker give a talk about the sequence of a key DNA sequence. The T-DNA or transferred DNA is a piece of bacterial DNA involved in the plant disease crown gall. Barker had almost single-handedly used the Maxam-Gilbert chemical method to determine a sequence of 24,595 nucleotide residues that encoded 14+ proteins. This was a tremendous feat at that time. My fellow grad students and I were awed, but we also fearfully joked that we hoped Barker had already sired his children because of all the radioactivity and nasty chemicals involved. Because of these risks, the Maxam-Gilbert chemical method lost out over time to an enzymatic method of DNA sequencing perfected by Fred Sanger and colleagues.

Sanger’s group built their enzymatic method around the way that cells naturally make new DNA strands by using the enzyme DNA Polymerase. This enzyme needs three components to build a new DNA strand. One, an old single-strand of DNA is needed as a template. The template strand is complementary to the new strand that will be made. By that I mean, A’s on the template strand will interact with T’s on the new strand and vice versa. G’s on the template strand will interact with C’s on the new strand and vice versa. Two, DNA Polymerase cannot start a new DNA strand from scratch, rather it has to add onto a pre-existing piece of single-stranded RNA in the cell or a piece of single-stranded DNA in the test tube. This starting piece is called a sequencing primer. By choosing the right sequencing primer, one can determine the sequence at different places along a large DNA strand. Three, DNA Polymerase catalyzes the formation of the new DNA strand using deoxyribonucleotides, the monomer subunits in a DNA strand polymer. In the Sanger enzymatic method, 4 tubes are set up with the same template DNA strand, the same starting complementary sequencing primer, and all 4 deoxyribonucleotides. The sequencing primer carried a radioactive label on one end. In the first tube, a little bit of a modified A nucleotide was added. The A was different in that once it was added onto a growing DNA strand, no more nucleotides can be added to it. So in this tube, the new DNA strands will each end with a modified A residue, but since the modified A is rare the termination of DNA strand grow will be rare and random in terms of which A is the termination point. In the second tube, a little bit of a modified C nucleotide was added. A little bit of modified G in tube 3 and a little bit of modified T in tube 4. Similar to the Maxam-Gilbert chemical method, the final labeled DNA strands in each tube are separated by size by running them through a jello-like sieving matrix called a sequencing gel. If we consider the same 24-base long DNA strand as before, ACGTACGTACGT…, then the lane on the sequencing gel using the results from tube 1 will show labeled fragments of the sequencing primer plus 1, plus 5, plus 9, plus 13, plus 17, and plus 21 in size. The lane for results from tube 2 will show fragments of the sequencing primer plus 2, plus 6, plus 10, plus 14, plus 18, and plus 22 in size. Eventually, the Sanger method became the method of choice for automated DNA sequencing machines and the radioactivity involved was replaced with four different fluorescent tags added to the four modified DNA terminating nucleotides. A laser at the end of the sequencing gel excites the fluorescent tag on each DNA fragment as it exits and the resulting fluorescence color tells us which nucleotide was at the end of the fragment.

In 1978, Sanger and coworkers published a paper reporting the complete sequence of the DNA virus phiX174, the first viral genome sequenced. The viral DNA of 5386 nucleotide residues encodes 10 proteins.

In 1982, the U.S. National Institutes of Health, NIH, established a public database for DNA and protein sequences that came to be called GenBank. By the end of 1982, there were 606 DNA sequences deposited in GenBank totaling over 680 thousand bases. Within a year, the amount of DNA available had tripled. By the end of 1987, there was over 10 million bases of DNA sequences deposited. By 1992, there was over 100 million bases available in GenBank. Most scientists sequenced just one to a few genes at a time, but change was coming and in 1995 it happened. More on that in later episodes. See you next time.

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Welcome to the podcast Genomics Revolution. I am Brad Goodner, Professor of Biology at Hiram College. This is a podcast about the biggest explosion in biological knowledge in human history and it is has been happening all around us over the last 30 years. The Genomics Revolution is based on 1) some basic knowledge about DNA – how it is structured and faithfully replicated, 2) an ambitious goal to fully understand the complete genetic basis of human biology, 3) new ways to store, collate, and compare incredibly large data sets, and 4) lots of determined biologists, chemists, computer scientists, and statisticians working together in new collaborations that have smashed holes in academic disciplines and forged new interdisciplinary/multidisciplinary academic departments and biotechnology companies. Genomics has and will continue to transform all aspects of biological understanding from individual cells to organisms to communities and ecosystems. Genomics has also begun to dramatically change medicine, forensics, agriculture, and even anthropology and paleontology.

Yet what is genomics? It is simply put the study of a genome or all the DNA of a cell or organism of interest. You and I have grown up knowing what DNA is, both as the hereditary material passed from one generation to the next and as a specific biological polymer – a double helix of strands each made up of units we know by their shorthand names – A’s, C’s, G’s, and T’s, linked together in a strand by strong bonds while the two strands interact to form the double helix using relatively weak bonds. You and I have also grown up knowing that what a gene is – a stretch of DNA sequence that tells a cell to do something. By “do something”, I mean that the DNA sequence of a gene tells a cell to make a copy of that DNA as RNA, a shorter-lived single stranded polymer where T’s are replaced by U’s. In most cases, the RNA made from a gene carries a code for making a very different polymer – a linear protein sequence made up of amino acid residues linked together. Proteins do most of the work of cells, but genes tell a cell what proteins to make. A genome is composed of all the genes of a given cell or organism along with some DNA that does not act as genes. That extra DNA is also important in other ways that we will discuss in a later episode.

So DNA makes up a gene and all the genes in a cell plus some extra DNA equals a genome. That is a lot of information, usually 400,000 base pairs of DNA or more in the genomes of cellular organisms and the more can range up into the billions of base pairs for organisms such as we humans or corn plants. In future episodes, we will see why scientists wanted all of information, how they figured out the most efficiently ways to obtain the information, and how they came up with different ways to analyze the information. We will also start to talk about specific genomes, why we care about them, what they teach us about how genomes and organisms work, and how we might use that information to solve problems in healthcare, agriculture, or bio-energy. We will also see how we can add layers of additional information onto a given genome. For example, all of the RNAs made from a genome through the cellular process of transcription is called the transcriptome, while all of the proteins made based on the genes in a genome are called the proteome. There are many other “-omes” out there to be explored. Finally, we will consider the combined genomes of many organisms, typically microbes, that live in a particular habitat or community. Such metagenomes, literally meaning “above one genome”, have identified new organisms we could not identify earlier because we could never grow them. All of this sounds complex, but we will break it down into understandable 10-20 minute segments.

I hope you keep listening to future episodes of Genomics Revolution. Talk to you again soon.