Genomics Revolution

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.

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.

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.

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.

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.

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

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

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.

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. 

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.