Genomics Revolution

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.

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.  

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

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.

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.

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

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

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.

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.

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.