A Brief Bit More on Reductive Evolution in M. leprae

In a previous post I discussed the evidence for reductive evolution in Mycobacterium leprae, an interesting obligate intracellular parasite.

At the 2008 ASM General Meeting, the Division U keynote lecture was headed by Tom Gillis of the National Hansen's Disease Program. His talk described the same work I cited in the previous article, which showed the immense amount of pseudogenes in the M. leprae genome.

Gillis was interested in elucidating the role of these pseudogenes. This included asking whether or not these genes are transcribed and translated. If these pseudogenes are not providing any function, then it stems that the cells will not put energy towards their expression.

The work he discussed showed that ~44% of all M. leprae transcription was due to pseudogene expression. There doesn't appear to be a locational bias for pseudogene transcription either. Looking closely at 10 pseudogenes downstream of full-length genes, only 8 produced full-length transcripts.

More indepth in silico analysis shows that all these pseudogenes are unilogs (no duplicates present in the M. leprae genome), the vast majority lack a strong Shine-Delgarno sequence upstream, ~75% lack a translational start codon, and ~98% have one or more in-frame stop codons inserted.

This indicates that a very small percentage of pseudogene transcripts actually create a full-length translational product. So, although the cells still create the transcript, few (if any) resources are put towards creating a functional (or detrimental) protein product.

I also picked up some interesting epidemiological facts of the M. leprae genome. For one, the global M. leprae population is nearly clonal (1 polymorphism to 20,000bp compared to 1:5000 for M. tb.). However, variation in SNPs can be seen in local populations. In looking at ~60 cases from a town in India, the bug had a higher rate of diversity than compared to 3 cases in the South Eastern US or to 20 wild armadillos. Furthermore, the US cases and the wild armadillo cases were strikingly similar on an SNP scale.

I think an important point to take home from this is that M. leprae is still an evolving organism, and we are only catching a snapshot in time. It is a prime example of a parasite that has come to depend greatly on its host and has lost the ability to function outside said host.

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Other articles of mine that may be of interest
Reductive Evolution in Mycobacterium Leprae
Evolution of Phage Capsid and Genome Size (Another 2008 ASM General Meeting Lecture)

Evolution of Phage Capsid and Genome Size

Viruses come in all shapes and sizes. From the very small, such as the picornaviruses or the parvoviruses, to the very large like mimivirus, or the herpesviruses, and poxviruses. These large viruses are not just large in physical size, but in the size of their genomes as well.

At the recent 2008 ASM General Meeting, Roger Hendrix of the University of Pittsburgh, laid forth a rather interesting hypothesis as to how large genomes, and the capsids that hold them came into existance and how they managed to be competitive in the gene pool.

Using phage P1 as an example, we know that larger capsids can be created "simply" by a single mutation allowing capsid subunits (capsomers) to come together in a quasi-equivilant matrix that is larger than the previous. This matrix follows the mathematical model set by Casper and Klug, and has discrete sizes (triangulation numbers, such as T=1,3,4,7,13). An increase in T number, as in our P1 example, causes a dramatic increase in capsid volume. Hendrix proposes that a mutation causing a such a shift acts as an evolutionary ratchet, and therefore smaller capsid sizers would no longer be available.

Now that we have a larger capsid, the phage now has the ability to package much more DNA. Not only does it have the ability, but in many cases, the phage MUST package DNA until its capsid is filled (headfull-packaging). With a larger capsid, phages who package via headful mechanisms now must package more DNA creating a greater amount of redundancy in its genome.

Hendrix explained that a greater amount of terminal redundancy leads to greater resistance from DNA damaging agents, specifically UV light. Although some in the session contended this, Hendrix described large amounts of genomic redundancy as an evolutionary advantageous trait for phages which live on the surface of the ocean and soil.

Furthermore, the extra space in the genome acts as a virtual genetic laboratory to aquire and mutate genes without disrupting the ability of the phage to survive. Gene aquisitions and subsequent mutations could create genes which provide some sort of marginal (or large) benefit to the phage or the host it infects.

With a simple click of the ratchet and a headfull of DNA, the role that large phages play in novel gene development are only now beginning to become clear.

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My posts on similar topics

Mosaicism: Life on a Small, Ever Changing Scale

The Definition of Life; and, Taxonomy as We Know It

Mosaicism: Life on a Small, Ever-Changing Scale (Part 2)

In the my last article, I briefly discussed the role of horizontal gene transfer in bacteria...specifically the development of mosaic pathogenicity islands in Escherichia coli. However, the formation of mosaics are not just limited to operons within bacterial genomes. In fact, we can see such events in phage and viral genomes. This article is part two in a brief series on genomic mosaicism.

Current research shows that illegitimate recombination events create mosaics of unique genes within the genome. Each gene then acts as an individual unit upon addition, whereby beneficial genes are selected for and remain in the genomes, and non-beneficial genes build up mutations or are dropped. Illegitimate recombination, by definition, occurs within gene boundaries, and as such, highly unique and variable regions within genomes are formed.

Looking within the sequenced genomes of the mycobacteriophages (an obvious interest of mine) we see a large amount of mosaicism. Small sets of genes, individual genes, and parts of genes are constantly being shuffled in, out, and around genomes. There are countless examples of gene insertions and deletions in the phage genomes, adding to the case that genomes are liquid and constantly changing. We see gene swaps not only between phages, but also between hosts.

These events show themselves by the presence of unique genes in genomes. For instance:
1) the presence of photosynthesis genes in many cyanophages
2) a portion of mycobacterial MetE in far right arm of the mycobacteriophage Giles genome (a conclusive example of illegitimate recombination between virus and host)
3) the protein Ro in mycobacteriophage Bxz1 (a eukaryotic protein implicated in the human disease lupus)
4) Bxz1 also contains a human peptide chain release factor
5) a portion of Bacillus VIP toxins in a mycobacteriophages PBI1 and PLot
6)Mycobacteriophage Che8 contains a human prion-like protien
7) motifs of resuscitation promoting factors in a wide variety of of mycobacteriophage tape measure proteins
8)not to mention the countless (30-50%) number of open reading frames with absolutely no homologs in the current databases or which only match other phage proteins.

(Yes, I did say that phage Bxz1 of the mycobacteria contains a human antigen gene, with significant sequence similarity, see here.....I'll leave this discussion for another day, mainly because as far as I know, no one has looked at such a seemingly odd phenomenon outside of bioinformatic comparison. How did a human gene sneak its way into a bacteriophage?)

What I am trying to drive home here, hopefully with some success, is that life at the small scale is ever fluctuating and massively interconnected. Nothing in biology is genetically isolated. We are the current homes of many genes, where they end up next is up to our viral and phage couriers.

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PEDULLA, M. (2003). Origins of Highly Mosaic Mycobacteriophage Genomes. Cell, 113(2), 171-182. DOI: 10.1016/S0092-8674(03)00233-2

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My Similar Articles
Mosaicism: The World of Horizontal Gene Transfer (Part 1)
The Definition of Life, and; Taxonomy as We Know It
ReQ's Role in Illegitimate Recombination
I'll Have My Bacteria Extra-CRISPR

Also, visit The Phamerator to explore the sequenced mycobacteriophage genomes on your own.

Mosaicism: The World of Horizontal Gene Transfer (Part 1)

Commonly, gene transfer is thought of as a vertical line from parent to offspring, along which all evolutionary traits are passed. However, as we began delving into genomic sequences, we found that this may not be true and that the lines between "species," especially on the microbial level, are quite fuzzy.

Horizontal gene transfer is the transfer of genetic elements between species. The microbial world is filled with examples of this phenomenon. This article is the first in a 3 part series that will explore the ever fluctuating genetic world of our microbial majority.

Escherichia coli is perhaps the most well studied and utilized bacterial organism on the planet. This bug also has the capability for a wide range of illness in humans including: non-pathogenic, enterohemorrhagic (such as popular strain 0157:H7), enterotoxigenic, and uropathogenic. The question is obviously raised--how can the same organism have such pathotypes, and what allows it to be versatile enough to thrive in such different environments as the urinary tract vs. the intestinal tract.

An article coming out of the University of Wisconsin-Madison shows that E. coli consists of a general common backbone that is nearly 100% identical across strains, yet only takes up 75%of the genome. However, they show that within this backbone are regions (islands) that are highly divergent between strains (See figure at left). It is no surprise that within these divergent regions contain the genes which allow for survival in different environments (pathogenicity islands)

These islands are distinct from each other, the surrounding genome, and the corresponding areas in other strains. However, the striking similarity between islands of different strains is that they all seem to be located in nearly the same locations.

Furthermore, strains of the same pathotype tend to have the same/similar genes located in islands (see figure below)--but not necessarily in the exact location or orientation. These islands tend to occur in areas at tRNA regions--commonly utilized by bacteriophages for integration.

Phages are clearly agents of horizontal gene transfer, further examination of these island regions show the presence of cryptic (non-viable) prophages. This indicates that at least 5 phages integrated, but subsequently lost genes required for the creation of viral progeny. It is possible that the other islands are also prophages, but gene loss has occurred to the point where they are no longer recognizable.

What this means as a whole to the definition of species is to me, yet, unclear. Strains of E. coli all share a distinct backbone of metabolic genes, but differ drastically in their specialized regions. How do we decide which belongs in a species?...especially when we differentiate humans from chimpanzees (only ~6% genetic differences).

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Welch, R.A. (2002). Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proceedings of the National Academy of Sciences, 99(26), 17020-17024. DOI: 10.1073/pnas.252529799

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My Similar Articles

The Definition of Life; and, Taxonomy as We Know It

Reductive Evolution in Mycobacterium leprae

I'll Have My Bacteria Extra CRISPR

The Definition of Life; and, Taxonomy as We Know It

In order to make sense of our surroundings, we, as humans, constantly group things into discrete catagories--states of matter, times of day, types of clothes, etc.

Since Linnaeus, we have consistantly grouped life in boxes-within-boxes, based on physical characteristics. The advent of nucleic acid sequencing changed some of these early notions of taxonomy. But now, as we discover more about life, specifically of our microbial counterparts and their viruses, we are coming to the realization that Linnaein taxonomy is not sufficient. Especially if our motivation for using taxonomy is to deduce evolutionary relationships.

Viral taxonomy has some very specific shortfalls. Researchers in this 2002 Journal of Bacteriology article, lay out these shortcomings. One striking point is that with viruses, structure alone is not sufficient to characterize a phage into species. The figure here shows two similar looking phages with very different DNA sequence. This concept could easily be applied to a variety of other viruses.

Another problem, is that species is defined as organisms that share a common gene pool. Viruses are constantly moving genetic information into and out of hosts. Many viruses have the ability to cross-infect more than one host as well. This means that the gene pool available to an organism crosses most borders of what we traditionally define as species.

The muddle within genetic taxonomy is getting a recent look. The discovery of MIcrobe MImic Virus (Mimivirus) is a startling look at how complex this tangled web is.

A recent article in Nature Microbiology, discusses not only the question of taxonomy, but challanges our definition of life. Their definition presupposes that viruses are more complex than merely "parasitic nucelic acids." They propose that the presence of either a capsid or ribosomes be the primary classification system for life and that genomic contents completely define an organism.

The proposal consists of defining capsid-encoding organisms (the viruses), ribosome-encoding organisms (eukarya, prokarya, and archea), and "orphan replicons" (plasmids and viroids).

Whether or not this classification scheme will take root remains to be seen. There will certainly be critics, however, I think that the evidence available strongly supports the classification of viruses as a segment of life.

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Raoult, D., Forterre, P. (2008). Redefining viruses: lessons from Mimivirus. Nature Reviews Microbiology, 6(4), 315-319. DOI: 10.1038/nrmicro1858

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Further Reading Possibilities
Liaisons of Life: From Hornworts to Hippos--How the Unassuming Microbe has Driven Evolution
Microcosmos: Four Billion Years of Microbial Evolution

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Reductive Evolution in Mycobacterium leprae

Mycobacterium leprae is quite an interesting bug. It is significantly related to M. tuberculosis (the causative agent of TB), yet leprosy is a very different illness.

Unlike other mycobacteria, M. leprae is unique in the fact that it is an obligate intracellular parasite. Replication of the bacterium cannot occur outside of host cells. To date, no synthetic media has been able to support M. leprae replication. Since M. leprae is required to parasitize, it stands to reason that discovering the genetic components of this lifestyle will provide possible venues for drug therapy against this debilitating illness.

It is clear that M. leprae is closely related to the other mycobacteria, this paperby Cole et al. analyzes the genetic sequence of M. leprae and compares it to M. tuberculosis. By doing this, we have the potential to deduce how M. leprae has evolved and which genes are essential for mycobacterial replication outside of host cells.

One of the first things we see is that M. leprae has a 1.6Mbp SMALLER genome than M. tb. (~3.2Mbp and ~4.4Mbp, respectively). Of this much smaller genome in M. leprae, only about 50% of it contains protein coding genes (compared to 90% in M. tb.). The rest of the M. leprae genome is 27% recognizable pseudogenes (with a number of 1,116 compared to only 6 pseudogenes in M. tb. ) and 23% non-coding sequence (possibly as regulatory elements or genes mutated beyond recognition).

It is clear that there has been a tremendous amount of downsizing in the M. leprae genome. The large loss of sequence, along with the large amount of pseudogenes, the low number of protein coding genes, and the huge amount of non-coding sequence all indicate that M. leprae has undergone a clear and classical example of reductive evolution.

The authors go into much specificity about which genes and pathways have been lost in M. leprae compared to M. tb. One highlight is that the central metabolism of M. leprae is much different than in other mycobacteria because of a variety of gene losses. M. leprae can not utilize acetate or galactose as a carbon source. Also, it has lost all enzymes needed to function in an anaerobic or microaerophilic environment. Even it’s aerobic electron transport chain has been truncated—indicating that it can not produce ATP from NADH, and it needs to rely on low energy gain pathways to recycle its reducing equivalents.

In looking at virulence genes, the authors found only one a laminin-binding protein which may cause a tropism for myelin-producing cells (which M. leprae has). However, M. tb. also has this gene, so it’s purpose is not yet known.

M. leprae also has a handful of genes that are not present in M. tb., including eukaryotic like adenylate cyclase and uridine phosphorylase. Also, two transport systems an ABC sugar transporter and a unique divalent metal ion transport system.

I am interested to see how this study affects future drug development for anti-leprosy drugs. Also, this study should help M. tb. researchers, due to the fact that essential genes are likely conserved in these two species. With the advent of MDR and XDR-strains of M. tb. and the persistence of M. leprae in our population, it is imperative that new methods of treatment be developed.

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Cole, S.T., Eiglmeier, K., Parkhill, J., James, K.D., Thomson, N.R., Wheeler, P.R., Honoré, N., Garnier, T., Churcher, C., Harris, D., Mungall, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R.M., Devlin, K., Duthoy, S., Feltwell, T., Fraser, A., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Lacroix, C., Maclean, J., Moule, S., Murphy, L., Oliver, K., Quail, M.A., Rajandream, M., Rutherford, K.M., Rutter, S., Seeger, K., Simon, S., Simmonds, M., Skelton, J., Squares, R., Squares, S., Stevens, K., Taylor, K., Whitehead, S., Woodward, J.R., Barrell, B.G. (2001). Massive gene decay in the leprosy bacillus. Nature, 409(6823), 1007-1011. DOI: 10.1038/35059006

I'll Have My Bacteria Extra-CRISPR

The microbial cold war between species has been widely studied and is, in my opinion, one of the most interesting topics in current biology.

Fungi make compounds to destroy bacteria, bacteria make compounds to destroy fungi. But it doesn't stop there. Phages mutate constantly to evade bacterial defenses. We've known about a handful of bacterial defenses against phage-- repressor systems, restriction enzymes, receptor modification, DNA modification, etc.

We have utlized these systems to our advantage--our first effective antibiotic (Penicillin G) came from the fungus Penicllium, a vital antifungal (cyclohexamide) comes from the bacteria Streptomyces, our ability to clone and subclone is only useful because we can specifically cut DNA with restriction enzymes, etc.....

But now, there is another form of bacterial defense. We have evidence for, what I will call, the "special forces" of bacterial defense systems. Heck, I'd liken this to a form of adaptive immunity.

CRISPR (clustered, regularly interspaced, short, palindromic repeats) sequences have been found in 40% of all sequenced Bacteria and 90% or sequenced Archaea. Essentially, you have an array of nearly identical repeats which form RNA stem-loops. Between the repeats, you find unique DNA that has sequence similarity to phages. There are also a handful of unique genes that are closely associated with the arrays.

The idea is that the bacteria are using an RNA interference mechanism. The CRISPR array gets transcribed and then spliced at the loop structures--forming small RNAs. These sRNAs associate with phage nucleic acids, recruit degradation genes, and subsequently denature the phage nucleic acid. See the figure at right taken from the review article.

You find a distinct correlation between the number of CRISPR sequences and resistance. You also can add specific phage sequence to the array and grant immunity. If you then take that sequence away, immunity is lost.

What is interesting (but not necessarily surprising)is that a small population of phage remained resistant to the system. It was found that these phage had a slightly different nucleic acid sequence at the site. BUT, given time, the bacteria can acquire CRISPR sequence against the phage.

It's an evolutionary arms race!

That we should be exploiting!There are a variety of ramifications that this system can and will have on the future of molecular biology.

For one, a system of spoligotyping could be developed based on the unique spacer regions in the CRISPR array.

Apparently a large problem in dairy industry (specifically yogurt and cheese) is that bacterial cultures get lost to phage. This system could help prevent that by allowing the development of ultra-resistant strains.

A more unique (and as far as I know, untested) use would be the development of a system to knock out genes in vivo. You could imagine setting up a CRISPR system on a plasmid with specificity for a specific gene (or multiple genes) you want to knock out. The CRISPR system would do what it does naturally and degrade the gene's transcript. Since these regions are only 26-72bp, it would certainly be an awesome tool to have--especially for organisms notoriously recalcitrant to genetic manipulation (like my favorite bugs: the Mycobacteria). Genes could be effectively silenced using only a plasmid, without the need for messy recombination systems.

Sorek, Kunin, and Hugenholtz. "CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea". Nature Reviews Microbiology. 6, 181-186 (March 2008)

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Sorek, R., Kunin, V., Hugenholtz, P. (2008). CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea. Nature Reviews Microbiology, 6(3), 181-186. DOI: 10.1038/nrmicro1793