RecQ's Role in Illegitimate Recombination

Genetic recombination is a ubiquitous event that occurs in every species; it is necessary for the production of unique gametes, it is involved in DNA damage repair, it provides a mechanism for evolution, and it is a required action for many viruses and phages to undergo nucleic acid replication.(My discussion here will focus entirely on recombination in prokaryotes and their viruses)

There are three different types of recombination: homologous recombination--where DNA of significant sequence similarity recombines, non-homologous recombination--where DNA without significant sequence similarity recombines (usually along gene boundaries), and finally there is illegitimate recombination--where DNA recombines randomly.

Although DNA is surprisingly fluid, there are enzymes that mediate recombination--by initiating DNA binding, strand invasion, and stabilizing ssDNA intermediates. Also, of important note, is that organisms have varying degrees of recombination levels. A classical example occurs within the Mycobacteria. Mycobacterium smegmatis has relatively low levels of illegitimate recombination (IR), while M. tuberculosis is notorious for high levels of IR compared to homologous recombination. This raises a question that can be phrased in a few ways. "What enzymes are responsible for IR?" or perhaps, "What enzymes for homologous recombination are lacking?"

I performed a cursory search of the Rec-type proteins in both M. smegmatis and M. tuberculosis, and found that M. smegmatis contained a RecQ homologue, while M. tuberculosis did not. A literature searched showed that RecQ is a DNA helicase that is involved in the suppression of IR (Hanada and colleagues in this paper published back in 1997) in E. coli.

The paper describes a rather elegant experiment to see the frequency of IR by utilizing a specialized transducing phage. Briefly, a specialized transducing phage is a lysogenic phage that, upon excision from the chromosome, accidentally packages host DNA located directly adjacent to the integrated. This event is usually very rare and is catalyzed by an IR event. Therefore, any changes that increase IR frequency will increase the frequency of transducing particles. In this instance, the authors used Lambda Phage, and a screened for the production of transducing particles.

The authors found that in a recQ minus strain, the number of transducing particles increased 10-100x higher than wild type (depending on conditions and marker used). This increase could be removed by the complementation of recQ. Furthermore, the authors found that this pathway was independent of the well characterized RecA pathway by creating a recQ / recA double mutant and finding no difference in IR from the recQ mutant.

They discuss further associations with the RecJOF pathways, however, delving further into a discussion on Rec pathways is beyond the scope of what I can discuss here. (More information can easily be found by searching Google for "Rec proteins")

Since this study was published back in 1997, it made me curious as to whether anyone has studied the lack of recQ in M. tuberculosis, its presence in M. smegmatis, and their drastic differences in IR. As it turns out, no one has (to my knowledge).

I am currently interested in examining recombination functions necessary for phage replication in the Mycobacteria. You can read about my current tuberculosis research here and I will post a recent update here (coming soon). I'm currently working on knocking out recQ in M. smegmatis and expressing recQ in M. tuberculosis and asking if there are effects on phage infection. There is already a system in place that overcomes IR in M. tuberculosis to allow allelic exchange, ( Nat Methods. 2007 Feb;4(2):147-52.) and I am testing this system as well.

For more information on Mycobacterial Genetics, I suggest the following book which is the most recent text on the subject.

Molecular Genetics Mycobacteria

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Hanada, K., Ukita, T., Kohno, Y., Saito, K., Kato, J., Ikeda, H. (1997). RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichiacoli. Proceedings of the National Academy of Sciences of the United States of America, 94(8), 3860-3865.

Myxoma virus: From Rabbits to Cancer

More than half a million individuals in the US died in 2004 from cancer and more than 10.8 million Americans are living with the disease.(CDC 2008) The number of cancer deaths each year continues to decline, due to advances in medical technologies. With the advent of a cervical cancer vaccine,Gardasil this number promises to continue to drop dramatically. Furthermore, there are new treatments in development that promise to revolutionize the effective treatment of cancer. One of these, which I am currently fascinated by, is oncolytic virotherapy. This is a summary of a review article by Grant McFadden at the University of Florida.

Oncolytic virotherapy is the use of live viruses to kill malignant cells in situ. The use of viruses holds potential to not only be a direct treatment of cancer, but also studies of viruses with cancer cell tropisms may help in the development of other treatments and vaccines.

There is much knowledge regarding the ways that viruses “push” host cells into appropriate stages of the cell cycle to allow a productive viral infection (Adenoviruses, Papillomaviruses, and Polyomaviruses all push non-replicating host cells into S-phase, allowing productive infection). These viruses, therefore, have the ability to not only cause cancer (by the unchecked growth), but also are more able to kill rapidly dividing cells—cancer cells are more susceptible.

A virus called “ONYX-015,” an adenovirus, was the first oncolytic virus tested. Lacking a gene called E1B-55K, this virus can only replicate in cancer cells. E1B-55K serves to block the p53 pathway, thereby allowing the host cell to enter S-phase unchecked. Without E1B-55k, p53 remains active. But in cancers, the p53 pathway is usually inactive; therefore these cells remain susceptible to infection.

This paper discusses the use of a more preferential type of oncolytic virus; one which does not normally infect human cells, but is able to enter productive infection in cancer cells. Specifically, this review describes the use of myxoma virus, a species of poxvirus.

Poxviruses have been known for ages, and have been studied as both vaccines and pathogens. These types of viruses posses a variety of features which are highly desirable to the development of oncolytic virotherapy. 1) Short (24hr) replication cycle 2) Large, easy-to-engineer genome 3)Exclusive replication in the cytoplasm—with no record of any chromosomal integration 4)Highly immunogenic, allowing strong immunity to be formed.

Myxoma virus causes the highly lethal “myxomatosis” in European rabbits (see photo at left) and is remembered by most as the infamous biocontrol method to decrease rabbit populations in Austrailia. This virus has a highly specific tropism for rabbits and is not known to infect any other vertebrate species, including humans.

Although myxoma virus (and most poxviruses) can attach and enter most mammal cells, it was shown to require the action of a specific pathway (type I IFN). This pathway is turned on specifically in cancer cells. Studies have shown that myxoma virus can infect and kill 70% of all cancer lines from the NCI collection—without a specific tumor cell-type tropism.

Further work still must be done to definitively show the pathway involved. Other work includes examining persistant infection, along with studying delivery methods. However, there is great hope for the use of this virus(and perhaps others down the line) to combat cancer.

Stanford, M.M., McFadden, G. (2007). Myxoma virus and oncolytic virotherapy: a new biologic weapon in the war against cancer. Expert Opinion on Biological Therapy, 7(9), 1415-1425. DOI: 10.1517/14712598.7.9.1415

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).

Clearly 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

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Read a breif bit more on M.leprae's reductive evolution here

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.

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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