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.

Recombineering: A Practical Application of Phage Biology

The most obvious use of phages is of course direct phage therapy. Although exciting, there are many other uses of phage that are just as revolutionary, but tend to slip by. In my last article, I hinted at a method in molecular biology called “recombineering.” (Short for homology-dependent, recombination-mediated, genetic engineering) This is a method whereby phage proteins are used to catalyze homologous recombination. The ramifications are huge. Mutations can be made; whole genes and operons can be knocked out with relative ease, and much more. In fact, some may say that this recently described process revolutionized E. coli genetics.

Since this system was so successful in E. coli, the Hatfull Lab at the University of Pittsburgh set out to develop this system in the Mycobacteria. Before this, genetic manipulation (especially gene knockouts) was highly difficult. M. tuberculosis is notoriously recalcitrant to genetic manipulation. This is due to a variety of factors including: its horribly slow growth rate, high pathogenicity, and its relatively high levels of illegitimate recombination compared to homologous recombination.

This paper, by vanKessel and Hatfull was published relatively recently (Jan 2007) and describes what is a great advance in mycobacterial genetics.

The first step was to identify potential recombination proteins encoded by phages. These proteins are presumed to be necessary for proper facilitation of lysogenic integration and DNA replication. Looking at the functions of the Lambda Phage “Red” recombination system, (used for recombineering) one can find functional homologs in other systems. The Red system uses three proteins: exo, beta, and gam. Exo acts as a 5’-3’ dsDNA dependant exonuclease, beta acts to bind ssDNA and promote annealing, and gam functions to inactivate an alternative host recombination pathway (the RecBCD system).

Using this as the basis for comparison, one first comes across the Rac prophage of E. coli. This prophage encodes two proteins called RecE and RecT that function identically to the exo and beta genes respectivly in the Red system, without significant sequence similarity. One then can look at the published mycobacteriophage genomes (30 at the time of publication, soon to be 50) and search for functional analogs of either the Red or Rac system. The authors found that in one phage of the 30, recET homologs were found. This phage, Che9c, contains two genes that are each around 30% identical to the Rac prophage RecE and RecT (at Che9c gp60 and 61).



Biochemical analysis showed that RecE and RecT function nearly identically to the Lambda phage exo and beta.

The authors placed Che9c gp60 and 61 onto a plasmid under an acetamide promoter (high expression of these genes under a constitutive promoter was determined to be toxic) and transformed into M. smegmatis (a fast growing, non-pathogenic relative to M. tuberculosis)

These cells could then be transformed with a dsDNA leuD knockout substrate containing 1,000bp homology on each side (500bp flanking sequence and 500bp leuD sequence) of a Hyg resistance cassette. Upon induction of the RecET homologs, allelic exchange was shown to occur at a measureable frequence (8x10^-4 recombinants per ug / cell competancy) by detecting the presence of leucine auxotrophs with hygromycin resistance. This event was shown to require both Che9c gp60 and gp61, indicating that both RecE and RecT functions were needed.

This method was shown to work not only in M. smegmatis, but also in M. tuberculosis and at different genetic loci.

Other work showed that 1,000bp homology was not necessarily needed and that substrates could have as little as 50bp (the current standard is now 500bp homology) flanking sequence to the knock out marker.

This method will certainly revolutionize the ability to genetically manipulate the Mycobacteria. The possibilities are endless. Not only can one create knockouts, but also insert point mutations, amber mutants, add frameshifts, and add His-Tags. This is not to mention that all these methods can be applied not only to the bacterial genome, but also phage.

This is a wonderful example of phage-based technology with a practical and immensely helpful function.

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van Kessel, J.C., Hatfull, G.F. (2007). Recombineering in Mycobacterium tuberculosis. Nature Methods, 4(2), 147-152. DOI: 10.1038/nmeth996

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Book Recommendations

For more information regarding how we work woth the Mycobacteria, this "handbook" describes all the major protocols.
Mycobacteria Protocols (Methods in Molecular Biology) (Methods in Molecular Biology)

As always, I recommend this book for anyone interested in a well described overview of mycobacterial genetics.
Molecular Genetics Mycobacteria

Finally, this is one of my favorite books on bacteriophages. It describes all the major topics in phage biology and their applications.
The Bacteriophages

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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.