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.

Research Synopsis

So my grad school application process continues. This is part of my personal statement....it is a synopsis of my research. Let me know what you think, any changes I should make, etc. Thanks in advance!

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For the past two and a half years, I have had the privilege to work as an undergraduate researcher in the lab of Dr. Graham Hatfull at the University of Pittsburgh. Dr. Hatfull is one of a unique group of researchers adamantly recruiting young students into biological research. Students as young as 6th and 7th grade are able to work in his lab through his Phagehunting program. Although I did not begin my work in the program at that young of an age, many would consider my start as a college freshman early compared to most.

I began my research in January of my freshman year. The first project was to use bioinformatic methods to analyze and annotate the genome of a Bacillus phage, MP15. Utilizing programs such as DNA Master, Glimmer, and GeneMark, I called the most likely protein coding regions in the genome. These putative genes were then compared to GenBank databases to assign potential functions and evaluate the novelty of the phage’s genome. Not surprisingly, this phage proved to be highly novel; its genome had a large amount of genes with no significant sequence similarity to those genes in the databases.

Having enjoyed my first research experience, I then continued work in the lab, moving from bioinformatics into wet lab projects. I became part of a larger project to isolate and characterize novel mycobacteriophages on Mycobacterium smegmatis. I isolated, amplified, and concentrated a novel mycobacteriophage named BPs. Using Sanger sequencing procedures, the phage’s genome was sequenced. The final sequence was analyzed and annotated. Lysogeny experiments showed BPs to form stable lysogens in M. smegmatis, likely integrating at a tRNA^Arg gene in the host. Furthermore, host range studies showed that BPs was able to infect not only its initial host, but also M. chelonae, M. bovis BCG, and notably, M. tuberculosis (M. tb.).

Following up this host range study of BPs, I began to characterize the ability of over two hundred mycobacteriophages (published and unpublished) to infect M. chelonae, M. bovis BCG, and M. tb. Dr. Hatfull approached me with a few ideas for projects that could stem from the large amount of host range work I was performing. One of these projects involved the discovery of a phage capable of performing generalized transduction in M. tb.





Generalized transduction occurs when a phage capsid accidentally packages host DNA, resulting in a viral particle capable of inserting that DNA into a new cell. This mechanism has been exploited in a variety of bacterial systems. Currently, there are only two published phages with the ability to transduce Mycobacteria; Bxz1 and I3. However, neither of these is able to infect or transduce M. tb. My goal was to find which of our published phages could infect M. tb. and which could function as transducing phages. I developed and performed transduction assays with two different genetic markers. At the same time, I finalized the host range of our published phages.

The results showed that 11 of our 30 published phages were able to transduce M. smegmatis and that 7 of these 30 were able to infect M. tb. However, no phages appear in both groups. Bioinformatic analysis of putative genes showed that there was no particular gene present only in the group of M. tb. infecting phages. Rather, the only genetic element common among M. tb. infectors is the presence of cohesive genetic termini. Those phages capable of mediating generalized transduction all have terminally redundant genetic termini. Currently, I am asking whether or not the presence of terminally redundant ends prevents phage from infecting M. tb.

The nature of a phage’s genomic ends determines how the DNA circularizes upon infection. Phages with cohesive termini circularize via complementation; while phages with terminal redundancy circularize by homologous recombination. M. tb. is notorious for their large amount of illegitimate recombination, which potentially may inhibit phages with terminal redundancy from circularizing and subsequently replicating. Previous studies have shown that recombination proteins are necessary for certain phages with terminal redundant ends, such as Salmonella phage P22, to circularize and replicate. Previous work with mycobacteriophages has discovered functionally analogous proteins for recombination in Mycobacteria. These proteins, encoded by phage Che12, have been successfully utilized to promote homologous recombination in both M. smegmatis and M. tb. My current work is establishing whether the presence of these proteins can overcome a recombination deficiency in M. tb. and allow terminally redundant phages cause infection. If this is true, generalized transduction in M. tb. may be a possibility.

Aside from learning laboratory techniques and critical thinking, this research experience has provided me with the opportunity to present my work at a wide variety of scientific meetings. These meetings include: the 2005 Molecular Genetics of Bacteria and Phages Meeting, the 106th and 107th Annual American Society for Microbiology General Meetings, along with meetings hosted by the University of Pittsburgh.

My work with phage has opened my mind to the world of microbial interactions. The constant flux between host and pathogen, on any scale, is vast and decidedly interesting. Studying these interactions can provide the focus for a wide range of projects. Microbial interactions have components in vaccine development, industrial fermentations, novel antimicrobial development, genetic tool creation, and evolutionary studies. It is my hope that graduate school will allow me to delve deeper into the interactions between microbes and humans, themselves, or their environment.

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This is the first 2/3 of a ~1000 word essay. The remaining will be geared towards which ever school I am applying too. Thanks for your opinions.