Bacteriophages are extremely abundant, and it has been estimated that the number of phage particles in the biosphere is approximately 1031
These phages are a source of high genetic diversity and are replete with genetic novelty.2
They have played key roles in the development of bacterial genetics and can provide effective solutions for the development of systems to manipulate genetically naïve bacteria.3
Genetic modification of phages can facilitate the study of these viruses and, in consequence, the development of new tools for bacterial manipulation.
In contrast to the range of tools that have been described for targeted mutagenesis of bacterial chromosomes, fewer methods have been implemented for targeted mutagenesis of lytically growing phages, because high frequency events are required in the absence of a selectable system.
Historically, construction of mutations in bacteriophages was accomplished by general mutagenesis using UV irradiation or chemical compounds that can generate DNA damage.
A homologous recombination approach has also been used to make gene deletions or recombinant phages,4,5
but the frequency is low and screening to find the desired mutant can be tedious and time consuming.
Recently, a new in vivo technology to introduce genetic changes in bacterial genomes has been developed. This technique—named “recombineering”—refers to the engineering of recombinant DNA by homologous recombination.6-8
Recombineering employs recombination systems encoded by bacteriophages to enhance the frequency of homologous recombination, allowing the construction of chromosomal gene knock outs, deletions, insertions, point mutations, in vivo cloning, mutagenesis of bacterial artificial chromosomes, phasmids and genomic libraries.6-15
The increased availability of bacterial genome sequences has facilitated the use of recombineering, because it is only possible to recombineer an organism whose sequence is known.
The first systems described employed recombination functions encoded by bacteriophage lambda and the Rac prophage, and were originally designed for modification of E. coli.
Subsequently, these were successfully employed in other Gram-negative bacteria, such as Salmonella
and further adapted for application to other more divergent bacteria, like the insect endosymbiont Sodalis glossinidius
and the promising subject for biotechnological exploitation, Pantoea ananatis
Double-stranded DNA recombineering in E. coli
using the lambda Red system involves three phage-encoded proteins, Exo, Beta and Gam. Exo is an exonuclease that degrades one strand of DNA from a double-stranded end to generate a ssDNA substrate. Beta is a DNA pairing enzyme that anneals the recombineering substrate to its chromosomal target. Gam inhibits the E.coli
RecBCD and SbcD enzymes, preventing degradation of the double-stranded DNA (dsDNA) substrate.20-23
For optimal dsDNA recombineering, RecBCD should be inactivated, either by mutation or by lambda gam.6,8,24
The original model proposed that after degradation by Exo, a 3′ single stranded DNA tail is exposed to which Beta can bind.8
Homologous recombination then occurs, following the strand invasion or DNA annealing model.25-28
Recently, a new model to explain the mechanism of dsDNA recombination mediated by lambda phage proteins has been proposed.29,30
In this alternative model, lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via β recombinase catalyzed annealing at the replication fork.
The Rac prophage encodes only RecE and RecT, which are functionally equivalent to lambda Exo and Beta, respectively.12,31
Beta belongs to a large family of ssDNA annealing proteins (SSAPs),32-34
and only this protein is necessary for single-stranded oligonucleotide recombination,7
used for the creation of point mutations and deletions. A strand bias in recombination levels has been observed when comparing two complementary ssDNAs. Higher frequencies are obtained when using an oligonucleotide that anneals to the DNA strand undergoing discontinuous synthesis (lagging strand). As a result of the replication process, transient regions of ssDNA may be accessible for Beta-mediated annealing of the oligo. The increased recombination efficiency of the ‘‘lagging strand’’ oligos may reflect the increased availability of single-stranded regions during lagging vs. leading strand synthesis.7,12,35
Regulated expression of the bacteriophage recombination system typically is required for recombineering. These functions can be integrated into the chromosome or contained within an extrachromosomal plasmid. A lambda lysogen with a defective prophage can be used to express the Red proteins from the strong lambda pL
The recombineering functions have also been transferred to a variety of different plasmids,36
including constructs in which the functions are under control of the arabinose promoter pBAD.14
Some plasmids carry a temperature-sensitive (ts
) origin of replication, so they can be cured from the cell after recombination. The prophage constructs contain the ts cI857
allele of the phage lambda repressor allowing protein expression to be controlled by temperature. At low temperature (30°C), recombineering functions are strongly repressed, and expression occurs after a shift to 42°C for only 15 min.
The efficiency of recombineering in E. coli
and the possibility of using DNA segments with short regions of homology (approx. 50 bp) to the target sequences has led to the search for proteins with similarity to the lambda/Rac encoded proteins in other phages or bacteria, especially because adapting the lambda/Rac systems for its use in other organisms, particularly those that are Gram-positive could not give optimal results.37-39
The complete genome sequences of more than 80 mycobacteriophage genomes40-45
has provided us with thousands of unique genes, making this an ideal reservoir in which to search for novel recombineering functions. Recently, RecE and RecT homologs—the products of genes 60
respectively—were identified in mycobacteriophage Che9c, the expression of which substantially enhances mycobacterial recombination frequencies. These proteins have been utilized to develop an efficient recombineering system, facilitating the construction of gene-knockouts and point mutations in the hard to manipulate and pathogenic bacterium Mycobacterium tuberculosis,
as well as in fast-growing related species like Mycobacterium smegmatis,
which is routinely use as a model system.46,47
Mycobacterial strains constructed for recombineering contain an extra chromosomal plasmid in which the phage recombination genes are under the control of the inducible acetamidase promoter.48
Similar to what has been shown in E. coli,
dsDNA recombineering in the mycobacteria requires both an exonuclease (gp60) and its associated recombinase (gp61),39
while recombination using ssDNA substrates requires only the recombinase.47
Targeted gene replacement mutants are engineered using linear dsDNA allelic exchange substrates (AESs), containing regions of homology upstream and downstream of the target gene flanking a cassette for antibiotic resistance, which are electroporated into either M. smegmatis
or M. tuberculosis
Substrates for ssDNA recombineering are short oligonucleotides—a minimum length of 50 bases is recommended—encoding the desired mutation, and this technique provides a simple and efficient method for constructing point mutations in mycobacterial genomes.47
The overall efficiency of ssDNA recombineering is substantially higher than with dsDNA substrates for both chromosomal and plasmid targets. However, optimal efficiencies are only obtained when using oligonucleotides that target, and anneal to, the DNA strand undergoing discontinuous synthesis (lagging strand template).47
These can display efficiencies that are up to 10,000-fold higher than oligonucleotides targeting the leading strand, which far exceeds the 2–50 fold strand biases observed in the λ Red system.7,35
It is not clear why the biases are so much larger in mycobacteria than in E. coli,
but this may reflect fundamental differences in the DNA replication systems and/or in how gp61 interacts with the replication machinery and supports the necessity of finding recombineering systems specific for each bacteria or related bacterial group.
Recombineering systems based on the mycobacteriophage Che9c-encoded proteins have provided new approaches to mycobacterial mutagenesis and greatly expanded the genetic toolbox available to study the pathogenic mycobacteria.
Datta et al.37
have also identified and characterized genes that are similar to Beta or RecT from Gram-positive and other Gram-negative bacteria and their phages. Nine genes, including Che9c gp61, were expressed in E. coli
under control of lambda pL, and protein activity was assayed using an oligo recombination system.49
Seven of these nine genes were adjacent to a known or putative exonuclease gene. In the E. coli
system, all the ssDNA binding proteins tested were able to catalyze oligo-mediated recombination but with variable efficiency; Che9c gp61 recombination levels were approximately 1000-fold lower than those observed with lambda β. When present with their canonical exonucleases, three of the four recombinase-exonuclease pairs tested were also able to carry out low level recombination with linear dsDNA, but not when coupled to lambda Exo; presumably because a physical and specific coupling is required between the cognate exonuclease and single strand annealing proteins. Thus, the exonuclease seems to be more species-specific, whereas the recombinase can function in diverse hosts.
After failure to observe lambda Red-mediated recombination in Pseudomonas syringae,
and based on the hypothesis that recombinases found in, or associated with, particular species would be more likely to function in close relatives than distantly species,32,50,51
Swingle et al.38
used a combination of bioinformatic tools to identify proteins homologous to RecE/RecT in Pseudomas syringae pv syringae
B728a. The so-called recTEPSY
genes are located in a 72 kb region that has been horizontally acquired in Pseudomas syringae pv syringae
B728a. This region contains 86 annotated open reading frames (ORFs), the majority of which encode hypothetical proteins. Thirteen ORFs in this locus are annotated as phage-related proteins, and it is likely this region corresponds to a prophage or a remnant of a prophage containing the recombination functions. Depending on the oligo concentration, up to a 450-fold difference in recombination frequency between the RecTPSY
expression and vector control strains was observed. Additionally, dsDNA recombination was observed when RecTPSY
was assisted by RecEPSY
Recombineering can be used to target the bacterial chromosome or extrachromosomal replicating molecules. As a corollary to this, it also can be extended for efficient and straightforward modification of bacteriophage genomes. Here, we will discuss the generation of E. coli
phage mutants using recombineering with lambda Red proteins, as well as the construction of mycobacteriophage mutants using BRED (Bacteriophage Recombineering of Electroporated DNA).52