Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags.

The complete genome sequences of over 220 mycobacteriophages reveal them to be highly diverse, with numerous types sharing little or no nucleotide sequence identity with each other. We have determined the preferences of these phages for M. tuberculosis and for other strains of M. smegmatis, and find there is a correlation between genome type (cluster, subcluster, singleton) and host range. For many of the phages, expansion of host range occurs at relatively high frequencies, and we describe several examples in which host constraints occur at early stages of infection (adsorption or DNA injection), and phages have the ability to expand their host range through mutations in tail genes. We present a model in which phage diversity is a function of both the ability of phages to rapidly adapt to new hosts and the richness of the diversity of the bacterial population from which those phages are isolated.

Investigation of the human microbiome has revealed diverse and complex microbial communities at distinct anatomic sites. The microbiome of the human sebaceous follicle provides a tractable model in which to study its dominant bacterial inhabitant, Propionibacterium acnes, which is thought to contribute to the pathogenesis of the human disease acne. To explore the diversity of the bacteriophages that infect P. acnes, 11 P. acnes phages were isolated from the sebaceous follicles of donors with healthy skin or acne and their genomes were sequenced. Comparative genomic analysis of the P. acnes phage population, which spans a 30-year temporal period and a broad geographic range, reveals striking similarity in terms of genome length, percent GC content, nucleotide identity (>85%), and gene content. This was unexpected, given the far-ranging diversity observed in virtually all other phage populations. Although the P. acnes phages display a broad host range against clinical isolates of P. acnes, two bacterial isolates were resistant to many of these phages. Moreover, the patterns of phage resistance correlate closely with the presence of clustered regularly interspaced short palindromic repeat elements in the bacteria that target a specific subset of phages, conferring a system of prokaryotic innate immunity. The limited diversity of the P. acnes bacteriophages, which may relate to the unique evolutionary constraints imposed by the lipid-rich anaerobic environment in which their bacterial hosts reside, points to the potential utility of phage-based antimicrobial therapy for acne.

A characteristic feature of bacteriophage genomes is that they are architecturally mosaic, with each individual genome representing a unique assemblage of individual exchangeable modules. Plausible mechanisms for generating mosaicism include homologous recombination at shared boundary sequences of module junctions, illegitimate recombination in a non-sequence-directed process, and site-specific recombination. Analysis of the novel mycobacteriophage Giles genome not only extends our current perspective on bacteriophage genetic diversity, with more than 60% of the genes unrelated to other mycobacteriophages, but offers novel insights into how mosaic genomes are created. In one example, the integration/excision cassette is atypically situated within the structural gene operon and could have moved there either by illegitimate recombination or more plausibly via integrase-mediated site-specific recombination. In a second example, a DNA segment has been recently acquired from the host bacterial chromosome by illegitimate recombination, providing further evidence that phage genomic mosaicism is generated by nontargeted recombination processes.The total estimated number of 10 31 bacteriophage particles in the biosphere suggests that phages represent a majority of biological entities (14). The complete genome sequences of approximately 400 double-stranded DNA tailed phages have been determined, which show, together with viral metagenomic studies (1,5,8), that the phage population has very high genetic diversity and is replete with genetic information that either is very distant from other known sequences or has a distinct evolutionary origin. Furthermore, the completely sequenced phage genomes have characteristic mosaic architectures, so that modules, composed of either single genes or several genes, are shared between individual genomes (6, 15, 18, 28); each phage genome can thus be considered a unique assemblage of individual modules. While the number of different modules remains ill-defined, the combinatorial possibilities of these modules far exceeds the estimated total number of phage particles in the biosphere (12).The evolution of mosaic genomes requires the creation of novel junctions at module boundaries. These module junctions often correspond closely with gene boundaries, and two distinct models have been proposed to explain this mosaicism. The first was proposed by Susskind and Botstein (34) and supposes that there are short conserved sequences at gene boundaries that act as recombination targets. Such boundary sequences have been described in some Escherichia coli phages (6), but these appear to be exceptional rather than typical examples. The second model proposes that module junctions are a result of illegitimate recombination events that occur without DNA sequence identity (beyond a few nucleotides in length), accompanied by selection for gene function and genome length of a packageable size (15). While such events are likely to be rare, the large population size, a long evolutionary history, and active p...

Bacteriophages are central components in the development of molecular tools for microbial genetics. Mycobacteriophages have proven a rich resource for tuberculosis genetics, and the recent development of a mycobacterial recombineering system based on phage Che9c-encoded proteins offers new approaches to mycobacterial mutagenesis. Expression of the phage exonuclease and recombinase substantially enhances recombination frequencies in both fast-and slow-growing mycobacteria, facilitating construction of both gene knockout and point mutants; it also provides a simple and efficient method for constructing mycobacteriophage mutants. Exploitation of host-specific phages thus provides a general strategy for recombineering and mutagenesis in genetically naive systems.