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1.  Functional requirements for bacteriophage growth: Gene essentiality and expression in Mycobacteriophage Giles 
Molecular microbiology  2013;88(3):577-589.
Summary
Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions – such as virion proteins and repressors – cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny.
doi:10.1111/mmi.12210
PMCID: PMC3641587  PMID: 23560716
Bacteriophage; Transcription; RNAseq
2.  Recombineering mycobacteria and their phages 
Nature reviews. Microbiology  2008;6(11):851-857.
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.
doi:10.1038/nrmicro2014
PMCID: PMC3503148  PMID: 18923412
3.  Propionibacterium acnes Bacteriophages Display Limited Genetic Diversity and Broad Killing Activity against Bacterial Skin Isolates 
mBio  2012;3(5):e00279-12.
ABSTRACT
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.
IMPORTANCE
Propionibacterium acnes is a dominant member of the skin microflora and has also been implicated in the pathogenesis of acne; however, little is known about the bacteriophages that coexist with and infect this bacterium. Here we present the novel genome sequences of 11 P. acnes phages, thereby substantially increasing the amount of available genomic information for this phage population. Surprisingly, we find that, unlike other well-studied bacteriophages, P. acnes phages are highly homogeneous and show a striking lack of genetic diversity, which is perhaps related to their unique and restricted habitat. They also share a broad ability to kill clinical isolates of P. acnes; phage resistance is not prevalent, but when detected, it appears to be conferred by chromosomally encoded immunity elements within the host genome. We believe that these phages display numerous features that would make them ideal candidates for the development of a phage-based therapy for acne.
doi:10.1128/mBio.00279-12
PMCID: PMC3448167  PMID: 23015740
4.  Recombineering 
Bacteriophage  2012;2(1):5-14.
Recombineering, a recently developed technique for efficient genetic manipulation of bacteria, is facilitated by phage-derived recombination proteins and has the advantage of using DNA substrates with short regions of homology. This system was first developed in E. coli but has since been adapted for use in other bacteria. It is now widely used in a number of different systems for a variety of purposes, and the construction of chromosomal gene knockouts, deletions, insertions, point mutations, as well as in vivo cloning, mutagenesis of bacterial artificial chromosomes and phasmids, and the construction of genomic libraries has been reported. However, these methods also can be effectively applied to the genetic modification of bacteriophage genomes, in both their prophage and lytically growing states. The ever-growing collection of fully sequenced bacteriophages raises more questions than they answer, including the unknown functions of vast numbers of genes with no known homologs and of unknown function. Recombineering of phage genomes is central to addressing these questions, enabling the simple construction of mutants, determination of gene essentiality, and elucidation of gene function. In turn, advances in our understanding of phage genomics should present similar recombineering tools for dissecting a multitude of other genetically naïve bacterial systems.
doi:10.4161/bact.18778
PMCID: PMC3357384  PMID: 22666652
bacteriophage; BRED; recombineering
5.  Mycobacteriophages BPs, Angel and Halo: comparative genomics reveals a novel class of ultra-small mobile genetic elements 
Microbiology  2009;155(Pt 9):2962-2977.
Mycobacteriophages BPs, Angel and Halo are closely related viruses isolated from Mycobacterium smegmatis, and possess the smallest known mycobacteriophage genomes, 41 901 bp, 42 289 bp and 41 441 bp, respectively. Comparative genome analysis reveals a novel class of ultra-small mobile genetic elements; BPs and Halo each contain an insertion of the proposed mobile elements MPME1 and MPME2, respectively, at different locations, while Angel contains neither. The close similarity of the genomes provides a comparison of the pre- and post-integration sequences, revealing an unusual 6 bp insertion at one end of the element and no target duplication. Nine additional copies of these mobile elements are identified in a variety of different contexts in other mycobacteriophage genomes. In addition, BPs, Angel and Halo have an unusual lysogeny module in which the repressor and integrase genes are closely linked. The attP site is located within the repressor-coding region, such that prophage formation results in expression of a C-terminally truncated, but active, form of the repressor.
doi:10.1099/mic.0.030486-0
PMCID: PMC2833263  PMID: 19556295
6.  Mycobacteriophages BPs, Angel and Halo: comparative genomics reveals a novel class of ultra-small mobile genetic elements 
Microbiology (Reading, England)  2009;155(Pt 9):2962-2977.
Mycobacteriophages BPs, Angel and Halo are closely related viruses isolated from Mycobacterium smegmatis, and possess the smallest known mycobacteriophage genomes, 41 901 bp, 42 289 bp and 41 441 bp, respectively. Comparative genome analysis reveals a novel class of ultra-small mobile genetic elements; BPs and Halo each contain an insertion of the proposed mobile elements MPME1 and MPME2, respectively, at different locations, while Angel contains neither. The close similarity of the genomes provides a comparison of the pre- and post-integration sequences, revealing an unusual 6 bp insertion at one end of the element and no target duplication. Nine additional copies of these mobile elements are identified in a variety of different contexts in other mycobacteriophage genomes. In addition, BPs, Angel and Halo have an unusual lysogeny module in which the repressor and integrase genes are closely linked. The attP site is located within the repressor-coding region, such that prophage formation results in expression of a C-terminally truncated, but active, form of the repressor.
doi:10.1099/mic.0.030486-0
PMCID: PMC2833263  PMID: 19556295
7.  BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes 
PLoS ONE  2008;3(12):e3957.
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.
doi:10.1371/journal.pone.0003957
PMCID: PMC2597740  PMID: 19088849
8.  Genomic Characterization of Mycobacteriophage Giles: Evidence for Phage Acquisition of Host DNA by Illegitimate Recombination▿ †  
Journal of Bacteriology  2008;190(6):2172-2182.
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.
doi:10.1128/JB.01657-07
PMCID: PMC2258872  PMID: 18178732

Results 1-8 (8)