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We report the complete genome sequence of Arthrobacter sp. ATCC 21022, a strain maintained by ATCC and a commonly used host for bacteriophage isolation and genomic analysis. The strain is prophage-free and CRISPR-free but codes for two predicted restriction-modification systems.

Mycobacteriophages represent a genetically diverse group of viruses that infect mycobacterial hosts. Although more than 80 genomes have been sequenced, these still poorly represent the likely diversity of the broader population of phages that can infect the host, Mycobacterium smegmatis mc 2 155. We describe here a newly discovered phage, Marvin, which is a singleton phage, having no previously identified close relatives. The 65,100-bp genome contains 107 predicted protein-coding genes arranged in a noncanonical genomic architecture in which a subset of the minor tail protein genes are displaced about 20 kbp from their typical location, situated among nonstructural genes anticipated to be expressed early in lytic growth. Marvin is not temperate, and stable lysogens cannot be recovered from infections, although the presence of a putative xis gene suggests that Marvin could be a relatively recent derivative of a temperate parent. The Marvin genome is replete with novel genes not present in other mycobacteriophage genomes, and although most are of unknown function, the presence of amidoligase and glutamine amidotransferase genes suggests intriguing possibilities for the interactions of Marvin with its mycobacterial hosts.

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (ϕ 2 GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing ϕ 2 GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. ϕ 2 GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of ϕ 2 GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with ϕ 2 GFP10. Fluorophage ϕ 2 GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.

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.

We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ∼2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved. Fil: Rondon Salazar, Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: Jacobs Jr., William R.. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: De Waard, Jacobus. Hospital Vargas; Venezuela Fil: Hatfull, Graham F.. University of Pittsburgh; Estados Unidos Fil: Takiff, Howard Eugene. Instituto Venezolano de Investigaciones Científicas; Venezuela

Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani. This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.

Viruses are powerful tools for investigating and manipulating their hosts, but the enormous size and amazing genetic diversity of the bacteriophage population have emerged as something of a surprise. In light of the evident importance of mycobacteria to human health—especially Mycobacterium tuberculosis, which causes tuberculosis—and the difficulties that have plagued their genetic manipulation, mycobacteriophages are especially appealing subjects for discovery, genomic characterization, and manipulation. With more than 70 complete genome sequences available, the mycobacteriophages have provided a wealth of information on the diversity of phages that infect a common bacterial host, revealed the pervasively mosaic nature of phage genome architectures, and identified a huge number of genes of unknown function. Mycobacteriophages have provided key tools for tuberculosis genetics, and new methods for simple construction of mycobacteriophage recombinants will facilitate postgenomic explorations into mycobacteriophage biology.

The cell envelope is a crucial determinant of virulence and drug resistance in Mycobacterium tuberculosis. Several features of pathogenesis and immunomodulation of host responses are attributable to the structural diversity in cell wall lipids, particularly in the mycolic acids. Structural modification of the α-mycolic acid by introduction of cyclopropane rings as catalyzed by the methyltransferase, PcaA, is essential for a lethal, persistent infection and the cording phenotype in M. tuberculosis. Here, we demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Interestingly, α-mycolic acid cyclopropanation was inducible in cultures grown at 25 °C. The growth temperature modulation of the cyclopropanating activity was determined by high resolution magic angle spinning NMR analyses on whole cells. In parallel, quantitative reverse transcription-PCR analysis showed that MSMEG_1351 gene expression is up-regulated at 25 °C compared with 37 °C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of α-mycolates. The functional equivalence of PcaA and MSMEG_1351 was established by cross-complementation in the MSMEG_1351 knock-out mutant and also in a ΔpcaA strain of Mycobacterium bovis BCG. Overexpression of MSMEG_1351 restored the wild-type mycolic acid profile and the cording phenotype in BCG. Although the biological significance of mycolic acid cyclopropanation in nonpathogenic mycobacteria remains unclear, it likely represents a mechanism of adaptation of cell wall structure and composition to cope with environmental factors.

International audience; Mycobacterial species, like other microbes, spontaneously form multicellular drug-tolerant biofilms when grown in vitro in detergent-free liquid media. The structure of Mycobacterium tuberculosis biofilms is formed through genetically programmed pathways and is built upon a large abundance of novel extracellular free mycolic acids (FM), although the mechanism of FM synthesis remained unclear. Here we show that the FM in Mycobacterium smegmatis biofilms is produced through the enzymatic release from constitutively present mycolyl derivatives. One of the precursors for FM is newly synthesized trehalose dimycolate (TDM), which is cleaved by a novel TDM-specific serine esterase, Msmeg-1529. Disruption of Msmeg-1529 leads to undetectable hydrolytic activity, reduced levels of FM in the mutant, and retarded biofilm growth. Furthermore, enzymatic hydrolysis of TDM remains conserved in M. tuberculosis, suggesting the presence of a TDM-specific esterase in this pathogen. Overall, this study provides the first evidence for an enzymatic release of free mycolic acids from cell envelope mycolates during mycobacterial growth.

Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of sixty – all infecting a common bacterial host – provides further insight into their diversity and evolution. Of the sixty phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, five of which can be further divided into subclusters; five genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the six genomes in cluster D share more than 97.5% average nucleotide similarity with each other. In contrast, similarity between the two genomes in Cluster I is barely detectable by diagonal plot analysis. The total of 6,858 predicted ORFs have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit smaller average size than genes of their host (205 residues compared to 315), phage genes in higher flux average only ∼100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.

When asked how they became involved in science, it is not unusual to hear microbiologists say that it was their early exposure to research that proved crucial. Doing research enhances science education and can promote a lifelong interest in science. However, academic institutions often select only the most gifted to do research. This bias is troubling because the skills needed to do research can be different from those used to measure academic success. Thus, while conformity and memorization are key at the high school and undergraduate levels, research success depends on challenging what we think we know.

Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan-peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an {alpha}/{beta} hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles {Delta}lysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan-peptidoglycan layer.

The world of prokaryotic viruses, including the ‘‘traditional’’ bacteriophages and the viruses of Archaea, is currently in a period of renaissance, brought about largely by our new capabilities in (meta)genomics and by the isolation of diverse novel virusehost systems. In this review, we highlight some of the directions where we believe research on the prokaryotic virosphere will lead us in the near future. 2008 Elsevier Masson SAS. All rights reserved.

The recognition of the vast numbers of bacteriophages in the biosphere has prompted a renewal of interest in understanding their morphological and genetic diversity, and elucidating the evolutionary mechanisms that give rise to them. We have approached these questions by isolating and characterizing a collection of mycobacteriophages that infect a common bacterial host, Mycobacterium smegmatis. Comparative genomic analysis of 50 mycobacteriophages shows that they are highly diverse, although not uniformly so, that they are pervasively mosaic with a multitude of single gene modules, and that this mosaicism is generated through illegitimate recombination.

Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-specific recombination event, in which the sites generated by integration, attL and attR , are used as substrates to regenerate attP and attB . Excision is catalyzed by integrase but also requires a phage-encoded recombination directionality factor (RDF). The Bxb1 recombination sites, attP and attB , are small (attL - and attR -recombinant products that are asymmetric but similar to each other, each being composed of B- and P-type half-sites. We show here that the determination of correct excision products is a two-step process, with a presynaptic RDF-dependent step that aligns attL and attR in the correct orientation and a postsynaptic step in which the nonpalindromic central dinucleotide confers identity to attL and attR and prevents each from recombining with itself.

Virology encompasses a broad spectrum of topics touching upon many aspects of our everyday lives. However, appreciation of this impact is too often restricted to those who have specialized training and participate in virology research. The Phage Hunters Integrating Research and Education (PHIRE) program and the This Week in Virology (TWiV) podcast seek to bring virology to new audiences through two different approaches—direct involvement of undergraduates in discovering and genomically characterizing bacteriophages (PHIRE) and clear, accessible, and free discussions among experts of all topics in virology (TWiV). Here we discuss these two high-impact programs, the audiences that they serve, their broader impacts, and their future potential.

Many species of mycobacteria form structured biofilm communities at liquid-air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 microM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes - especially those involved in siderophore synthesis and iron uptake - are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 microM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron-exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

Cyanobacteriophage Syn9 is a large, contractile-tailed bacteriophage infecting the widespread, numerically dominant marine cyanobacteria of the genera Prochlorococcus and Synechococcus. Its 177,300 bp genome sequence encodes 226 putative proteins and six tRNAs. Experimental and computational analyses identified genes likely involved in virion formation, nucleotide synthesis, and DNA replication and repair. Syn9 shows significant mosaicism when compared with related cyanophages S-PM2, P-SSM2 and P-SSM4, although shared genes show strong purifying selection and evidence for large population sizes relative to other phages. Related to coliphage T4 - which shares 19% of Syn9's genes - Syn9 shows evidence for different patterns of DNA replication and uses homologous proteins to assemble capsids with a different overall structure that shares topology with phage SPO1 and herpes virus. Noteworthy bacteria-related sequences in the Syn9 genome potentially encode subunits of the photosynthetic reaction centre, electron transport proteins, three pentose pathway enzymes and two tryptophan halogenases. These genes suggest that Syn9 is well adapted to the physiology of its photosynthetic hosts and may affect the evolution of these sequences within marine cyanobacteria.

Mycobacteriophage Tweety is a newly isolated phage of Mycobacterium smegmatis. It has a viral morphology with an isometric head and a long flexible tail, and forms turbid plaques from which stable lysogens can be isolated. The Tweety genome is 58 692 bp in length, contains 109 protein-coding genes, and shows significant but interrupted nucleotide sequence similarity with the previously described mycobacteriophages Llij, PMC and Che8. However, overall the genome possesses mosaic architecture, with gene products being related to other mycobacteriophages such as Che9d, Omega and Corndog. A gene encoding an integrase of the tyrosine-recombinase family is located close to the centre of the genome, and a putative attP site has been identified within a short intergenic region immediately upstream of int. This Tweety attP-int cassette was used to construct a new set of integration-proficient plasmid vectors that efficiently transform both fast- and slow-growing mycobacteria through plasmid integration at a chromosomal locus containing a tRNA(Lys) gene. These vectors are maintained well in the absence of selection and are completely compatible with integration vectors derived from mycobacteriophage L5, enabling the simple construction of complex recombinants with genes integrated simultaneously at different chromosomal positions.

Mycobacteriophages Cambiare, FlagStaff, and MOOREtheMARYer are newly isolated phages of Mycobacterium smegmatis mc 2 155 recovered from soil samples in Pittsburgh, PA. All three genomes are closely related to cluster G mycobacteriophages but differ sufficiently in nucleotide sequence and gene content to warrant division of cluster G into several subclusters.

Bacteriophages are the dark matter of the biological universe: the population is vast and replete with novel genes whose function is unknown. The genomic insights such as the mosaic architecture gleaned from perhaps 2,000 currently sequenced bacteriophage genomes is far from representative of the total number phage particles in the biosphere - about 10ˆ31. The recent comparative analysis of 627 mycobacteriophages isolated on Mycobacterium smegmatis mc2 155 is the most extensive examination yet in pursuit of this question.

Mycobacteriophage ShedlockHolmes is a newly isolated phage infecting Mycobacterium smegmatis mc 2 155. It has a 61,081-bp genome containing 99 predicted protein-coding genes and one tRNA gene. ShedlockHolmes is closely related to mycobacteriophages Pixie, Keshu, and MacnCheese and is a new member of subcluster K3.

Det7 is a Myoviridae bacteriophage that gains entry into its Salmonella enterica serovar Typhimurium host cells by adsorbing to O-antigen polysaccharide. We report here the complete 157,498-bp sequence of its genome. Det7, together with its Vi01-like relatives, are distantly related to phage T4.

Mycobacteriophage Phayonce is a newly isolated phage recovered from a soil sample in Pittsburgh, PA, using Mycobacterium smegmatis mc 2 155 as a host. Phayonce's genome is 49,203 bp long and contains 77 protein-coding genes, 23 of them having predicted functions. Phayonce shares a strong similarity in nucleotide sequence with phages of cluster P.

AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo are newly isolated phages of Mycobacterium smegmatis mc 2 155 discovered in Pittsburgh, Pennsylvania, USA. All five phages share nucleotide similarity with cluster B mycobacteriophages but span considerable diversity with Corofin and OrangeOswald in subcluster B3, AlanGrant and Vincenzo in subcluster B4, and Baee in subcluster B5.

Luchador and Nerujay are two newly isolated mycobacteriophages recovered from soil samples using Mycobacterium smegmatis . Their genomes are 53,387 bp and 53,455 bp long and have 96 and 97 predicted open reading frames, respectively. Nerujay is related to subcluster A1 phages, and Luchador represents a new subcluster, A14.

Mycobacteriophages provide an abundance of systems for use in mycobacterial genetics, including manipulation of Mycobacterium tuberculosis. Because of the dearth of antibiotic resistance cassettes and biosafety concerns in constructing recombinant virulent M. tuberculosis strains, we developed the use of mycobacteriophage-encoded repressor genes that can be selected in the presence of lytic versions of their cognate phages. The phage Adephagia repressor gene (43) was identified through its ability to confer immunity to Adephagia superinfection, together with the mapping of mutations in gene 43 that confer a clear-phage phenotype. Plasmid transformants containing either Adephagia 43 or the previously identified BPs repressor 33 can be readily selected following electroporation using engineered lytic derivatives of Adephagia and BPs, respectively. Selection is as efficient as antibiotic selection, can be used with either single-copy integration vectors or with extrachromosomal vectors, and works similarly in both Mycobacterium smegmatis and M. tuberculosis.

Momo is a newly discovered phage of Mycobacterium smegmatis mc 2 155. Momo has a double-stranded DNA genome 154,553 bp in length, with 233 predicted protein-encoding genes, 34 tRNA genes, and one transfer-messenger RNA (tmRNA) gene. Momo has a myoviral morphology and shares extensive nucleotide sequence similarity with subcluster C1 mycobacteriophages.

Bacteriophages are the most abundant biological entities in the biosphere, and this dynamic and old population is, not surprisingly, highly diverse genetically. Relative to bacterial genomics, phage genomics has advanced slowly, and a higher-resolution picture of the phagosphere is only just emerging. This view reveals substantial diversity even among phages known to infect a common host strain, but the relationships are complex, with mosaic genomic architectures generated by illegitimate recombination over a long period of evolutionary history.

Genetic dissection of M. tuberculosis is complicated by its slow growth and its high rate of illegitimate recombination relative to homologous DNA exchange. We report here the development of a facile allelic exchange system by identification and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli. Identifiable recombination proteins are rare in mycobacteriophages, and only 1 of 30 genomically characterized mycobacteriophages (Che9c) encodes homologs of both RecE and RecT. Expression and biochemical characterization show that Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates recombination facilitating allelic exchange in both M. smegmatis and M. tuberculosis. Mycobacterial recombineering thus provides a simple approach for the construction of gene replacement mutants in both slow- and fast-growing mycobacteria.

The predominant morphotype of mycobacteriophage virions has a DNA-containing capsid attached to a long flexible non-contractile tail, features characteristic of the Siphoviridae. Within these phage genomes the tape measure protein (tmp) gene can be readily identified due to the well-established relationship between the length of the gene and the length of the phage tail – because these phages typically have long tails, the tmp gene is usually the largest gene in the genome. Many of these mycobacteriophage Tmp's contain small motifs with sequence similarity to host proteins. One of these motifs (motif 1) corresponds to the Rpf proteins that have lysozyme activity and function to stimulate growth of dormant bacteria, while the others (motifs 2 and 3) are related to proteins of unknown function, although some of the related proteins of the host are predicted to be involved in cell wall catabolism. We show here that motif 3-containing proteins have peptidoglycan-hydrolysing activity and that while this activity is not required for phage viability, it facilitates efficient infection and DNA injection into stationary phase cells. Tmp's of mycobacteriophages may thus have acquired these motifs in order to avoid a selective disadvantage that results from changes in peptidoglycan in non-growing cells.

Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku's and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections.

SummaryMycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential—presumably providing the housekeeping chaperone functions—while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates—long-chain fatty acid components of the mycobacterial cell wall—specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56–C68) fatty acids, and strains with altered mycolate profiles—including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA—are defective in biofilm formation.

Recombination by site-specific recombinases is a highly concerted process that requires synapsis of the correct pair of DNA substrates. Phage-encoded serine-integrases are unusual among the serine-recombinase family, which includes transposon resolvases and DNA invertases, in that they utilize two simple but different DNA substrates (attB and attP) and do not require accessory sites, additional proteins, or DNA supercoiling. Synapsis must therefore be directed solely by integrase-DNA interactions. We show here that the Bxb1 serine-integrase binds as a dimer to its two DNA substrates (attB, attP) and recombinant products (attL, attR) with similar affinities. However, synapsis occurs only between attP and attB, and not between any of the other nine possible site combinations. The Bxb1 integrase domain structure, the unusual DNA-binding properties of the integrase, and the characterization of a mutant protein with altered site-discrimination, are consistent with synaptic selectivity being derived from DNA sequence-induced changes in the conformations of integrase-DNA complexes.

Summary In August, more than 350 conferees from 24 countries attended the ASM Conference on the New Phage Biology, in Key Biscayne, Florida. This meeting, also called the Phage Summit, was the first major international gathering in decades devoted exclusively to phage biology. What emerged from the 5 days of the Summit was a clear perspective on the explosive resurgence of interest in all aspects of bacteriophage biology. The classic phage systems like λ and T4, reinvigorated by structural biology, bioinformatics and new molecular and cell biology tools, remain model systems of unequalled power and facility for studying fundamental biological issues. In addition, the New Phage Biology is also populated by basic and applied scientists focused on ecology, evolution, nanotechnology, bacterial pathogenesis and phage-based immunologics, therapeutics and diagnostics, resulting in a heightened interest in bacteriophages per se, rather than as a model system. Besides constituting another landmark in the long history of a field begun by d’Herelle and Twort during the early 20th century, the Summit provided a unique venue for establishment of new interactive networks for collaborative efforts between scientists of many different backgrounds, interests and expertise.

The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.

This chapter focuses on a discussion of recent developments in mycobacteriophage research with an emphasis on how these have advanced one’s understanding of tuberculosis. Mycobacteriophages have been isolated from rather exotic locations, such as the zebra pits at the Bronx zoo and the grounds of the tuberculosis hospital in Chennai, but they can be isolated from most soil and compost samples. The overall diversity of the mycobacteriophage population can also be examined by assessing the proportion of gene products that are unique and do not match any other mycobacteriophage genes. It is sometimes stated that the average genome size for double-stranded DNA (dsDNA) tailed phages is about 50 kbp, perhaps based largely on the well characterized and much utilized phage lambda. One of the most evident features of the mycobacteriophage genomes is their pervasive mosaicism, a common feature of phage genomes. This mosaicism is found in other phage genomes but is particularly pervasive in the mycobacteriophages; it is notable in that the mosaic elements are frequently single genes rather than large sets of genes. Examination of mycobacteriophage tape measure protein (Tmp) reveals the presence of small sequence motifs that are related to Mycobacterium tuberculosis proteins. Investigators may be discouraged from using phage-based delivery tools for mycobacterial genetics due to the perceived complexity of constructing the necessary recombinant phages.

Integration of the mycobacteriophage Bxb1 genome into its host chromosome is catalyzed by a serine-integrase, a member of the transposon-resolvase family of site-specific recombinases. These enzymes use a concerted mechanism of strand exchange involving double-stranded cleavages with two-base extensions, and covalent protein-DNA linkages via phosphoserine bonds. In contrast to the resolvase/invertase recombination systems--where there are strict requirements for a specific synaptic complex within which the catalytic potential of the enzyme is activated--synapsis of attP and attB by Bxb1 integrase is completely promiscuous, aligning the sites with equal proclivity in parallel and antiparallel alignments. Moreover, the catalytic potential of Bxb1 integrase is fully active in either alignment. As a consequence, the nonpalindromic central dinucleotide (5'-GT) at the center of attP and attB is the sole determinant of Bxb1 prophage orientation, and a single base pair substitution in the two sites is sufficient to eliminate orientation control.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3' end of the groEL1 gene such that integration results in alteration of the C-terminal 21 amino acid residues. An integration-proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1-integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high-energy cofactors, divalent metals, DNA supercoiling or additional proteins.

Comparative genomic analysis of the tailed bacteriophages shows that they are genetically mosaic with respect to each other, implying that horizontal exchange of sequences is an important component of their evolution. Horizontal exchange occurs intensively among closely related phages but also at reduced frequency across the entire population of tailed phages. It results in exchange of homologous functions, exchange of analogous but non-homologous functions as with the prophage integrases, and introduction of novel functions into the genome as with the morons. Extrapolation of these processes back in evolutionary time leads to a speculative model for the origins and early evolution of phages.

Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.

Salmonella bacteriophage χ is a member of the Siphoviridae family that gains entry into its host cells by adsorbing to their flagella. We report the complete 59,578-bp sequence of the genome of phage χ, which together with its relatives, exemplifies a largely unexplored type of tailed bacteriophage.

Our understanding of the biological principles of mycobacterial tolerance to antibiotics is crucial for developing shorter anti-tuberculosis regimens. Various in vitro approaches have been developed to identify the conditions that promote mycobacterial persistence against antibiotics. In our laboratories, we have developed a detergent-free in vitro growth model, in which mycobacteria spontaneously grow at the air–medium interface as self-organized multicellular structures, called biofilms. Mycobacterial biofilms harbor a sub-population of drug tolerant persisters at a greater frequency than their planktonic counterpart. Importantly, development of these structures is genetically programmed, and defective biofilms of isogenic mutants harbor fewer persisters. Thus, genetic analysis of mycobacterial biofilms in vitro could potentially be a powerful tool to unravel the biology of drug tolerance in mycobacteria. In this chapter we describe a method for screening biofilm-defective mutants of mycobacteria in a 96-well format, which readily yields a clonally pure mutant for further studies.

Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection—lysogenic or lytic growth—as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33–34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

The genomes of Mycobacterium tuberculosis H37Rv and CDC1551 each contain two prophage-like elements, phiRv1 and phiRv2. The phiRv1 element is not only absent from Mycobacterium bovis BCG but is in different locations within the two sequenced M. tuberculosis genomes; in both cases phiRv1 is inserted into a REP13E12 repeated sequence, which presumably contains the bacterial attachment site, attB, for phiRv1. Although phiRv1 is probably too small to encode infectious phage particles, it may nevertheless have an active integration/excision system and be capable of moving from one chromosomal position to another. We show here that the M. tuberculosis H37Rv phiRv1 element does indeed encode an active site-specific recombination system in which an integrase of the serine recombinase family (Rv1586c) catalyses integration and excision and a small, basic phiRv1-encoded protein (Rv1584c) controls the directionality of re-combination. Integration-proficient plasmid vectors derived from phiRv1 efficiently transform BCG, can utilize four of the seven REP13E12 sites present in BCG as attachment sites, and can occupy more than one site simultaneously.

We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time. The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis that shares a similar genome organization to mycobacteriophage L5, although the two phages are heteroimmune. We have investigated the regulatory circuitry of Bxb1 and found that it encodes a repressor, gp69, which regulates at least two promoters, an early lytic promoter, Pleft, and the divergent promoter, Pright. Bxb1 gp69 is 41% identical to the L5 repressor (gp71) and binds to repressor binding sites that conform to a similar, but distinct, 13 bp asymmetric consensus sequence to that for the L5 gp71 binding sites. The two phage repressors have a strong preference for their cognate binding sites, thus accounting for their immunity phenotypes. The Bxb1 genome contains 34 putative repressor binding sites located throughout the genome, but situated within short intergenic spaces and orientated in only one direction relative to the direction of transcription. Comparison with the locations of repressor binding sites within the L5 genome provides insights into how these unusual regulatory systems evolve.