Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution. © 2013 Pope et al

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotopes

Genomic analysis of a large set of phages infecting the common hostMycobacterium smegmatis mc2155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode

Propionibacterium acnes bacteriophages display limited genetic diversity and broad killing activity against bacterial skin isolates.

UnlabelledInvestigation 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.ImportancePropionibacterium 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 about 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

Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. © 2009 Piuri et al

A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells

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. © 2006 The Authors

Analysis of Novel Mycobacteriophages Indicates the Existence of Different Strategies for Phage Inheritance in Mycobacteria

Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100?400 nm, genome size in the 50?70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30uC, eight of them failed to propagate at 37uC. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.Fil: Stella, Emma Julieta. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Tasselli, Sabrina Emilse. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia

Cluster J Mycobacteriophages: Intron Splicing in Capsid and Tail Genes

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous freestanding HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution

Functional analysis of lysB gene from the lysis module of mycobacteriophage Ms6

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2012Most described bacteriophages end their replication cycle by lysing their hosts. This characteristic has attracted researchers all over the world aiming to develop new alternatives to fight bacterial infectious diseases. All double stranded DNA phages achieve lysis by synthesizing two essential proteins: an endolysin, a protein with peptidoglycan hydrolase activity, and a holin, a small membrane protein that disrupts the cytoplasmic membrane and allows the access of the endolysin to its target or its activation. Holins have been described as essential to determine the optimum timing of lysis, so that the phage release is productive for phage survival. In addition to the endolysin and holin genes, new genes have been identified in the lysis cassette of bacteriophages that infect Gram-negative bacteria. These genes, exemplified by the λ Rz and Rz1 encoded proteins that compromise the stability of the outer membrane, eliminating the last barrier to the release of the progeny virions. No homologues of Rz/Rz1 genes have been identified in mycobacteriophage genomes. The lytic cassette of mycobacteriophage Ms6, a phage that infects Mycobacterium smegmatis cells, comprises, in addition to an endolysin and a holin, three accessory lysis proteins encoded by genes gp1, gp3 (lysB) and gp5. Despite being classified as Gram-positive bacteria, mycobacteria have a complex cell wall. This cell wall consists of peptidoglycan covalently linked to arabinogalactan, which is in turn esterified to a variety of long chain (C60–C90), α-branched, β-hydroxy fatty acids (mycolic acids). The present work describes the characterization of the protein encoded by the mycobacteriophage Ms6 gene lysB and its role in mycobacteria lysis. A BLAST search within the sequences of protein databases revealed similarities to other putative proteins encoded by mycobacteriophages. After His6-LysB protein production in E. coli, it was possible to test its activity in several lipolytic enzymes substrates. Bioinformatics analysis revealed the presence of a conserved motif (GYSQG), characteristic of enzymes with lipolytic activity. The results show that LysB is indeed a lipolytic enzyme showing higher affinity towards substrates of longer chain length (C16 and C18). It is demonstrated that the natural substrates of LysB are mycobacterial lipids containing mycolic acids and that that its main target is mycolyl-arabinogalactan peptidoglycan complex, as it was shown to release mycolic acids from mAGP. Additionally, LysB is also able to hydrolyze the trehalose 6,6’-dimycolate (TDM) from both fast and slow growing mycobacteria species. Construction of a mycobacteriophage Ms6 ΔlysB demonstrated that the gene product of lysB is nonessential for phage viability, but is involved in the lysis mechanism. Given the complexity of the mycobacterial cell envelope, it is easy to understand why mycobacteriophages have evolved new lysis strategies by acquiring them through their evolution.A maioria dos bacteriófagos descritos na literatura são capazes de terminar um ciclo de replicação lisando as suas células hospedeiras. Esta característica tem atraído os investigadores no sentido de explorar esta propriedade para desenvolver novas alternativas para combater doenças infecciosas de origem bacteriana. Todos os bacteríofagos de cadeia dupla de DNA atingem a lise através da síntese de 2 proteínas essenciais: uma endolisina, uma proteína com actividade de hidrólise, e uma holina. Proteína de pequenas dimensões que destrói a membrana citoplasmática permitindo que a endolisina aceda ao seu substrato ou seja activada. As holinas estão descritas como sendo essenciais para determinar o tempo óptimo da lise, de modo a que a libertação de fagos seja produtiva para a sobrevivência do fago. Para além destas duas proteínas essenciais, têm sido identificados novos genes nas cassetes de lise de bacteriófagos que infectam bactérias Gram-negativa. Estes genes, exemplificados pelas proteínas Rz e Rz1 codificadas pelo bacteriófago λ, comprometem a estabilidade das proteínas da membrana externa, eliminando a última barreira para a libertação dos viriões produzidos. Até à data nunca foram identificados homólogos destes genes em micobacteriófagos. A cassete lítica do micobacteriófago Ms6, fago que infecta Mycobacterium smegmatis, para além de codificar as duas proteínas essenciais, compreende três proteínas acessórias à lise codificadas pelos genes gp1, gp3 (lysB) e gp5, restritos aos micobacterófagos. Apesar de serem classificadas com bactérias Gram-positivas, as micobactérias apresentam uma parede celular complexa. Esta parede celular consiste num peptidoglicano ligado covalentemente ao arabinogalactano, que por sua vez se encontra esterificado a uma variedade de ácidos gordos de cadeia longa (ácidos micólicos). - A presente dissertação caracteriza a proteína codificada pelo gene lysB do micobacteriófago Ms6 e o seu papel durante a lise. A análise bioinformática revelou a presença de um motivo conservado (GYSQG), característico de enzimas com actividade lipolítica. Usando a sequência de aminoácidos de LysB, foi realizada uma procura de similaridades nas bases de dados, através do programa BLAST, que identificou um grande número de proteínas similares. Após produção de uma proteína recombinante, His6-LysB, em E. coli foi possível testar a sua actividade em diferentes substratos de enzimas lipolíticas. Os resultados demonstraram que LysB apresenta uma maior afinidade para substratos de cadeia longa (C16 e C18). Foi possível demonstrar que o alvo de Ms6 LysB é a membrana externa de Mycobacterium smegamtis, ao clivar a ligação ester que liga os ácidos micólicos ao arabinogalactano no complexo micolil-arabinogalactano peptidoglicano. No entanto este substracto não é único uma vez que LysB também é capaz de hidrolisar o 6,6’-trealose dimicolato (TDM) de diferentes espécies de micobactérias. A construção de um micobacteriófago delecionado no gene lysB, demonstrou que o produto deste gene não é essencial - xv - para a viabilidade do fago, mas participa no mecanismo de lise. Tendo em conta a complexidade do envelope das micobactérias, é fácil entender a necessidade dos micobacteriófagos adquirirem, durante a sua evolução, genes que lhes conferem uma vantagem evolutiva sobre aqueles que não os adquiriram de modo a conseguirem atingir uma lise mais rápida e eficiente.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/29167/2006, PTDC/SAL-FCF/73017/2006

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists