Cluster K Mycobacteriophages: Insights into the Evolutionary Origins of Mycobacteriophage TM4

Five newly isolated mycobacteriophages –Angelica, CrimD, Adephagia, Anaya, and Pixie – have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them – with the exception of TM4 – form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species

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

Genomic analysis of a large set of phages infecting the common host Mycobacterium 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

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

Genomic analysis of a large set of phages infecting the common host Mycobacterium 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

Cluster k mycobacteriophages: Insights into the evolutionary origins of mycobacteriophage tm4

Five newly isolated mycobacteriophages -Angelica, CrimD, Adephagia, Anaya, and Pixie - have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them - with the exception of TM4 - form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species. © 2011 Pope et al.Fil: Pope, Welkin H.. University of Pittsburgh; Estados UnidosFil: Ferreira, Christina M.. University of Pittsburgh; Estados UnidosFil: Jacobs Sera, Deborah. University of Pittsburgh; Estados UnidosFil: Benjamin, Robert C.. University of North Texas; Estados UnidosFil: Davis, Ariangela J.. Calvin College; Estados UnidosFil: DeJong, Randall J.. Calvin College; Estados UnidosFil: Elgin, Sarah C. R.. Washington University in St. Louis; Estados UnidosFil: Guilfoile, Forrest R.. University of Pittsburgh; Estados UnidosFil: Forsyth, Mark H.. The College Of William And Mary; Estados UnidosFil: Harris, Alexander D.. Calvin College; Estados UnidosFil: Harvey, Samuel E.. The College Of William And Mary; Estados UnidosFil: Hughes, Lee E.. University of North Texas; Estados UnidosFil: Hynes, Peter M.. Washington University in St. Louis; Estados UnidosFil: Jackson, Arrykka S.. The College Of William And Mary; Estados UnidosFil: Jalal, Marilyn D.. University of North Texas; Estados UnidosFil: MacMurray, Elizabeth A.. The College Of William And Mary; Estados UnidosFil: Manley, Coreen M.. University of North Texas; Estados UnidosFil: McDonough, Molly J.. The College Of William And Mary; Estados UnidosFil: Mosier, Jordan L.. University of North Texas; Estados UnidosFil: Osterbann, Larissa J.. Calvin College; Estados UnidosFil: Rabinowitz, Hannah S.. Washington University in St. Louis; Estados UnidosFil: Rhyan, Corwin N.. Washington University in St. Louis; Estados UnidosFil: Russell, Daniel A.. University of Pittsburgh; Estados UnidosFil: Saha, Margaret S.. The College Of William And Mary; Estados UnidosFil: Shaffer, Christopher D.. Washington University in St. Louis; Estados UnidosFil: Simon, Stephanie E.. University of North Texas; Estados UnidosFil: Sims, Erika F.. Washington University in St. Louis; Estados UnidosFil: Tovar, Isabel G.. University of North Texas; Estados UnidosFil: Weisser, Emilie G.. Washington University in St. Louis; Estados UnidosFil: Wertz, John T.. Calvin College; Estados UnidosFil: Weston-Hafer, Kathleen A.. Washington University in St. Louis; Estados UnidosFil: Williamson, Kurt E.. The College Of William And Mary; Estados UnidosFil: Zhang, Bo. Washington University in St. Louis; Estados UnidosFil: Cresawn, Steven G.. James Madison University; Estados UnidosFil: Jain, Paras. Albert Einstein College Of Medicine Of Yeshiva University; Estados UnidosFil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh; Estados UnidosFil: Jacobs, William R.. Albert Einstein College Of Medicine Of Yeshiva University; Estados UnidosFil: Hendrix, Roger W.. University of Pittsburgh; Estados UnidosFil: Hatfull, Graham F.. University of Pittsburgh; Estados Unido

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