Welkin H. Pope, Christina M. Ferreira, Deborah Jacobs-Sera, Robert C. Benjamin, Ariangela J. Davis, Randall J. DeJong, Sarah C. R. Elgin, Forrest R. Guilfoile, Mark H. Forsyth, Alexander D. Harris, Samuel E. Harvey, Lee E. Hughes, Peter M. Hynes, Arrykka S. Jackson, Marilyn D. Jalal, Elizabeth A. MacMurray, Coreen M. Manley, Molly J. McDonough, Jordan L. Mosier, Larissa J. Osterbann, Hannah S. Rabinowitz, Corwin N. Rhyan, Daniel A. Russell, Margaret S. Saha, Christopher D. Shaffer, Stephanie E. Simon, Erika F. Sims, Isabel G. Tovar, Emilie G. Weisser, John T. Wertz, Kathleen A. Weston-Hafer, Kurt E. Williamson, Bo Zhang, Steven G. Cresawn, Paras Jain, Mariana Piuri, William R. Jacobs, Roger W. Hendrix, Graham F. Hatfull

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.