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.

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.

Melanins are common feather pigments that contribute to signaling and crypsis. Melanins may also help feathers resist feather-degrading bacteria (FDB). Two recent studies (Goldstein et al. 2004, Grande et al. 2004) tested the resistance of melanized versus unmelanized feathers to FDB using in vitro experiments, but draw opposite conclusions. Goldstein et al. (2004) concluded that melanized feathers resist FDB more than unmelanized feathers, while Grande et al. (2004) concluded that unmelanized feathers resist FDB more than melanized feathers. To resolve this conflict in the literature, we replicated previous studies but included additional tests not previously used. We inoculated melanized and unmelanized feathers of domestic geese Anser anser domesticus, with the FDB Bacillus licheniformis and measured bacterial activity every two days over two weeks. Three metrics of bacterial activity on feathers were measured: soluble protein content around feathers in solution, bacterial growth on feathers, and loss of feather mass. The latter two metrics were not considered in the aforementioned studies, which indirectly measured bacterial activity. We conducted two trials, one in which feathers were sterilized by autoclaving before inoculation (Goldstein et al. 2004, Grande et al. 2004, and a second in which feathers were sterilized by ethylene oxide gas. This allowed us to test whether autoclaving, done in previous studies, influences bacterial activity on feathers and could confound results. In both trials, unmelanized feathers degraded earlier, supported greater bacterial growth, and lost more mass than melanized feathers. These results support the findings of Goldstein et al. (2004); melanized feathers are more resistant to FDB than unmelanized feathers. Thus, using direct metrics of bacterial activity, we resolve a current conflict in the literature. We also found that autoclaving feathers influences FDB activity on them, and thus autoclaving should be avoided in future studies.