Genomics and Proteomics of Mycobacteriophage Patience, an Accidental Tourist in the Mycobacterium Neighborhood

Pope, Welkin H.; Jacobs-Sera, Deborah; Russell, Daniel A.; Rubin, Daniel H. F.; Kajee, Afsana; Msibi, Zama N. P.; Larsen, Michelle H.; Jacobs, William R.; Lawrence, Jeffrey G.; Hendrix, Roger W.; Hatfull, Graham F.

Patience is the first phage to our knowledge for which the proteomic profile in infected cells has been examined by mass spectrometry. The approach is highly informative, providing strong evidence that many of the annotated reading frames are expressed-including 29 that are shorter than 120 codons-and providing support for many of the translational start sites, as well as revisions of start sites of several genes. Moreover, a previously unannotated gene was identified (gene 111) containing only 37 codons, and the revision of start codons indicates that two pairs of genes have significant overlaps (Ͼ60 bp). Phage genome annotation is generally more error-prone than that of other genomes because of the abundance of small open reading frames and relatively small gene size (average mycobacteriophage gene length is 640 bp). HPLC-MS/MS adds confidence to genome annotation, especially for phages such as Patience, whose coding potential does not closely match its bacterial host. It is surprising to find that at least two reading frames embedded out of frame within annotated genes are also expressed. These ORFs are generally not conserved and may not express functional products, but an intriguing possibility is that this expression is a consequence of movement into higher-GC hosts, presenting a small reservoir of new products available for selection and further adaption at little evolutionary cost. We note that ribosomal profiling of phage suggests that previously unannotated genes are expressed from its genome (26) .