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.

First, we note that peptides at or near the N terminus confirm the previously annotated translation start site for 54 proteins but also identify seven (gp4, gp17, gp29, gp47, gp53, gp89, and gp101) for which revisions of the annotated start sites are supported. For 16 proteins, the peptide coverage is insufficient to be informative about start site usage. Interestingly, seven proteins, all of them particle associated (gp1, gp20, gp21, gp26, gp31, gp37, and gp79), are acetylated at their N terminus (following methionine loss), although the functional significance-if any-is not known. Six are acetylated at an N-terminal threonine following methionine removal (the seventh is a serine acetylation), although not all proteins with N-terminal threonine residues are acetylated. gp21 is unusual in that the N-terminal-most peptides start at residue 35 of the annotated product following a glycine in the Ϫ1 position, indicating that they were not generated either by tryptic digestion or by translation initiation. Other processes such as posttranslational processing or intron splicing prior to translation may be involved. We also note that gp24 is present in the particles but that the peptide coverage (62 total spectra) is restricted to the N-terminal 50% of the protein, whereas in infected cells, the spectra reflect 100% coverage of the protein (149 total spectra). This could be explained by assembly-associated protein processing. Surprisingly, we identified several peptides corresponding to translation of regions wholly embedded within annotated genes. Two of these were identified only with peptides for which we have lower confidence and will not be considered further. The evidence supporting translation of the other two is, however, quite strong. The first of these is a 372-bp open reading frame transcribed on the same strand, in a different reading frame, within gene 91, the DNA polymerase III catalytic subunit (see Fig. S1A and S2A in the supplemental material). A total of 17 instances of seven different peptides were identified, and the spectra and fragmentation tables strongly support the peptide assignments (see Text S1 in the supplemental material). Moreover, 10 peptides correspond to the extreme N terminus with the methionine present, and there is a strong ribosome binding site upstream (Fig. S2) . Although the protein is seemingly expressed, the functional relevance is unclear. The predicted product has no close database relatives, and the evidence for conservation is ambiguous (Fig. S1A ). Barnyard contains a similar open reading frame within its polymerase, and the products share 30% amino acid identity, whereas the corresponding segments of the polymerases share only 53% identity. This region of the polymerase is more distantly related in Konstantine (40% identity), and the second open reading frame is not conserved (Fig. S1A) .