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.

Of the other 65 Patience ORFs, only seven can be assigned putative functions, most of which are involved in DNA metabolism (helicases, RecA, DNA polymerase III [Pol III] alpha subunit, The three most abundant proteins likely correspond to the major tail subunit (gp31), the capsid subunit (gp23), and gp15, with predicted molecular masses of 30.5 kDa, 35.3 kDa (after processing), and 32 kDa, respectively. The major tail subunit may migrate slower than its predicted mass, as observed in some other phages (37) . Numbers at right are molecular masses in kilodaltons. Table 1 ]). These all have homologues in a variety of mycobacteriophages as well as in phages of Gordonia and Corynebacterium. One (gene 103) encodes a MazG-like protein, a putative nucleoside triphosphate pyro-phosphohydrolase that is common in a variety of phage genomes (20) . Patience has a single tRNA gene, and the tRNA is predicted to be charged with glutamine and has the anticodon 5=-UUG [i.e., tRNA Gln (UUG)]. It is unclear how the Patience genes are tran- (35) and the database Mycobacteriophage_285, containing 285 complete genome sequences, and aligned by the left ends of the tape measure gene. Maps are displayed in three tiers with pairwise nucleotide sequence similarities displayed in spectrum coloring, with violet being the most similar and red the least similar (minimal BLASTN cutoff E value is 10 Ϫ4 ). Genes are shown as colored boxes, with colors reflecting pham assignments for each gene (gene members of a phamily have the same color). Genes shown in white are orphams and have no mycobacteriophage relatives with greater than 32.5% amino acid identity or a BLASTP E value lower than 10 Ϫ50 . Phamily assignments with the number of phamily members in parentheses are above each gene. scribed, and there are no strongly predicted SigA-like promoters, similar to other mycobacteriophages such as Giles (21), even though SigA-like promoters are active in other mycobacteriophages (22) (23) (24) . Table S1 in the supplemental material). Using stringent criteria for peptide identification, we identified 79 of the 109 previously annotated predicted gene products (82 different gene products, including previously unannotated genes) present in at least one of the samples, including 34 of the 35 putative virion structure and assembly proteins (genes 5 to 39), 27 of which are particle associated (Fig. 5) . Although gp9 was not detected, particle-associated peptides corresponding to a previously unannotated 37-codon gene between genes 8 and 9 were identified, which we designate gene 111 (Fig. 5) . Particles contain four additional products, gp1, gp4, gp79, and gp86, although gp86 is abundant in lytic growth and the few particle-associated peptides could be contaminants in the phage preparation (Table S1 ). The capsid subunit (gp23) appears to be proteolytically cleaved between residues 82 and 83 to generate a 35.3-kDa mature protein product; that and the gp31 major tail subunit (30.5 kDa) and gp15 (32 kDa) likely correspond to the three major species observed by SDS-PAGE (Fig. 1B) . It is noteworthy that-with the exception of 31-residue gp9 -all of the genes corresponding to insertions within the otherwise canonically syntenic virion structure and assembly operon are present in Patience particles, although only 3 spectra of gp21 were identified and could represent contaminants from the lysate (Fig. 5 ; Table S1).