Virion morphology and virion PAGE analysis.

Mycobacteriophage Patience is morphologically a member of the Siphoviridae with an isometric head and a long flexible tail (Fig. 1A). The diameter of the heads averages 58 nm, and the tails average 358 nm in length, among the longest of the mycobacteriophages described to date, similar to those of the cluster M phages (348 nm [8]); other phages with notably long tails are those in cluster H (approximately 290 nm) and cluster R (approximately 288 nm) (13). SDS-PAGE analysis of Patience particles shows three abundant proteins and at least 12 other lower-abundance proteins between the sizes of 15 and 150 kDa (Fig. 1B).

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FIG 1 

Mycobacteriophage Patience virions. (A) Electron micrograph of Patience virions. Bar, 100 nm. (B) SDS-PAGE of Patience virions (V) and marker proteins (M). 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.

Relationship of Patience to other mycobacteriophages.

The Patience genome is not closely related to other phage genomes, although dot plot analysis shows weak similarity to cluster H phages (Fig. 2). The related segments span only 14%, 8%, and 6% of genome lengths with Barnyard, Predator, and Konstantine genomes, respectively (Fig. 2), and the closest matching segment of Patience and Barnyard is a 1,091-bp region (73% nucleotide identity) corresponding to the capsid subunit genes. Alignment of the genome maps of Patience, Barnyard, Konstantine, and Predator in Phamerator shows their architectural relationships (Fig. 3), and although the nucleotide sequence similarity is minimal, 37% of Patience genes have homologues in cluster H phages (Fig. 3). However, more than half of the Patience ORFs are orphams (genes with no close mycobacteriophage relatives [Fig. 3; Table 1]).

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FIG 2 

Dot plot analysis of Patience and cluster H phages. Dot plot analysis was performed using Gepard (31), and the designation of subcluster H1 and H2 genomes is shown.

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FIG 3 

Alignment of genome maps of mycobacteriophages Patience, Barnyard, Konstantine, and Predator. Maps were generated using Phamerator (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 10⁻⁴). 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 10⁻⁵⁰. Phamily assignments with the number of phamily members in parentheses are above each gene.

The overall GC content of the Patience genome is 50.3% and is maintained almost throughout the genome (Fig. 4A). There are two notable departures suggesting recent acquisitions by horizontal exchange. One is within Patience gene 37, where the 3′ end has 81% nucleotide identity to Rosebush gene 32 (subcluster B2) and the elevated percent GC reflects that of the B2 phages (Fig. 4B). Patience gp37 is related to tail fibers of many other mycobacteriophages that are implicated in host range determination (4). The second example is gene 47 (62% GC [Fig. 4C]), although it is an orpham with no close mycobacteriophage relatives and is of unknown function.

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FIG 4 

Distribution of percent GC in the Patience genome. (A) Percent GC scan of the Patience genome. The dotted horizontal line indicates the average GC content of 50.3%. (B) Percent GC plot of the Patience minor tail protein genes. Genes 34 to 39 encoding putative minor tail proteins have GC contents similar to those of the genome as a whole, but a segment of gene 37 with nucleotide similarity to other mycobacteriophages (coordinates 31172 to 32559; blue bar) corresponds with an increase in percent GC (55.6%). (C) Variation in percent GC about gene 47.

Patience genome organization.

Genome annotation of Patience indicates that all open reading frames and one tRNA gene are transcribed in the same direction (Fig. 5; Table 1). The overall coding capacity is 94.9%, and there are no intergenic spaces greater than 250 bp. The recognizable virion structure and assembly functions lie within the gene 4 to 39 region, and the siphoviral syntenic arrangement of terminase, portal, protease, capsid, head-tail connector proteins, major tail subunit, tail assembly chaperones, tape measure, and minor tail protein genes is observed, spanning 32 kbp of the genome (14). This is atypically long not only because of the long tape measure gene corresponding to the long phage tail but because of a dozen genes of mostly unknown function located between the terminase large subunit (gene 6) and portal (gene 8) genes, between the portal (gene 8) and protease (gene 19) genes, and between the MuF-like gene (gene 20) and the putative scaffolding gene (gene 22) (Fig. 5); most of these are orphams (i.e., they do not have a close mycobacteriophage relative), with the exceptions of genes 10 and 18 (Fig. 5). Gene 7 encodes a putative Endo VII protein and has weak similarity (<30% identity) to genes in phages Konstantine and Predator (subcluster H1), as well as Dori (singleton). The gene upstream of the terminase large subunit gene encodes a protein with similarities to putative HNH homing nucleases (Fig. 5) but may function as part of the DNA packaging machinery as described for HK97 gp74 (15). The four leftmost genes (genes 1 to 4) are of unknown function.

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FIG 5 

Genome map of mycobacteriophage Patience. The map was generated using Phamerator (35) and displayed as described for Fig. 3. Putative functional assignments are shown. Identification of the gene product by mass spectrometry in particles (P) or early (E)- or late (L)-infected samples is shown above each gene.

The lysis cassette is located downstream of the virion structural genes and contains an endolysin gene (gene 41, lysin A) and a putative mycolylarabinogalactan esterase (gene 42, lysin B) (16, 17). Genes 43 and 44 code for proteins containing two and four predicted transmembrane domains, respectively, that may act as holins or lysis chaperones (18). The lysis cassette is not closely related to that of the cluster H phages, and lysin A is most closely related to the corresponding genes of cluster M phage PegLeg gp35 and Bongo gp35 (35% identity), with an Org-U domain organization (19). In contrast, lysin B is most closely related to phages in subcluster B3 (66% identity to Gadget gp48), illustrating the mosaic nature of the lysis cassette (19). Patience gp43 has no homologues, but gp44 has distant relatives in the cluster D phages (<30% identity), where the genes are located between lysin A and lysin B.

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, RuvC, and a DNA primase/polymerase [Fig. 4; 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 pyrophosphohydrolase 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 transcribed, 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,–24).

Identification of Patience proteins by HPLC-MS/MS.

To determine which Patience genes are expressed in M. smegmatis, we analyzed three samples by high-pressure liquid chromatography–tandem mass spectrometry (HPLC-MS/MS): purified Patience particles and whole-cell extracts of Patience-infected samples at 30 min and 150 min after infection (Fig. 5; see also 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).

Of the total of 81 Patience proteins identified in infected cells, only one—gp47—was not identified in the late-infected (2.5-h-postinfection) sample. Patience gp47 was identified in the early-infected sample and is presumably not expressed late in infection but also is turned over rapidly (Fig. 5; see also Table S1 in the supplemental material). Many of the virion proteins (genes 1 to 40) are expressed at higher levels (using peptide spectral count as a surrogate for expression) in the late-infected sample, although there is also substantial expression of many of these at the early time. Of the 29 annotated proteins not identified in any of the samples, 11 are predicted to be smaller than 10 kDa and may have escaped detection if they are not expressed at high levels. Twelve are predicted to be membrane or wall associated and are likely excluded from the soluble fractions used for analysis. Peptide abundance in infected cells is expected to generally correlate with expression, although other factors such as protein size and trypsin cleavage efficiencies influence peptide detection.

Unusual expression events revealed by HPLC-MS/MS.

Identification of a large number of Patience proteins provides several new insights into gene expression and posttranslational events. 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 −1 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).

A similar scenario is seen within Patience gene 20, where two peptides corresponding to a 171-bp open reading frame on the same strand were identified with high confidence (see Fig. S1B in the supplemental material). Peptides corresponding to the predicted N terminus were not identified, although a start codon with a strong ribosome binding site is present (Fig. S2B). The corresponding segment of Patience gp20 is conserved in Barnyard gp18 and Konstantine gp17, but the second open reading frame is not. Presumably, either expression of embedded out-of-frame genes is more common than anticipated or these reflect expression artifacts resulting from the use of noncognate host transcription and translation systems.

Codon usage and codon selection.

Codon usage resulting from mutational biases was estimated using the collection of all genes from a genome. The fraction (f) of each codon for a given amino acid was calculated as the ratio of the codon count to the amino acid count. Relative synonymous codon usage (RSCU) normalizes codon frequencies so that the sum of RSCU for codons of each amino acid is equal to the number of synonymous codons for that amino acid.

To measure codon selection, a second codon usage table (f_o) was tabulated, limited to genes experiencing strong codon selection. For bacterial genomes, this table was constructed from homologues of Sharp’s set of 40 genes whose products participate in translation (36). For Patience, this table was constructed in three steps. First, a codon usage table was generated from 20% of the genome using the genes with the most extreme value of codon usage bias as determined by χ² (where expected codon usage is calculated from the nucleotide composition). Next, adaptive codon enrichment (ACE_u) values (25) are calculated for all genes as described previously (25), using this table to represent codon frequencies under codon selection (f_o) and the frequencies of codons among all genes in the Patience genome to represent codon frequencies expected from mutational processes alone (f_N). High ACE_u values are shown by genes which favor codons which are overrepresented in the f_o table relative to the f_N table. Those genes with the highest ACE_u values were used to construct another codon table; this process was repeated 50 times, reducing the size of the table to 5,000 codons total. Codon selection was measured as δ values (25), where δ = f_o/f_N for each codon. Preferred codons show δ values greater than 1.0.

Bioinformatic analyses used DNAMaster (http://cobamide2.bio.pitt.edu/), Gepard (31), ARAGORN (32), tRNAscan (33), HHpred (34), and Phamerator (35).

Electron microscopy.

CsCl gradient-purified Patience particles were applied to glow-discharged Formvar- and carbon-coated copper grids (400 mesh) (Ted Pella). They were stained with 1% uranyl acetate and imaged with a Morgagni 268 transmission electron microscope fitted with a Hamamatsu Orca HR side-model digital camera and AMT540 software.

SDS-PAGE.

Patience particles were concentrated and purified via CsCl gradient and ultracentrifugation. The visible phage band was dialyzed against two changes of phage buffer; 500 µl of the dialyzed CsCl band was pelleted by a 30-min spin at 14,000 rpm in a microcentrifuge. The pellet was resuspended in 75 µl of 20 mM dithiothreitol (DTT), and then 2 µl of 0.5 M EDTA and 1 µl of 1 M MgSO₄ were added. The phage was disrupted by being heated to 75°C for 2 min and then sonicated on ice six times for 30 s to disrupt the DNA. The sample was then mixed with 25 µl of 4× SDS sample buffer and heated in a boiling bath for 3 min at 95°C. The sample was electrophoresed through a 12% polyacrylamide gel containing SDS and stained with Coomassie brilliant blue in methanol.

This work was supported in part by a grant to the University of Pittsburgh by the Howard Hughes Medical Institute (HHMI) in support of G.F.H. under HHMI’s Professorship program; from the Howard Hughes Medical Institute to William R. Jacobs, Jr.; and by National Institutes of Health grants GM093901; to G.F.H., AI26170; to W.R.J., GM077548; to J.G.L., and GM47795 to R.W.H. We thank the KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) for support of the mycobacterial genetics workshop.

Phage Patience was isolated during a 2-week mycobacterial genetics workshop at the University of KwaZulu-Natal in 2009, and the genome was annotated in the 2011 offering of the same workshop. We thank the KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) and the University of KwaZulu-Natal for hosting the workshops and the participants and instructors for their efforts.