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Division of Dermatology, Department of Medicine and Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine University of California Los Angeles, Los Angeles, CA, USA
Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions – such as virion proteins and repressors – cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny.
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The size, age and dynamic nature of the bacteriophage population contribute to their vast genetic diversity (Hatfull and Hendrix, 2011). Not only do phages infecting hosts of different bacterial genera typically share little or no nucleotide sequence similarity, but phages infecting the same specific bacterial strain can also encompass large genetic diversity (Hatfull and Hendrix, 2011; Krupovic et al., 2011). Moreover, it is common for large proportions of phage genes (> 75%) to fail to have significant sequence similarity to genes outside of the close phage relatives, and given the massive size of the population, bacteriophages likely represent the largest reservoir of unexplored sequences in the biosphere (Mokili et al., 2012). A central challenge in phage biology is thus to elucidate the functions of these unknown genes.
Mycobacteriophages are viruses of mycobacterial hosts including Mycobacterium tuberculosis and M. smegmatis. Comparative analysis of over 220 completely sequenced genomes shows that they are mosaic, with DNA segments corresponding to single genes being pervasively exchanged among phages in the environment (Pedulla et al., 2003; Hatfull, 2010). All of these genomes infect a single common host strain, M. smegmatis mc2155, and span considerable diversity and host range profiles (Jacobs-Sera et al., 2012). To simplify the genomic analysis, closely related genomes (nucleotide sequence similarity spanning more than 50% of genome length) are grouped in clusters (Cluster A, B etc.), with some clusters being further divided into subclusters reflecting genome nucleotide variation. Currently, the ∼ 220 sequenced mycobacteriophage genomes deposited in GenBank are grouped into 15 clusters, and eight singletons, i.e. phages for which close relatives have yet to be identified (Pope et al., 2011a,b; Hatfull, 2012a).
Giles is a singleton mycobacteriophage that is temperate in M. smegmatis, and contains a 53 746 bp genome with 14 bp 3′ single-stranded extensions (Morris et al., 2008); the DNA is presumably packaged by a cos-packaging mechanism. Twelve virion structure and assembly genes were identified but more than 50% of its predicted genes encode proteins with no close sequence similarity (> 32.5% identity) to other mycobacteriophages and the functions of fewer than 30% of its genes can be predicted (Fig. 1) (Morris et al., 2008). The Giles genome was established as a good substrate for development of the Bacteriophage Recombineering of Electroporated DNA (BRED) system that enables simple construction of phage mutants (Marinelli et al., 2008; 2012), and this was used to demonstrate roles of the LysA and LysB lysins (Marinelli et al., 2008; Payne et al., 2009). The full extent of the Giles host range is not known, but it forms plaques on other strains of M. smegmatis at a greatly reduced efficiency of plating (Jacobs-Sera et al., 2012). It does not infect M. tuberculosis, but plaques can be recovered as infectious centres on lawns of M. smegmatis following introduction of Giles genomic DNA into M. tuberculosis by electroporation (Jacobs-Sera et al., 2012).
Mycobacteriophages represent a rich resource of tools for mycobacterial genetics as well as novel strategies for rapid TB diagnosis and drug susceptibility testing (Jacobs et al., 1993; Piuri et al., 2009; Hatfull, 2012b). Mycobacterial-specific recombineering systems have been derived from mycobacteriophage Che9c (van Kessel and Hatfull, 2007; 2008), and a variety of integration-proficient vectors have been described, including those generated from phage Giles (Lee et al., 1991; Pham et al., 2007; Morris et al., 2008; Pope et al., 2011a). Further exploitation of mycobacteriophage genomes is limited by our poor understanding of the overall patterns of gene expression, regulation, gene function and gene essentiality.
Using a combination of transcriptomic and functional genomic approaches we describe the transcription patterns of mycobacteriophage Giles and show that at least 35 of its 78 predicted genes are non-essential for lytic growth, including three virion-associated proteins. A small non-coding RNA (ncRNA) is expressed at high levels both in lysogeny and in late lytic growth, but is non-essential and has no known function.
Giles structural proteins
Mycobacteriophage Giles has a siphoviral morphology and a genome architecture sharing features with the large group of siphoviral phages including phage λ (Morris et al., 2008); the attP attachment site is located near the centre of the genome and defines the left and right arms (genes 1–28 and 29–78 respectively). The left arm encodes the rightwards-transcribed virion structure and assembly functions, interrupted by three leftwards-transcribed genes between the terminase small and large subunit genes (Fig. 1). Of the 11 virion proteins identified previously (Morris et al., 2008) one (gp36) is unusually encoded within the genome right arm (Fig. 1). Mass spectrometry of whole virion particles identified a total of 20 proteins (Table 1), 18 of which are encoded in the left arm, (Fig. 1) as well as a second right-arm encoded protein, gp37 (Table 1, Fig. 1). Although only one peptide of gp37 was identified, the predicted protein is small (50 aa) and generates only two possible peptides of significant complexity (> 7 amino acids) by trypsin digestion (DVTNSQWTAHTQQMNR and LLEAEGLQQTGK). The latter peptide was identified with a 99% confidence from its mass spectrum and represents 24% of the protein sequence. The peptide is complex and the fragmentation pattern of b and y ions closely matches that expected from the sequence. The presence of both protease (gp7) and scaffold (gp8) suggests that these are incompletely removed from proheads following DNA packaging, although we cannot rule out contamination of the phage preparation with incompletely assembled particles.
Table 1. Giles virion proteins determined by mass spectrometry
No. of peptide hits (> 95% confidence)
aThis is a longer peptide with a unique sequence that is well represented by y and b ions in the MS/MS spectra. It is identified with a > 99% confidence level.
Most of the products encoded in the left arm (1–28) are virion associated, although seven are not, including the putative small and large terminase subunits (gp1 and gp4 respectively, which are required for packaging), and gp17, gp18 and gp19, which may function in tail assembly (Morris et al., 2008). However, the organization of the region between the Giles major tail subunit (15) and tape measure (20) genes is a departure from that of other phages where two ORFs [presumed to be Giles 16 and 17 (Morris et al., 2008)] coding for tail assembly chaperones are expressed by a programmed translational frameshift (Hatfull and Sarkis, 1993; Xu et al., 2004). We also did not find gp23 or gp26, which are encoded among the other tail genes (Fig. 1).
Giles genes required for lytic growth
We determined which Giles genes are required for lytic growth using the BRED strategy described previously (Marinelli et al., 2008). In this method, phage genomic DNA is co-electroporated into a recombineering strain of M. smegmatis with a DNA substrate (typically about 200 bp long) that contains the mutant allele – either a specific gene deletion or a point mutation – and plaques recovered on M. smegmatis plating cells after a short recovery period. Each plaque is thus derived from a single cell that has taken up phage DNA, and at least 10% of these typically contain a mixture of the wild-type and mutant alleles, from which a homogenous mutant of a non-essential gene (i.e. a gene that is not required to form a visible plaque) can be recovered after further purification. Because the overall process is efficient, mutants can be identified by physical characterization (PCR) without the need for selection. If a mutation is deleterious to lytic growth, then a mixed primary plaque can usually be recovered – because of complementation by wild-type particles in the same plaque – but cannot be recovered after subsequent purification.
Of the 78 predicted Giles genes, we selected 54 for deletion avoiding most of the virion structure and assembly genes, which are expected to be essential for lytic growth (i.e. is required to form a visible plaque) (Hendrix et al., 1983). Of the 54 genes tested, 35 (65%) were determined to be non-essential for lytic growth (and can thus be isolated as a homogenous mutant population; Table 2, Figs 1 and 2A–C). Most of these form normal sized plaques containing similar numbers of particles as wild-type Giles (Table 2; Fig. S2); several mutants show mild losses in fecundity but only Δ50 shows a large reduction (Table 2), suggesting it plays an important role, even though it is non-essential for lytic growth.
Table 2. Giles genes essential for lytic and lysogenic growth
Nineteen ORFs are predicted to be essential for lytic growth (and cannot be recovered as a homogenous mutant population), and we attempted to recover each of the mutants using plasmid-mediated complementation. For two of these (genes 31 and 64) the complementation was successful [the gene 31 deletion was reported in a previous study (Payne et al., 2009)], and the mutant was isolated and purified in the complementing strain (Fig. 2D–G). The Δ64 mutant was shown to only form plaques when plated on the complementation strain, but not on a wild-type strain. Giles gene 64 is thus essential for lytic growth (Fig. 2G). The remaining 17 mutants identified in initial BRED platings could not be further propagated even when plasmid-encoded genes were provided (Table 2), and for some we constructed plasmids with pairs of complementing genes (such as 62 and 63) but still failed to recover the mutant. Complementation fails presumably due to poor expression of the complementing gene or expression at inappropriate stoichiometry, loss of an essential cis-acting element, or because of genetic polarity. Nonetheless, the simple conclusion from the BRED approach is that each of these genes is required for lytic growth.
In one case, we were able to isolate mixed primary plaques for a gene 67 deletion but a pure mutant was difficult to isolate. As this gene was likely to be required for lytic growth, a complementation plasmid was constructed and a pure mutant was isolated on the complementing stain. We found that the Δ67 mutant phage does form plaques on M. smegmatis mc2155 in the absence of complementation, but the plaques are extremely small and barely visible (Fig. S1). The gene is therefore designated as being non-essential for lytic growth, although it is clearly important. Two of the deletion mutants (Δ32 and Δ29) required removal of ∼ 1.2 kbp DNA representing a reduction of genome size of about 2%. Because these were successfully generated, this reduction has no significant impact on DNA packaging. We note that we were able to successfully generate a double mutant that removes both gene 73 and 74; other double mutants were not attempted.
Finally, some genes might appear to be essential in this assay as a consequence of misannotation of reading frames that are adjacent to essential components, including cis-acting elements. One example of this emerged through the failure to construct a deletion mutant of gene 75. However, there is a short non-coding gap between genes 74 and 75, which reduces confidence in the choice of the translation initiation codon in the current annotation (Fig. 1), and RNAseq data suggests that this is incorrect (see below). We therefore used an alternative substrate to remove the 3′-half of the originally annotated gene 75, assuming use of an alternative initiation codon at co-ordinate 52 251. This mutant was created successfully, and we conclude that the revised 75 is non-essential. A second example is a gene originally annotated as 48, which we were unable to delete. However, functional characterization of the flanking genes 47 and 49 coupled with transcriptomic analysis (see below) showed that the 48 open reading frame lies in an intergenic regulatory region, accounting for the inability to remove it. We have thus removed gene 48 from the genome annotation. These corrections are included in an updated GenBank file (accession number EU203571.3).
Roles of Giles genes 50, 64 and 67 in DNA replication
Although few genes in the Giles right arm have known functions, it is likely that at least some are involved in phage DNA replication. Because the Δ64 phage could be constructed and propagated on a complementing strain, we tested whether it is defective in DNA replication in a non-complementing host. Using qPCR, we observed no replication of Δ64 phage DNA following infection of a wild-type host, and the defect was largely restored in the complementing strain (Fig. 3A). This is consistent with gp64 having weak but significant similarity (Probability = 95.18, E-value = 0.025) to the G39P helicase loader of Bacillus phage SPP1 shown by HHPred (Soding et al., 2005). The higher level of Giles DNA replication in strain mc2155pRMD1 compared with mc2155 could be a consequence of a higher level of gp64 expression. This gene is not being controlled in its native state (in the phage) and its dysregulation might contribute to elevated DNA replication. In contrast, DNA replication of the Δ67 mutant was observed in a wild-type strain although it is reduced from the parent phage and is only modestly enhanced by complementation (Fig. 3B). This is consistent with the predicted role of gp67 as a RuvC-like protein involved in Holliday Junction (HJ) resolution. Giles Δ50 is viable on a non-complementing strain but shows a marked defect in fecundity and produces small plaques (Table 2) and like Δ64, it too shows a strong defect in DNA replication (Fig. 3B). There are no bioinformatic clues as to its specific function.
Identification of the repressor
Although Giles is a temperate phage, and its integration system has been characterized (Morris et al., 2008), its repressor has not been identified. Because wild-type Giles plaques are only lightly turbid, making it hard to distinguish clear plaque mutant phenotypes, we screened each of the deletion mutants for the ability to form lysogens on phage-seeded plates (Table 2). We observed significant defects in lysogeny in only two mutants, Δ47 and Δ49 (Fig. 4A and Table 2), and in both cases, lysogeny was reduced to below 0.01%. To determine which of these encodes the repressor, 47 and 49 were cloned, expressed and tested for the ability to confer immunity to Giles superinfection (Fig. 4B). Gene 47 confers strong immunity and we conclude that gp47 is the phage repressor. We note that gp47 has no close homologues and does not contain any readily identifiable DNA binding motifs. Gene 49 does not confer immunity and its role is unclear, although it could regulate repressor expression, similar to λ cII; gp49 has no close homologues but HHPred predicts a small zinc finger domain and it is likely a DNA binding protein.
Transcription of the Giles genome
We used RNAseq to determine transcription profiles in early and late Giles infections, 30 min and 2.5 h after adsorption, respectively, according to the infection patterns described previously (Payne et al., 2009) as well as in a Giles lysogen, and compared these to uninfected M. smegmatis (Figs 1, 6 and 7). Several datasets were generated to optimize the number of non-rRNA sequence reads, and all are in general agreement but differ significantly in overall quality (see Experimental procedures). We also used qRT-PCR to amplify each of the gene junctions in lysogeny and lytic growth (Fig. 5).
The transcription profile of a Giles lysogen is relatively simple. The strongest signal spans gene 47 with transcription initiating in the 47–49 intergenic region (Figs 5, 6 and 7G), consistent with gp47 being the phage repressor. The transcription start site was determined by 5′ RACE (Fig. 4C), showing that initiation occurs at co-ordinate 36 674, in strong agreement with the RNAseq data. The promoter does not obviously correspond to σ-70 like promoters described in other mycobacteriophages and presumably a different sigma factor is used. The transcriptional level of the repressor is unusually high, with expression equivalent to the top 0.5th percentile of host genes, similar to ribosomal protein L2 and RpoC, in notable contrast to the relatively low but autoregulated levels of cI transcription in a λ lysogen (Ptashne et al., 1980). Fusion of this promoter to an mCherry reporter gene shows strong expression in lysogens but little or no expression in wild-type cells, indicating that it strongly requires activation (data not shown). There is some transcription of the genes downstream of 47, and qRT-PCR indicates genes 44–46 are also expressed in the lysogen (Figs 5, 6 and 7G). Strong transcription also is observed in a short (∼ 100 bp) non-coding segment between genes 74 and 75, and is discussed in further detail below (Fig. 7H). A modest level of genes 2–4 transcription was also observed (Figs 5, 6 and 7A).
Only modest levels of Giles transcription were observed during early lytic growth. The strongest transcription initiates between co-ordinates 36 975 and 36 990 in front of the rightwards-transcribed gene 49, and gradually diminishes across the downstream operon up to gene 59, at which point transcription is barely detectable. Both the repressor (47) and integrase (29) are expressed at low or barely detectable levels, although several other leftwards-transcribed genes are expressed, including genes 43 and 44, 39–41, and 2–3 (Figs 5, 6 and 7F). Transcription initiates within the putative RDF gene, suggesting an alternative start site, (30; co-ordinate ∼ 26 649; Figs 7D and S3) but diminishes within lysin A (31). There are barely detectable levels of expression of any of the virion structure and assembly genes at this time. qRT-PCR analyses (Fig. 5) are in general agreement with the RNAseq data but good signals are observed all the way through to the end of the right end of the genome, suggesting that RNAseq data may somewhat under represent regions at the 3′ end of long operons.
Late in infection the transcription pattern is markedly different. Interestingly, much of the early transcription pattern is retained, and the early and late levels from gene 49 to the right end of the genome are almost superimposable (Fig. 6). The notable exception is the very high level of transcription of the 74–75 intergenic region, a similar segment to that transcribed from the prophage (see below).The virion structure and assembly genes are expressed at high levels although with some apparent variation across the left arm (Fig. 6). Very high levels of expression begin with the portal gene (6) but diminish at the tape measure gene (20), return to high levels at gene 26 (Figs 6 and 7B–D), and stop near the factor-independent terminator following gene 28. The right-arm genes 30–37 – including the two virion gene 36 and 37 and the lysis cassette – are also expressed late in lytic growth, but transcription rises sharply between genes 30 and 31 (Figs 6 and 7E–F).
These transcriptomic data suggest there are at least three early lytic promoters, upstream of genes 4, 30 and 49, (Figs 1 and 6). Transcription initiates upstream of gene 49 at around 36 973, although there are no obvious σ-70 like sequences and presumably another sigma factor is used (Fig. 4C); presumably this promoter is directly regulated by the gp47 repressor as it is not active in a lysogen. These same observations apply to a probable promoter upstream of gene 30. There is a predicted σ-70 leftwards promoter upstream of gene 4 with a start site at co-ordinate 1828, which is the first base of the ATG start codon, and other examples of leaderless mRNAs have been reported (Broussard et al., 2012). There are several plausible promoters active in late lytic growth located upstream of genes 78, 6, 26, 31, and transcribed rightwards, and one for expression of 37 and /or 38, although it is unclear which strand is being transcribed. None of these contain an obvious σ-70 like promoter and we assume that transcription from these requires an as yet unidentified Giles-encoded transcriptional activator.
Expression and role of a small non-coding RNA
High levels of a small (∼ 100 nucleotide) transcript are observed from the 74–75 intergenic region in both late lytic and lysogenic growth (Figs 6 and 7H). This was confirmed by qRT-PCR which also demonstrated that it is expressed in the rightwards direction (Fig. 8A). To determine if it is required for lysogeny or lytic growth, we using BRED mutagenesis to delete co-ordinates 51 631–51 728 such that the flanking genes were not interrupted. The mutant was readily constructed, and we have been unable to identify any defect in either lytic growth or lysogeny. Fusion of the upstream region (co-ordinates 51 565–51 620) to a mCherry reporter gene showed an active promoter, although it is about 10-fold more active in a Giles lysogen than a non-lysogen (Fig. 8B). Metabolomic comparisons of lysogens carrying the deletion mutation and wild-type Giles revealed no significant differences in growth. We also found no evidence of the RNA being incorporated into Giles particles.
The massive increase in bacteriophage genomics over the past 10 years has revealed their enormous genetic diversity and a rich abundance of novel gene sequences. While a great deal is known about the detailed biology of a small number of phages such as λ, T4 and T7, the question arises as to what extent this knowledge applies to the broader population of phages, especially those that infect hosts other than Escherichia coli. Moreover, methods for determining phage gene functions and transcriptomic profiles are not well established. Giles presents an excellent model system for functional genomic analysis as most Giles genes have no close relatives and few functions can be predicted. BRED mutagenesis provides a simple method for determining gene essentiality for a large proportion of its genome, but is likely to be effective not only for other mycobacteriophages, but also for phages of any other host in which recombineering is available.
The proportion of Giles genes required for lytic growth was not predictable bioinformatically. In phage λ at least 18 genes are non-essential – including nine in the b2 region (Hoess and Landy, 1978; Hendrix et al., 1983) – and it is surprising that as many as 35 Giles genes – 45% of its genome – are dispensable for lytic growth. Other than the three virion-associated proteins and lysin B (Payne et al., 2009) it is unclear what roles these play, and bioinformatic analyses provide little insight. The one exception is gp67, a putative RuvC-like HJ resolvase, but loss of 67 leads to barely viable phage suggesting that unresolved HJs interfere with DNA packaging. This indicates that recombination is active in Giles replication, and the essentiality of the RecE/T-like recombination proteins (gp52 and gp53) suggests that recombination is required, as in T4 (Kreuzer and Brister, 2010). Nonetheless, if extrapolated to the larger collection of 220 sequenced mycobacteriophages, these data suggest there are over 11 300 non-essential genes in almost 1500 unique sequence phamilies (Hatfull, 2012a).
All of the non-essential genes are expressed either in lytic or lysogenic growth. The most dramatic perhaps is the ncRNA between 74 and 75, expressed at high levels both in a lysogen and in late lytic growth. This ncRNA is not required for either lytic growth or lysogeny and its role remains unclear. However, this situation is not common, as we have also identified many protein-coding genes that are also not required for either lytic or lysogenic growth and for which a function has not yet been assigned. One attractive explanation is that at least some of these RNA and protein functions could confer protection from competing phage infections, as seen with restriction-modification and rogue immunity acquisition (Pope et al., 2011b). However, this is difficult to examine directly unless an excluded phage can be identified, and because mycobacteriophage diversity is extremely high, such phage(s) may not as yet have been isolated. Alternatively, these genes could mediate changes to host gene expression, although there are only subtle differences between the lysogenic and non-lysogenic transcriptomes. We note that genes 2–4, which are expressed in both lytic and lysogenic growth, share a similar location to gene 2 of Streptomyces phage ϕC31, between the small and large terminase subunits and oriented in the opposite direction to the structural genes (Smith et al., 1999), so perhaps these have similar functions.
Nineteen ORFs are essential and the requirement for WhiB is surprising as the WhiB-like protein of phage TM4 is non-essential (Rybniker et al., 2010). However, many mycobacteriophages encode WhiB-like proteins – some with several copies – and they are a highly diverse perhaps providing a variety of particular functions. Because the DNA methylase is essential, it is plausible that it provides a modification component of a restriction system, although the best candidate for a restriction endonuclease (based on HHPred analysis, which also shows similarity to an HNH nuclease and an HJ resolvase) is gp76, which is, however, also essential. HHPred also suggests that the essential gp42 is implicated in replication initiation, and that gp40 (with a C-terminal DNA binding motif) may be a regulator.
The identification of gp47 as the phage repressor – it is well-expressed in a lysogen and is both required for lysogeny and for superinfection immunity – is surprising given its complete lack of known DNA binding motifs. This illustrates the amazing genetic diversity of the phage population in that even this thoroughly well-studied class of proteins cannot be readily predicted bioinformatically in phage genomes. Presumably, gp47 regulates the rightwards early lytic promoter upstream of 49, and identifying what class of promoter is used and how it is regulated will be of interest.
Bacterial strains and media
Mycobacterium smegmatis mc2155, lysogens and recombinants were grown and recombineering cells prepared as described previously (Marinelli et al., 2008; Morris et al., 2008). Tween was omitted and 1 mM CaCl2 was included in all media for phage infections. Plasmids used are listed in Table S1 and oligonucleotides in Table S2. A revised mycobacteriophage Giles GenBank file has been submitted to NCBI under accession number EU203571.3. See Supporting Information for further details regarding annotation revisions.
Construction of gene deletions
Giles deletions were constructed using BRED as described previously (Marinelli et al., 2008). Briefly, PCR was used to produce 200 bp substrates containing 100 bp of homology to the upstream and downstream regions of the gene to be deleted. A 9-base unique bar coding ‘tag’ was inserted in place of the deleted coding region to facilitate mutant identification. Giles DNA and the target substrate were electroporated into M. smegmatis recombineering cells (van Kessel and Hatfull, 2007) and plated in an infectious centre assay. In general, flanking primer (FP) PCR worked well in identifying mixed plaques and pure mutants, but tag-specific PCR was important to identify hard-to-isolate mutants in which FP PCR generated weak signals. In general this strategy was effective, although there was one example (gene 77) for which we were not able to recover a mixed primary plaque unless a substrate was used that lacked the 9-base tag.
For each mutant construction, we typically screened 16–20 primary plaques and mixed plaques were identified from 3% to 94%; there was no obvious correlation between the number of mixed plaques isolated and the essentiality of the gene. Secondary plaques were screened similarly, picking 16–20 for PCR validation. When a pure mutant could not be recovered from the secondary plating, several pools of 5–10 plaques were screened, and if this still did not verify that a mutant was present, several mixed plaque lysate dilutions were screened. If a mutant band was present in all lysate dilution plates, suggesting that the mutant was present and non-essential, then further single plaque PCR screenings were performed. For essential genes, mutant plaques were only seen at the lowest dilution of the mixed plaque lysate and not the higher dilutions. This suggested that the mutant could not survive without the presence of wild-type phage particles acting as helpers. Primers are listed in Table S2.
For complementation, mc2155 containing plasmids were grown to OD600 = 0.4, ε-caprolactam (Sigma) added at varying concentrations (0.2–1%), and cultures grown for 3 h at 37°C. Following infection with mixed primary plaques, plaques were recovered and screened by PCR.
Lysogeny frequencies were measured by plating dilutions of M. smegmatis (104–107 cfu) on agar plates seeded with 109 pfu of each phage, and determining the number of colonies relative to non-seeded plates.
Total RNA was isolated from a log-phase culture of an OD600 = 1.0. For every 500 μl of culture, 1 ml of RNAprotect reagent (Qiagen) was added. Samples were pelleted and the RNeasy Mini Kit (Qiagen) was completed. Cells were broken in Matrix B (MP Biomedicals) using a Beadbeater twice for 45 seconds on max speed with 1 min incubation on ice in between. After DNase I treatment (Invitrogen) the samples regularly contained 1 μg μl−1 of RNA. RiboZero (Epicentre) rRNA removal kit was used according to the manufacturers instructions; mRNA was then retreated with DNaseI. Removal of rRNA and concentrations of mRNA were confirmed using Agilent 2100 Bioanalyzer. Purified samples were stored at −80°C. Samples were prepared for RNA-seq using the TruSeq RNA Sample Preparation kit (Illumina #15026495) according to the manufacturer's instructions. Samples were sent to Tufts Genomics Core Facility (Tufts University, MA, USA) where single-end libraries were subjected to 50 bp read Hi-Seq Illumina sequencing. Data was analysed using Galaxy (Penn State University). First, Bowtie was used to map the RNAseq reads to the reference genome. This file was then filtered of unmapped reads (Filter SAM), and converted into a BAM file format (SAM-to-BAM). The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) accessible through GEO Series accession number GSE43434.
Primers for qRT-PCR were generated using PrimerExpress software (Applied Biosystems). cDNA was generated using random hexamers and Maxima reverse transcriptase (Fermentas), and qRT-PCR performed using the Maxima SYBR Green qPCR Master Mix (Fermentas). The Applied Biosystems 7300 Real-Time PCR System was the instrument used with cycling conditions of: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s, 60°C for 1 min. Dissociation curves were produced to verify amplification. Raw data were analysed using ABI 7300 software and other calculations were performed with Microsoft Excel. RT-PCR primers were designed manually and standard 50 μl PCR reactions were used with 10 ng of RT product as template. Thermocycler conditions were: 95°C for 5 min, 20 cycles of 95°C for 30 s, 61.7°C for 30 s, and 72°C for 1 min, followed by 72°C for 7 min and a final hold at 4°C.
mRNA from uninfected, early and late infected, and lysogen samples were treated with Tobacco Acid Pyrophosphatase for 30 min at 37°C. The samples were extracted with phenol/chloroform and precipitated with EtOH and 0.3 M sodium acetate. A 5′ RNA adaptor (5′-AUAUGCGCGAAUUCCUGUAGAACGAACACUAGAAGAAA-3′) was ligated with T4 RNA ligase (Fermentas) at 17°C overnight. Again, samples were extracted with phenol/chloroform and precipitated with EtOH and 0.3 M sodium acetate. The samples were reverse transcribed using a gene 49 specific oligo (5′-CAGGTACTTATCGCGGTG-3′) and Maxima RT (Fermentas). Samples were treated with RNase H for 20 min at 37°C. PCR amplification was completed using an adaptor specific oligo (5′-GCGCGAATTCCTGTAGA-3′) and a gene 49 specific oligo (5′- CAGGTACTTATCGCGGTG-3′) at the following conditions: 95°C for 10 min, 35 cycles of 95°C for 40 s, 58°C for 40 s, and 72°C for 40 s, 72°C for 7 min and 4°C hold. PCR products were gel purified and subjected to TOPO-TA cloning (Invitrogen). E. coli transformation candidates were verified to contain an insert by PCR and sequenced (Genewiz) using T7 and T3 primers.
DNA replication analyses
Mycobacterium smegmatis infected with phages at a multiplicity of infection of 1 were incubated at room temperature for 15 min; Tween-80 added to a final concentration of 0.1%, and cells were shaken for 1 min. Infected cells were centrifuged and the supernatant containing unadsorbed phage was removed. The pellet was washed once with 7H9/ADC/Tween 0.1% and once with 7H9/ADC/Tween 0.05%. Cells were resuspended in 7H9/ADC, incubated at 37°C, and sample taken every hour for qPCR analysis.
Sample trypsinization and LC-MS/MS analysis
Sample preparation was performed as described in Guttman et al. (2009). Mycobacteriophage Giles CsCl2 preparations were concentrated to > 1 × 1011 phage ml−1 using a Speed Vac for in solution protein digestion. RapiGest SF reagent (Waters Corp.) was added to the 0.1 ml phage sample to a final concentration of 0.1% and samples were boiled for 5 min. TCEP [Tris (2-carboxyethyl) phosphine] was added to 1 mM (final concentration) and the samples were incubated at 37°C for 30 min. Subsequently, the samples were carboxymethylated with 0.5 mg ml−1 of iodoacetamide for 30 min at 37°C followed by neutralization with 2 mM TCEP (final concentration). Proteins samples prepared as above were digested with Promega sequencing grade modified trypsin (trypsin : protein ratio – 1:50) overnight at 37°C. RapiGest was degraded and removed by treating the samples with 250 mM HCl at 37°C for 1 h followed by centrifugation at 15 800 g for 30 min at 4°C. The soluble fraction was then added to a new tube and the peptides were extracted and desalted using a 1 ml SepPak C18 solid phase extraction columns (Waters).
Trypsin-digested peptides were analysed by high pressure liquid chromatography (HPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-spray ionization as described by McCormack et al. (1997) with these changes. The nano-spray ionization experiments were performed using a QSTAR-Elite hybrid mass spectrometer (ABSCIEX) interfaced with nano-scale reversed-phase HPLC (Tempo) using a 10 cm-100 micron ID glass capillary packed with 5 μm C18 ZorbaxTM beads (Agilent Technologies, Santa Clara, CA). Peptides were eluted from the C18 column into the mass spectrometer using a linear gradient (5–60%) of ACN (Acetonitrile) at a flow rate of 400 μl min−1 for 1 h. The buffers used to create the ACN gradient were: Buffer A (98% H2O, 2% ACN, 0.2% formic acid and 0.005% TFA) and Buffer B (100% ACN, 0.2% formic acid and 0.005% TFA). MS/MS data were acquired in a data-dependent manner in which the MS1 data was acquired at m/z of 400–1800 Da and the MS/MS data was acquired from m/z of 50–2000 Da. Finally, the collected data were analysed using MASCOT® (Matrix Sciences) and Protein Pilot 4.0 (ABSCIEX) for peptide identifications. The LC MS/MS analysis was performed in the UCSD Biomolecular and proteomics Mass Spectrometry Facility by Majid Ghassemian.
A promoterless mCherry vector was digested with NotI and KpnI. Primers were designed to amplify the region of interest from the Giles genome and contained NotI and KpnI restriction sites. After amplification of the target region, the reaction was cleaned (Qiagen), digested and cloned upstream of the reporter gene mCherry. Clones were verified by sequencing and transformed into wild-type mc2155 and the Giles lysogen. Liquid cultures were analysed using an Image Reader FLA5000.
Wild-type mc2155 and the Giles lysogen were sent to Biolog, Inc. (Hayward, CA) for phenotypic microarray services. A small change in menadione resistance was noted in mc2155(Giles), but was not reproducible upon further testing. Nonetheless, the small ncRNA deletion lysogen was tested for resistance to menadione, but none was observed.
We thank Christina Ferreira and Carlos Guerrero for excellent technical assistance and Daniel Russell for assistance with RNA-seq data processing. We also thank Amrita Balachandran and the 2008 Gene Team (University of Pittsburgh) for isolating mixed plaques of mutants Δ29, Δ51 and Δ61, Anna Mansueto for isolating a mixed plaque of mutant Δ68 and constructing its complementation plasmid, and Chiara Ricci-tam for protein sample preparation. Greg Broussard provided comments on the manuscript. This work was supported by a National Institutes of Health training grant fellowship 5T32AI049820 to R.M.D., and Grant GM093901 to G.F.H. The authors have no conflict of interest to declare.