Functional requirements for bacteriophage growth: Gene essentiality and expression in Mycobacteriophage Giles

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 center 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.

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 & 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, 50Figs. 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 Δ shows a large reduction (Table 2) suggesting it plays an important role, even though it is non-essential for lytic 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 coordinate 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 to 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 disregulation 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. GilesΔ50 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 for 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 minutes and 2.5 hours 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, 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 Materials and Methods). 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 (Fig. 5, 6, 7G), consistent with gp47 being the phage repressor. The transcription start site was determined by 5′ RACE (Fig. 4C), showing that initiation occurs at coordinate 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 (Fig. 5, 6, 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, 7A).

Only modest levels of Giles transcription were observed during early lytic growth. The strongest transcription initiates between coordinates 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, 7F). Transcription initiates within the putative RDF gene, suggesting an alternative start site, (30; coordinate ~26,649; Fig. 7D, 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 (Fig. 6, 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 (Fig. 6, 7E–F).

These transcriptomic data suggest there are at least three early lytic promoters, upstream of genes 4, 30, and 49, (Figs 1, 6). Transcription initiates upstream of gene 49 at around 36,973, although there are no obviousσ-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 coordinate 1,828, 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 (Fig. 6, 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 coordinates 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 (coordinates 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.

Bacterial strains and media

M. smegmatis mc²155, lysogens, and recombinants were grown and recombineering cells prepared as described previously (Morris et al., 2008, Marinelli et al., 2008). Tween was omitted and 1mM 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 200bp substrates containing 100bp 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 & Hatfull, 2007) and plated in an infectious center 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 nonessential, 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 hrs at 37°C. Following infection with mixed primary plaques, plaques were recovered and screened by PCR.

Lysogeny Assays

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.

RNA analyses

Total RNA was isolated from a log-phase culture of an OD₆₀₀= 1.0. For every 500ul 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 1min incubation on ice in between. Afer DNase I treatment (Invitrogen) the samples regularly contained 1 ug/ul 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 manufacturers 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 analyzed 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′, 95°C for 10′, and 40 cycles of 95°C for 15″, 60°C for 1′. Dissociation curves were produced to verify amplification. Raw data was analyzed using ABI 7300 software and other calculations were performed with Microsoft Excel. RT-PCR primers were designed manually and standard 50ul PCR reactions were used with 10ng of RT product as template. Thermocycler conditions were: 95°C for 5′, 20 cycles of 95°C for 30″, 61.7°C for 30″, and 72°C for 1′, followed by 72°C for 7′ and a final hold at 4°C.

5′ RACE

mRNA from uninfected, early and late infected, and lysogen samples were treated with Tobacco Acid Pyrophosphatase for 30min at 37°C. The samples were extracted with phenol/chloroform and precipitated with EtOH and 0.3M 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 0.3M 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 20min 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′, 35 cycles of 95°C for 40″, 58°C for 40″, and 72°C for 40″, 72°C for 7′ 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

M. smegmatis infected with phages at a multiplicity of infection of 1 were incubated at room temperature for 15min; tween-80 added to a final concentration of 0.1%, and cells were shaken for 1min. Infected cells were centrifuged and the supernatent 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. (Guttman et al., 2009). Mycobacteriophage Giles CsCl₂ preparations were concentrated to >1 × 10¹¹ phage/ml 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 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 analyzed by high pressure liquid chromatography (HPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano-spray ionization as described by McCormack et al (McCormack et al., 1997) with these changes. The nanospray 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 Zorbax^™ 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 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 to 1800 Da and the MS/MS data was acquired from m/z of 50 to 2,000 Da. Finally, the collected data were analyzed 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.

mCherry fusions

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 analyzed using an Image Reader FLA5000.

Metabolic analysis

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.