A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

ABSTRACT

Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc²155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ΔDRKIN, a strain derived from mc²155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ΔDRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2.

IMPORTANCE Mycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state.

INTRODUCTION

Members of the Y family DNA polymerases are capable of translesion synthesis (TLS) that allows them to catalyze the insertion of deoxyribonucleotide triphosphates (dNTPs) opposite potentially lethal replication-blocking lesions (1). The ability of these polymerases to bypass such lesions helps the cell to survive under DNA-damaging conditions. However, survival comes at a cost. Mutations are introduced more frequently than under normal conditions, as these polymerases function in an error-prone manner (2). In Escherichia coli, DinP/DinB (DNA polymerase IV [Pol IV]) and UmuC (DNA Pol V) are the two Y family polymerases that mediate TLS (2). Mutations in the genes that encode UmuC and a related protein, UmuD, result in a UV-nonmutable (umu) phenotype. In contrast, mutation of dinB, the gene that codes for the Pol IV enzyme DinB, does not lead to an apparent phenotype, and therefore, the function of this protein remains enigmatic. The expression of the E. coli dinB gene, also known as dinP (3), can be induced through the SOS pathway. Additionally, it can also be induced in response to general stress through the involvement of the alternative σ factor σ^s (RpoS) (4, 5). The increased expression of dinB (dinP) results in a hypermutable phenotype (3). Accumulation of mutations under stress conditions can be beneficial, as the resulting variants may be more fit to survive under such conditions than their predecessors (1, 6). DinB, however, may have other roles to play that are not directly related to error-prone repair. It has been found that overexpression of dinB inhibits fork progression and is lethal (7, 8). Thus, it has been proposed that DinB possibly acts as a brake for DNA Pol III, thereby slowing down fork progression (7). Under stress conditions, slowing down of fork progression may protect E. coli from genome instability. In Mycobacterium tuberculosis, two DinB orthologs are found, DinB1 (Rv1537) and DinB2 (Rv3056) (9). The corresponding E. coli counterparts are DinX and DinB/DinP, respectively. In Mycobacterium smegmatis, the MSMEG_3172 and MSMEG_2294 genes encode the DinB1 and DinB2 orthologs, respectively. A third ortholog of DinB has been found in M. smegmatis (DinB3 [MSMEG_6443]), which appears to be more related to DinB1 than to DinB2 (10). The M. smegmatis protein encoded by MSMEG_2294, henceforth referred to only as DinB2, has been characterized biochemically, and it has been demonstrated that it can function as an “unfaithful” DNA polymerase (10–12). However, the in vivo role of DinB2 is unclear, as deletion of the genes encoding DinB2 orthologs did not affect either the spontaneous (9) or DNA damage-induced (13) mutation rate in M. tuberculosis. Thus, the M. tuberculosis DinB2 homologs do not appear to be involved in error-prone repair, although they are biochemically capable of doing so. Error-prone DNA repair was found to be mediated by DnaE2 and not DinB2 in M. tuberculosis (13). Thus, as far as mycobacterial DinB2 is concerned, very little is known about how it functions, even though it is expressed at a higher level than either DinB1 or DnaE2 (9). The interesting part is that although the dinB2 genes are known to be DNA damage inducible in other organisms, they were not found to be so in mycobacteria (14). The general impression derived from research done on mycobacterial DinB polymerases is that they do not behave like their counterparts from E. coli or other organisms (9).

The Y family polymerases UmuC and DinB have the intrinsic ability to incorporate ribonucleotides into the DNA chain in a templated manner. However, this ability is controlled by a single amino acid residue, which is referred to as the “steric gate.” In wild-type versions of these proteins, the steric gate amino acid residues are bulky in nature, a result of which is that ribonucleotides become excluded (15, 16). However, if these bulky residues are replaced by smaller ones, sugar selectivity becomes relaxed, and the mutants develop the capability of incorporating ribonucleotides into DNA efficiently. In the case of DinB of E. coli, it has been found that the steric gate has a role to play in controlling the ability of the enzyme to execute TLS over N²-deoxyguanosine adducts (17). Interestingly, in DinB2 of mycobacteria, the steric gate amino acid is a leucine residue (Leu14) and not the bulky aromatic amino acid phenylalanine (Phe13) that is found in its E. coli counterpart. Therefore, mycobacterial DinB2 is sugar unselective, and hence, it is naturally capable of catalyzing the incorporation of ribonucleotides into DNA (10, 12). Whether this property of DinB2 is beneficial to mycobacteria under special circumstances is an interesting issue that remains to be explored.

One of the consequences of DNA damage is that fork movement comes to a halt, as the DNA polymerases, particularly the accurate ones, cannot replicate past the lesions. Fork movement can be stalled due to not only DNA damage but also deoxyribonucleotide pool imbalances, such as those caused by treatment with hydroxyurea (HU), a class I ribonucleotide reductase (RNR) blocker (18). In E. coli, a mutant version of Pol V, UmuC (UmuC122), has been shown to be capable of repairing stalled forks induced by HU (19). Whether the wild-type counterpart can perform the same function is not clear, as deletion of the gene encoding the wild-type protein had no effect. In E. coli, the Pol IV class DNA polymerase DinB by itself did not play any significant role in the restart of replication from HU-induced stalled forks. However, its presence was found to be necessary for the fork-repairing activity of the UmuC variant UmuC122. Unlike E. coli, mycobacterium species such as M. smegmatis do not possess UmuC, and hence, how these bacteria cope with stalled forks remains unresolved.

M. smegmatis strain ΔDRKIN is a mutant (20) derived from mc²155, a strain used widely to study mycobacterial molecular biology. The parental strain mc²155 harbors a 56-kb chromosomal duplication. One copy of this duplicated region is missing in ΔDRKIN. All the genes, including dinB2, that are associated with the 56-kb region are thus present as two copies in mc²155 but as one copy in ΔDRKIN. While investigating the function of a class II ribonucleotide reductase encoded by mycobacteriophage D29 (D29Gp50), it was observed that overexpression of the gene encoding it in ΔDRKIN resulted in dTTP-limiting conditions, which in turn led to growth arrest. However, the arrested state could be overcome by increasing the level of DinB2. The results indicate that this DNA polymerase can bring about DNA replication in vivo under dTTP-limiting conditions. Thus, for the first time, a role has been found for the enigmatic mycobacterial Y family DNA polymerase DinB2.

MATERIALS AND METHODS

Bacterial strains, bacteriophages, and plasmids.

E. coli strain XL1-Blue was used for basic cloning and expression purposes. M. smegmatis strains mc²155 and ΔDRKIN (20) were used for expression of genes in mycobacteria. Mycobacterium bovis BCG was used as a representative of slow-growing mycobacteria. The BCG strain and not the wild type was used so as to minimize biohazards. Mycobacteriophage D29 (21) was obtained as a gift from Ruth McNerney. Overexpression in E. coli XL1-Blue cells was done by using the pQE30 vector system (Qiagen). Mycobacterial expression was performed by using either the acetamide-inducible vector pLAM12 (22) or the tetracycline-inducible vector pMind (23).

Chemicals and reagents.

Restriction endonucleases/DNA-modifying enzymes were obtained from Thermo Scientific or New England BioLabs. Luria-Bertani (LB) broth and Middlebrook 7H9 (MB7H9) broth were obtained from Himedia and Difco (BD), respectively. Acetamide was purchased from Merck, and anhydrotetracycline (ATc) was purchased from Clontech. Anthranilic acid (AA), nicotinic acid (NA), and diammonium hydrogen citrate (DAHC) were obtained from Fluka (Switzerland). All other chemicals/reagents for protein purification and analysis were of the highest purity and obtained from Sigma, SRL, or Merck (India).

Bacterial and bacteriophage growth conditions and DNA isolation.

E. coli cells were cultured in LB broth, and mycobacteria were cultured in MB7H9 broth supplemented with 0.25% bovine serum albumin (BSA), 0.2% glycerol, and 0.05% Tween 80 at 37°C. The antibiotic used was either kanamycin (20 or 50 μg/ml), hygromycin (50 or 200 μg/ml), or both if needed. Phage was grown to confluence and then extracted by diffusion into phage dilution buffer (22). After concentration by ultracentrifugation at 1,00,000 × g for 2 h, phage was finally suspended in a minimum amount of phage dilution buffer. Phage DNA was isolated by using a Qiagen lambda phage DNA isolation kit. Bacterial genomic DNA was isolated by using the HiPurA bacterial genomic DNA purification kit (Himedia, India).

Cloning and expression of recombinant genes.

The gene (gene 50) encoding the mycobacteriophage D29 class II ribonucleotide reductase enzyme (D29Gp50) (24) was amplified from D29 phage DNA and cloned in the mycobacterial expression vector, either pLAM12 or pMind, to generate pSG6 or pSG19, respectively (see Fig. S1 in the supplemental material). A mutation that alters the catalytically important cysteine residue, C375S (25), was introduced by site-directed mutagenesis (SDM) using a commercially available kit (Stratagene) to generate pSG6A (see Fig. S1 in the supplemental material). For the construction of an integrative vector corresponding to pSG6, a PciI-XbaI fragment encompassing the origin of replication (oriM) was deleted from the plasmid, and an integration cassette derived from mycobacteriophage L5 was inserted in its place (pSG6B) (see Fig. S2 and S3 in the supplemental material). The dinB2 gene of M. smegmatis (MSMEG_2294) (20) and D29 gene 48, which codes for D29Gp48, a thymidylate synthase (TS) (24), were amplified by using specific primers (see Tables S1 and S2 in the supplemental material) and cloned into the multiple-cloning site (MCS) of the pMind vector to generate pSG16 and pSG17, respectively (see Fig. S1 in the supplemental material). In order to construct a M. smegmatis strain in which only dinB2 is duplicated, a HindIII-NheI fragment spanning the dinB2 sequence in pSG16 was cloned into pSG6B to generate an integration vector, pSG18 (see Fig. S2 and S4 in the supplemental material). All the PCR-amplified gene inserts and mutations were confirmed by DNA sequencing using a 3130X genetic analyzer (Applied Biosystems).The recombinant vectors were introduced into electrocompetent mycobacterial cells by using standard protocols (26), and transformants were selected by using the appropriate antibiotics, either kanamycin or hygromycin. Integration into the host genome following transformation was confirmed by monitoring antibiotic resistance and also PCR using primers derived from the genomic and phage-derived DNA sequences.

For induction of gene expression in M. smegmatis with acetamide, cells were grown overnight to an optical density at 600 nm (OD₆₀₀) of 0.4 and then induced with 0.2% acetamide for 3 h (27). In the case of the tetracycline-inducible system, ATc was used at a concentration of 50 ng/ml (23) or as indicated. To prepare cell extracts, 20 ml of cell suspension was pelleted at 9,300 × g for 10 min, washed twice with Tris-sorbitol buffer, resuspended in 1 ml of phosphate-buffered saline (pH 7.5), and lysed by sonication. To confirm that the target genes were successfully expressed, Western blot analysis was performed with antisera raised in rabbits, essentially as reported previously (24).

Growth monitoring experiments.

The viability of mycobacterial cells was monitored by measuring the OD₆₀₀ of the culture medium spectrophotometrically or by counting the CFU after plating the cells onto MB7H9 agar. To monitor the effect of target gene overexpression on the growth of mycobacterial cells, a saturated culture (OD₆₀₀ of ∼3) was used to inoculate MB7H9 broth in such a way that the initial OD₆₀₀ became ∼0.02. The cells were grown overnight (∼16 h) in MB7H9 broth under shaking conditions, (200 rpm) to an OD₆₀₀ of ∼0.3. The culture thus obtained was divided into two equal parts, one of which was induced and the other of which was kept uninduced. The time point at which the inducer was added was considered time zero. In particular experiments where marker frequencies were compared (see below), the cultures that were growing exponentially were diluted from time to time with fresh medium to make certain that the saturation stage was never reached. The dilutions were performed by discarding a certain volume of the culture, usually one-fourth, and replenishing the culture with fresh medium. In some cases where the expression of dual genes was needed, the inducers were added one after the other, allowing a gap of 4 h in between, so that the product of the first gene reached the maximum level before the induction of the second gene. For the isolation of genomic DNA, 2-ml aliquots were recovered at the desired time points.

Determination of Ori/Ter ratio (marker frequency analysis).

For marker frequency analysis, a real-time PCR (RT-PCR)-based technique was used. For this technique, two sets of primers were designed (see Table S1 in the supplemental material), one amplifying the origin (Ori) region of the M. smegmatis genome (28) and the other amplifying the terminus (Ter). The cells were allowed to grow under a given set of conditions. Aliquots were removed at the desired time points, and DNA was extracted. RT-PCR was set up by using Power Sybr green PCR master mix (Applied Biosystems), 2 pmol of each primer, and 1 μl of the DNA sample. The reaction was done by using the 7500 Fast real-time PCR system (Applied Biosystems). The Ori/Ter ratio of DNA extracted from cells aliquoted at a particular time point was calculated from the difference in the threshold cycle (C_T) values obtained by using the respective probes. The arithmetic difference of 1 unit between the C_T(Ori) and C_T(Ter) values translates to an Ori/Ter ratio of 2 (2^ΔCT). The Ori/Ter ratio thus obtained was normalized to that corresponding to time zero. In some experiments involving the determination of Ori/Ter ratios, the cultures were pretreated with the antibiotic rifampin (60 μg/ml) (29).

Isolation and analysis of the nucleotide pool.

Growth of mycobacterial cells and induction of gene synthesis were done as mentioned above. Induction of the test gene was done for 6 h, after which aliquots of both uninduced and induced cells were poured into tubes containing formic acid at a final concentration of 1 M. The tubes were immediately frozen in liquid nitrogen and stored at −80°C. At the 6-h time point, the OD₆₀₀ was in the range of 0.5 to 0.8, corresponding to mid-log phase, in the cases of both uninduced and gene 50-induced cultures. The frozen cell samples were thawed at 37°C for 30 min and immediately placed on ice, with mild vortexing at intervals for ∼30 min. Thawed cells were centrifuged at 7,000 × g for 10 min, filtered, and diluted with high-performance liquid chromatography (HPLC)-grade water 20-fold. The filtrate was passed through a Q-Sepharose Fast Flow column (15-mm diameter) with a bed volume of 3 ml. The nucleotides were then eluted with 2 bed volumes of 1 M ammonium formate (30). The eluate was then dialyzed, using dialysis tubing with a 100-Da cutoff (Spectrum), against deionized water overnight. The dialyzed fractions were then frozen and lyophilized.

Nucleotide pool analysis was performed by using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. For analysis, a matrix solution comprising 45 mM AA, 45 mM NA, and 55 mM DAHC in 45% acetonitrile was prepared, and 1 μl of this solution was pipetted onto an Anchor Chip target plate (Bruker Daltonics, Germany) and dried. Subsequently, the lyophilized sample was resuspended in HPLC-grade water, 1 μl of which was pipetted onto the crystallized matrix. Analysis was performed by using an Autoflex II MALDI-TOF mass spectrometer (Bruker Daltonics, Germany), using the negative-reflection mode. Laser attenuation was set at 50 to 100 Hz, and 100 shots were used for each mass spectrum. The instrument was calibrated by using peptide calibration standard II (Bruker Daltonics, Germany). Prior to analysis of the test sample, commercially acquired dNTPs (Sigma), which were highly pure, were analyzed individually. The monoisotopic masses derived by using the commercial dNTP samples were used as reference standards to identify the peaks corresponding to dNTPs present in the experimental sample. Once identified, the peaks were annotated, and their heights (intensities) were determined. The peak height corresponding to each dNTP was considered to be a measure of its abundance. The experiments were performed in triplicate by inoculating three flasks in parallel to begin with. The heights obtained from the replicates were averaged and expressed as means ± standard deviations (SD).

RESULTS

Expression of gene 50 in mycobacteria and its effects.

Gene 50 of mycobacteriophage D29 and related phages encodes a class II ribonucleotide reductase (RNRII). In a previous study, this protein was biochemically characterized (24). In order to investigate its function, an attempt was made to express this gene in M. smegmatis strain ΔDRKIN, in which one copy of a 56-kb duplication found in M. smegmatis mc²155 is missing (20). As a result, it has a reduced dosage of genes associated with the 56-kb region. One such gene is nrdE, which codes for the host class I ribonucleotide reductase. Since this mutant has only one copy of nrdE, it is therefore highly sensitive to the well-known class I RNR inhibitor HU (31). To investigate whether the deficiency of RNR activity caused by the loss of one nrdE copy could be compensated for by expressing the mycobacteriophage D29-derived gene encoding Gp50, a plasmid (pSG6) that expresses this gene under the control of an acetamide-inducible promoter was introduced into the ΔDRKIN strain. A similar construct, pSG6A, from which a mutant version of the protein, Gp50(C375S), is synthesized, was also introduced separately into the same strain. The C375S mutation alters a crucial Cys residue (Fig. 1B) that is considered to be necessary for RNR activity (25). Induced expression of the wild-type and mutant alleles in either M. smegmatis mc²155, ΔDRKIN, or both was confirmed by Western blotting using antisera against D29Gp50 (Fig. 1A). In order to determine whether the expression of gene 50 has any effect on the growth rates of bacteria, the OD₆₀₀s of the cultures at different time points following induction of expression were determined. The results indicate that although induced expression of gene 50 did not have any effect on M. smegmatis mc²155 growth, it had an adverse effect in the case of ΔDRKIN (Fig. 1C and D, respectively). The observed phenomenon was further confirmed by performing viability assays (Fig. 1E and F, respectively). When the gene encoding the C375S mutant of D29Gp50 was expressed by using plasmid pSG6A, no significant difference in growth kinetics before and after induction was observed (Fig. 1G and H). This observation indicated that the inhibitory effect produced by D29Gp50 was specifically due to its ribonucleotide-reducing function. The same experiment was repeated with M. bovis BCG. In this slow-growing mycobacterium, the genes corresponding to those found within the 56-kb duplicated region of M. smegmatis mc²155 are all present in single copies. Overexpression from pSG6 (Fig. 1I) but not pSG6A (Fig. 1J) led to growth inhibition in M. bovis. The results thus obtained indicate that the induced expression of gene 50 is growth inhibitory in cells that have single copies corresponding to genes associated with the 56-kb duplication found in M. smegmatis mc²155.

FIG 1.

Effect of overexpression of D29 gene 50 in mycobacterial cells. (A) Western blot analysis to detect synthesis of D29Gp50 in the indicated M. smegmatis strains, either mc²155 or ΔDRKIN, which were transformed with plasmids, either pSG6 (for synthesis of D29Gp50) or pSG6A [for synthesis of the D29Gp50(C375S) mutant]. U and I represent uninduced and induced conditions, respectively. In lane C, a purified sample of E. coli expressing D29Gp50 (24) was loaded to serve as a control. (B) Clustal W alignment of D29Gp50, the class II ribonucleotide reductase from D29, and that derived from Lactobacillus leichmannii (Lle). Only a part of the alignment spanning a conserved motif (highlighted in red) within the active site is shown. The arrow points to a redox-active Cys residue (C375 in Gp50 and C408 in the L. leichmannii enzyme), which was changed to serine, giving rise to the Gp50(C375S) mutant. (C to H) Growth patterns of ΔDRKIN (D, F, and H) and mc²155 (C, E, and G) expressing the gene for either D29Gp50 or its mutant (C375S) from plasmid pSG6 or pSG6A, respectively, as indicated. Growth was monitored indirectly either by determining the optical density (OD₆₀₀) of the culture medium or by obtaining viable counts (CFU per milliliter), as indicated. (I and J) Similar experiments were performed by using M. bovis transformed with either pSG6 (I) or pSG6A (J). The color of the connecting lines denotes the expression status, with blue for uninduced and red for induced conditions. In panel E, the blue line is merged with the red line and therefore is invisible. Each data point represents the mean ± SD of data from three biological replicate experiments performed in parallel. In the figures presented here, the error bars are often not visible as they are too small compared to the symbols.

Marker frequency analysis.

Marker frequency analysis (32) was performed by determining the relative Ori/Ter ratios at each time point during the growth of M. smegmatis cells in the presence or absence of inhibitory conditions. The principle behind this analysis is that in an actively replicating chromosome, the markers located close to the origin replicate at a higher frequency than those that are located at a distance. If a circular chromosome is in the process of being replicated in the theta mode, the frequency at which a marker located close to the origin (Ori) is replicated should be at least twice (assuming the activation of a single fork) that of a marker which is located at the diametrically opposite end (Ter). If the chromosome is not being replicated, then the ratio will be 1. The experiment was performed by using the M. smegmatis ΔDRKIN::pSG6B strain, a recombinant which harbors an integrated copy of gene 50 in its genome, the expression of which can be induced by the addition of acetamide (see Fig. S2 and S3 in the supplemental material). The results indicated that the Ori/Ter ratio increased by 8-fold (Fig. 2B, blue trace) in cells that were growing in an uninhibited manner (uninduced cells) (Fig. 2A, blue trace). In cells in which growth had stopped (Fig. 2A, red trace), the Ori/Ter ratio did not increase with time (Fig. 2B, red trace). On the contrary, a minor dip was observed. For the sake of control, a similar experiment was repeated with M. smegmatis mc²155 cells in which growth inhibition was induced with rifampin, a well-known replication initiation inhibitor (29, 33), at a concentration of 60 μg/ml (Fig. 2C, red trace). The Ori/Ter ratio of rifampin-treated cells did not change over time (Fig. 2D, red trace), although in the case of the untreated ones, an increase of ∼8-fold was evident, as expected (blue trace).

FIG 2.

Comparison of Ori/Ter ratios for chromosomal DNA isolated from mycobacterial cells growing either normally (without any inhibition) (blue traces) or subjected to growth inhibition (red traces). (A and C) Growth inhibition was induced by either the overexpression of gene 50 (A) or the addition of rifampin at a 60-μg/ml concentration (C). (B and D) Corresponding Ori/Ter ratio profiles. The arrows in the growth profiles indicate the time points at which the cultures growing in an uninhibited manner (blue traces) were diluted to maintain the OD₆₀₀ at ∼0.7. Dilutions were done by discarding 5 ml from a 20-ml culture followed by replenishment with 5 ml of fresh medium. For estimating Ori/Ter ratios, cells were harvested at the specified time points (0, 3, 6, and 9 h), and DNA was extracted. Ori and Ter regions were PCR amplified by using specific primer pairs in real time, and the corresponding C_T values were determined. The difference between the C_T values was used to calculate the Ori/Ter ratios. The values thus obtained were further normalized with respect to the ones corresponding to time zero. Each data point represents the mean derived from three biological replicate experiments ± SD. In the case of the growth profile in panel A, the error bars are larger than those in panel C, as the individual assays were performed on different days.

The experiments performed here indicate that the Ori/Ter ratio reached a value of >2 in cells that were growing in an uninhibited manner. The observations thus obtained show that, unlike what has been claimed in several recent investigations (34–36), multifork replication occurs in M. smegmatis. Multifork replication is observed in bacteria in such cases where the mass doubling time is less than the sum of their chromosomal replication and cell division times, the C and D periods, respectively (37, 38). The C and D periods usually remain constant, but the mass doubling time tends to vary with the culture conditions. If the cells grow faster due to better culture conditions, the mass doubling time will become shorter than the (C + D) period. The differences in the observations made in this study and those that were reported in previous single-cell-based investigations (34–36) could be due to differences in culture conditions and/or experimental strategies used. In the case of single-cell experiments, growth is achieved on microscopic slides, within specialized chambers, whereas here the more traditional method of growing cells in culture flasks and under highly aerated conditions was used. Overall, the results indicate that whereas multiple forks formed in cells growing normally, none developed in cells that had stopped growing due to either the induction of gene 50 expression or, for that matter, treatment with rifampin.

Reversal of Gp50-induced growth inhibition by coexpression of D29 gene 48.

The results presented above indicate that the overexpression of mycobacteriophage D29 gene 50 in the ΔDRKIN strain led to growth inhibition. To understand the mechanism by which growth inhibition occurs, it was noted that ribonucleotide reductases can produce only three of the four deoxyribonucleotides, dATP, dCTP, and dGTP, necessary for DNA synthesis, while the fourth, dTTP, is synthesized by the action of thymidylate synthase (TS). Hence, if only ribonucleotide reductase is overproduced, the level of dTTP may become limiting. In various investigations, it has been shown that nucleotide pool imbalances could lead to genotoxicity and, eventually, cell death (39). A dTTP-deficient state may also be considered as one of nucleotide imbalance, and therefore, in such a situation, either growth inhibition or cell death can happen. Moreover, dTTP deficiency could specifically lead to what is known as “thymineless death” (TLD), which has been studied extensively in various organisms (40). The genomes of D29 and related phages possess genes encoding not only RNR (Gp50) but also TS (D29Gp48) (24). It was therefore hypothesized that if D29Gp48 is synthesized within the cell along with Gp50, the disparity in the dTTP level relative to the other dNTPs would be corrected, resulting in uninhibited cell growth. To test this possibility, gene 48 was cloned in the extrachromosomal tetracycline-inducible plasmid vector pMind (23) to create the recombinant vector pSG17 (see Fig. S1 in the supplemental material), which was then introduced into the M. smegmatis mc²155::pSG6B and ΔDRKIN::pSG6B integrants, both of which express an acetamide-inducible, genomically integrated copy of gene 50 (see Fig. S2 and S3 in the supplemental material). The results indicate that, as expected, overexpression of gene 50 resulted in growth inhibition in ΔDRKIN::pSG6B (Fig. 3A, compare red trace and blue trace) but not in mc²155::pSG6B (Fig. 3C). However, the retarding effect observed in ΔDRKIN::pSG6B could be reversed by the addition of ATc, which induces the expression of gene 48 from pSG17 (Fig. 3B). Hence, it appears that growth retardation induced in ΔDRKIN cells overexpressing gene 50 is due primarily to a decrease of the dTTP level in these cells relative to the levels of the other dNTPs.

FIG 3.

Effect of D29 gene 48 expression on survival of M. smegmatis strains overexpressing gene 50. M. smegmatis ΔDRKIN::pSG6B and mc²155::pSG6B integrants were further transformed with the extrachromosomal D29 gene 48-expressing vector pSG17. Expression of gene 48 in transformed cells was induced by using ATc (50 ng/ml) for 4 h prior to the induction of gene 50 expression using acetamide. Growth was monitored spectrophotometrically for both acetamide-treated (red traces) and untreated (blue traces) cells. Each data point in these experiments represents the mean of data from three biological replicates performed in parallel ± SD.

Evidence for dTTP deficiency in cells overexpressing gene 50.

In order to confirm that the alteration of the dTTP level is the reason behind the growth-inhibitory phenotype mentioned above, the nucleotide pool was isolated from ΔDRKIN cells expressing gene 50 (ΔDRKIN::pSG6B cells) and examined by using MALDI-TOF mass spectrometry. The results show that in the case of uninduced cells, which were growing exponentially, barring dCTP, the peak heights of the remaining dNTPs were comparable (Fig. 4A [representative profile] and B [graphical representation of the mean data from three experiments]). The peak height corresponding to dCTP was found to be consistently shorter than those of the other dNTPs in all the determinations. It could be that dCTP is indeed less abundant in mycobacterial cells. Alternatively, the observed lower intensity may be due to some limitation of the analytical methods used here. Following gene 50 overexpression, it was observed that the relative levels of all the dNTPs changed to some extent (Fig. 4A [pSG6B-induced compared to uninduced conditions], and compare Fig. 4B and C). However, the maximum change was observed in the case of dTTP, the level of which was reduced to such an extent that it was undetectable. The dTTP-deficient state could nevertheless be reversed by prior expression of the TS gene from plasmid pSG17 (Fig. 4A [pSG17 induced], and compare Fig. 4C and D). Thus, the results confirm that the root cause of growth inhibition was the drastic reduction of the dTTP level in the affected cells, as proposed above. The MALDI-TOF profile (Fig. 4A) is a representative one. These experiments were repeated at least three times, if not more. Since the dNTP profile for the gene 48 expression set (pSG17 induced) is of crucial importance, at least insofar as this study is concerned, the raw data corresponding to each of the three replicates used to calculate the means in Fig. 4D are therefore provided in the supplemental material (see Fig. S5 in the supplemental material).

FIG 4.

Effect of D29 gene 50 expression on the nucleotide pool balance in M. smegmatis cells. (A) MALDI-TOF analysis profiles of the nucleotide pool isolated from ΔDRKIN::pSG6B integrant cells transformed with pSG17, the gene 48-expressing plasmid (ΔDRKIN::pSG6B/pSG17). The cells were either uninduced (top) or induced with acetamide for gene 50 expression (middle) or with ATc for gene 48 expression prior to the addition of acetamide (bottom). (B to D) Intensities of dCTP (m/z 466), dTTP (m/z 481), dATP (m/z 490), and dGTP (m/z 506) isolated from uninduced ΔDRKIN::pSG6B/pSG17 cells (B), ΔDRKIN::pSG6B/pSG17 cells induced for gene 50 expression (C), and ΔDRKIN::pSG6B/pSG17 cells induced for both gene 48 and gene 50 expression (D). The y axis values represent intensities in arbitrary units. For the sake of clarity, the y axis scales in panel A were redrawn and positioned alongside the profiles. Each data point in these experiments represents the mean of data from three biological replicates performed in parallel ± SD. The y axis scales in panels B to D have been split into two segments to highlight the intensities of both highly as well as lowly abundant species in the same graph. The three individual profiles for panel D are given in the supplemental material (see Fig. S5 in the supplemental material).

Gp50-induced lethality is determined by dinB2 copy number.

The inhibitory effect of gene 50 overexpression was selectively observed in strain ΔDRKIN but not in mc²155. ΔDRKIN is known to possess only one copy of a 56-kb region that is duplicated in mc²155. We therefore hypothesized that a reduced copy number of genes associated with this segment could be responsible for the observed phenotype in ΔDRKIN. Since a large number of genes are present in this region (20), a knowledge-based approach was taken to identify the possible candidate(s). One of the genes present in the region is dinB2, which encodes the Y family polymerase DinB2. Considering previous reports (19) that the Y family polymerase UmuC can help in overcoming DNA replication defects, we hypothesized that a reduction of the DinB2 level in ΔDRKIN, caused by a copy number deficiency of its gene (dinB2), could be the determining factor. To test this possibility, the ΔDRKIN strain was modified to include a second chromosomal copy of dinB2 (Fig. 5A). The introduction of the second copy was done by using a bacteriophage L5-based integrative vector, pSG18 (see Fig. S2 and S4 in the supplemental material). The resulting integrant, ΔDRKIN::pSG18, which has two chromosomally carried copies of dinB2 (dinB2⁺/dinB2⁺), and also ΔDRKIN, in which one copy is missing (dinB2 negative/dinB2⁺), as well as mc²155, which has both copies (dinB2⁺/dinB2⁺), were transformed with pSG19 (see Fig. S1 in the supplemental material), a pMind-derived vector expressing gene 50 under the control of a tetracycline-inducible promoter. Following the induction of expression of gene 50, only the growth of the ΔDRKIN strain (Fig. 5C), and not the growth of ΔDRKIN::pSG18 (Fig. 5D) or mc²155 (Fig. 5B), was found to be adversely affected. The results confirm our hypothesis that the decreased dinB2 copy number is the sole reason why ΔDRKIN is highly susceptible to the lethal effects of gene 50 expression.

FIG 5.

Effect of dinB2 copy number duplication on survival of ΔDRKIN cells expressing gene 50. (A) Schematic diagram of the chromosome (thick line) of the ΔDRKIN::pSG18 integrant showing the locations of the two copies of dinB2, one in the 56-kb region (unshaded box on the right) and the other within the integrated copy of plasmid pSG18 (thin line bordered by hatched boxes representing the mycobacteriophage L5 chromosomal left and right attachment sites, attL and attR, respectively). The relative positions of the open reading frames present in the plasmid are shown. (B to D) Growth of pSG19-transformed mc²155 (B), ΔDRKIN (C), and ΔDRKIN::pSG18 (D) cells in the presence (red) or absence (blue) of ATc, the inducer of gene 50 expression from pSG19. Genetically, these strains have either two copies of dinB2 (dinB2⁺/dinB2⁺) or just one copy (dinB2 negative/dinB2⁺). Each data point in these experiments represents the mean of results from three biological replicates performed in parallel ± SD. The error bars in most cases are not visible, as they are too small compared to the symbol.

Ectopic expression of dinB2 reverses gene 50-induced lethality and restores Ori/Ter ratios.

To further test whether DinB2 has any role to play in the differential susceptibilities of the mutant and the parent strains, its gene was cloned in the extrachromosomal tetracycline-inducible plasmid vector pMind (23) to create the recombinant plasmid pSG16 (see Fig. S1 in the supplemental material), which was then introduced into the M. smegmatis mc²155::pSG6B and ΔDRKIN::pSG6B integrants, both of which carry an integrated copy of gene 50 in their chromosomes. Survivability under various inducible conditions was then determined. The results show that following acetamide-induced expression of gene 50, growth of ΔDRKIN::pSG6B came to a halt (Fig. 6C, compare red and blue traces), whereas the corresponding mc²155 construct continued to grow normally (Fig. 6A). Prior induction of DinB2 synthesis from pSG16 by the addition of ATc, however, allowed acetamide-treated ΔDRKIN::pSG6B cells to survive (Fig. 6D). Determination of the Ori/Ter ratio of DNA samples isolated from the gene 50 integrants transformed with pSG16, the dinB2-expressing plasmid, indicated that in the absence of any form of induction, the ratio increases in a time-dependent manner, as expected (Fig. 6E). Following the induction of gene 50 expression from the ΔDRKIN::pSG6B integrant using acetamide, no increase, rather a decrease, in the Ori/Ter ratio was observed (Fig. 6F). The addition of the inducer (ATc) for dinB2 to ΔDRKIN::pSG6B cells overexpressing gene 50 had no effect (mock experiment) on the Ori/Ter ratio, as the corresponding expression plasmid (pSG16) was not present in these cells (Fig. 6F). The Ori/Ter ratio of ΔDRKIN::pSG6B integrants, harboring dinB2-expressing plasmid pSG16, decreased following the selective induction of gene 50 by using acetamide (Fig. 6G). However, the decline in the ratio could be reversed by introducing ATc, a dinB2-specific inducer (compare Fig. 6H to G and F). The results indicate that the adverse effects of gene 50 overexpression on growth as well as the Ori/Ter ratio can be reversed by increasing the level of DinB2 in the affected cells. The finding that the DinB2-mediated reversal of the lethal phenotypes mentioned above is not due to an elevation of dTTP levels through some indirect mechanism was verified. It was found that cells overexpressing dinB2 in addition to gene 50 were as deficient of dTTP as those overexpressing gene 50 alone (see Fig. S6 in the supplemental material).

FIG 6.

Effect of dinB2 expression on growth of M. smegmatis expressing gene 50. (A to D) M. smegmatis mc²155::pSG6B (A and B) and ΔDRKIN::pSG6B (C and D) integrants were transformed with the extrachromosomal dinB2-expressing vector pSG16. The transformed cells were either treated with ATc for dinB2 overexpression (B and D) or left untreated (A and C). Both types of cells were then treated with acetamide to induce gene 50 expression. Growth in each of the cases was monitored spectrophotometrically by determining the OD₆₀₀. Each data point in these experiments represents the mean of results from three biological replicates performed in parallel ± SD. (E to H) Ori/Ter ratios of DNA isolated from ΔDRKIN::pSG6B cells harboring either no plasmid (F) or pSG16 (E, G, and H). The cells were either uninduced (E), induced for gene 50 expression (F and G), or induced for the expression of both gene 50 and dinB2 (H). Induction of gene 50 and dinB2 expression was done by using acetamide (Ace) and ATc, respectively. The uninduced state is shown as −, and the induced state is shown as +.

DISCUSSION

In the present study, we report the growth-inhibitory consequences of the expression of a mycobacteriophage D29-derived gene (gene 50) encoding a class II RNR in a particular strain of M. smegmatis known as ΔDRKIN. In this strain, there is only one copy of a 56-kb region, compared to two copies found in M. smegmatis mc²155, the strain from which it was derived. The growth-inhibitory effect is apparently due to alterations in the nucleotide levels that resulted following the induced expression of gene 50. In the altered scenario, the relative level of dATP was found to be increased, possibly due to the enhanced reduction of ATP to dATP by D29Gp50. However, the levels of dCTP and dGTP were found to be decreased, although they were still in the detectable range. The most-affected dNTP was dTTP, the level of which decreased to such an extent that it could not be detected. Coexpression of a gene encoding a TS, along with gene 50, resulted in the reversal of growth inhibition, confirming that a deficiency of dTTP is the primary reason for cell death under the given conditions. Ostensibly, the phenomenon appears to be related to thymineless death (TLD) (40), which has been studied extensively in E. coli (41) and Bacillus subtilis (42). TLD is a globally important issue, as it can be exploited for the development of antimicrobial as well as anticancer drugs (43).

It has been demonstrated using marker frequency determination experiments that gene 50 overexpression in ΔDRKIN cells leads to the cessation of DNA replication. A similar effect could be produced by treatment of cells with rifampin, a transcriptional inhibitor that can inhibit replication by preventing initiation (29, 33, 44). Given the similarity in the phenotypes observed between gene 50 expression and rifampin treatment, it is tempting to speculate that under dTTP-deficient conditions such as those described here, replication initiation in mycobacteria is inhibited. However, if dTTP is limiting, the elongation step will also be inhibited. Thus, in those copies of the genome in which replication initiation has already been initiated, no further replication fork progression is expected to take place following the expression of gene 50.

To the best of our knowledge, this is the first attempt to use marker frequency analysis to investigate DNA replication in mycobacteria. The markers chosen were a DNA sequence derived from the origin region of M. smegmatis (28), the proximal marker, and another from the diametrically opposite end, the distal marker. The proximal marker was designated Ori and the distal marker was designated Ter based on the Ori/Ter nomenclature used in the case of E. coli (45). In the case of mycobacteria, termination of replication is not known in detail, and therefore, the term Ter is used loosely to describe a site where the bidirectional forks possibly meet.

The experiments performed here indicate that the Ori/Ter ratio more than doubled (multifork) in cells that were growing in an uninhibited manner. The increase in Ori/Ter ratios in actively growing cells is a novel finding. This finding indicates that under conditions such as those mentioned here, multiple forks develop during mycobacterial DNA replication.

The results of the marker frequency analysis also show that unlike actively growing cells, the Ori/Ter ratio of growth-inhibited cells remains constant. Our initial expectation was that in retarded cells, Ori/Ter ratios should be higher, as was observed for “dGTP-less” death (45) in E. coli, a phenomenon that may have similarities with TLD. TLD and other related phenomena are very complex, and the exact mechanisms behind them are not fully understood. However, one consensus that has emerged is that under TLD-inducing conditions, origin sequences are lost, and as a result, marker frequency at the Ori region becomes apparently the same as that at Ter (40, 46, 47). The constancy of the Ori/Ter ratio in growth-retarded cells observed here could be due to either a lack of replication initiation or deletion of origin sequences resulting from aberrant initiation. A more detailed high-resolution marker frequency analysis may have to be done to come to a specific conclusion. Nevertheless, considering that the dTTP level was found to be low in the affected cells and that the region where replication initiates is A/T rich (28), it is perhaps not difficult to conclude that dTTP deficiency leads to some kind of a defect in origin function. The finding that the expression of gene 48, the TS-encoding gene, leads to recovery is an additional indication that such a hypothesis could be correct.

An important question may arise as to why ΔDRKIN, which retains one copy of dinB2, is unable to resist the toxic effects of gene 50 expression. Is gene dosage the only discriminating factor? The precise answer to this question is not known as of now. However, it may be noted that the dosage of genes associated with the 56-kb region that is duplicated in M. smegmatis mc²155 appears to have an important role to play in deciding the phenotypes of this organism. For example, as mentioned in Results, it has been demonstrated that a lower gene dosage of another 56-kb region-associated gene, nrdE, encoding a ribonucleotide reductase type I subunit, leads to increased susceptibility of the ΔDRKIN strain to the clastogen hydroxyurea (HU) (31). Therefore, the copy numbers of not only dinB2 but also the other genes present in this locus appear to have a role to play in determining the fate of M. smegmatis mc²155 under circumstances where their products are necessary. Why the single copies of the genes, be it dinB2 or nrdE, present in the ΔDRKIN strain are incapable of giving protection when needed is also an issue that needs to be addressed. The results presented in this study suggest that it is the copy number alone that is important. However, it is possible that in a duplicated context, the genes are expressed more efficiently for some unknown reasons.

How the higher level of DinB2 influences the growth properties of ΔDRKIN cells is another important issue. Recent biochemical studies have indicated that DinB2, but not the other two mycobacterial DinBs, has the ability to scavenge dNTPs and also misincorporate them (10, 12) under limiting conditions. Moreover, DinB2 has low sugar selectivity and thus appears to be naturally capable of incorporating ribonucleotides during templated DNA synthesis (10). The observations made in this study may be explained by considering that under dTTP-limiting conditions created by the overexpression of gene 50, synthesis of DNA at the origin is hampered (40), which results in the formation of single-stranded gaps. The creation of such gaps could trigger the subsequent generation of lethal double-stranded DNA breaks. DinB2 can help in rectifying the situation by ensuring that such gaps are “patched up” through the incorporation of the ribonucleotide UTP (12). The “ribopatches” may be subsequently removed through ribonucleotide excision repair (RER) mechanisms, which have been characterized in E. coli (48, 49) but, to the best of our knowledge, not in mycobacteria.

The ability of a Pol IV, DinB2 being one, to execute TLS depends on its ability to compete with the replicative DNA Pol III in a concentration-dependent manner (50). At low concentrations, both polymerases remain associated with the β clamp in an arrangement that has been compared to a “tool belt.” Such an arrangement allows efficient polymerase “switching” from Pol III to Pol IV and back. At higher concentrations, such as those observed following the induction of the SOS pathway, Pol IV displaces Pol III from the clamp and takes over the role of synthesizing DNA in a templated manner. The observation that the lethal effect of gene 50 expression in ΔDRKIN cells can be overcome only in cells having a relatively high concentration of DinB2 suggests the involvement of the second mechanism, although the first may also be operative. By displacing Pol III and/or taking over its functions, at least to a limited extent, DinB2 may aid the survival of cells under dTTP-limiting conditions.

The results presented here indicate that DinB2 may have a role to play in the resistance of mycobacterial cells to drugs, particularly those that damage DNA. Moreover, the gene 50 expression-dependent growth inhibition phenomenon reported here could serve as a surrogate TLD model for mycobacteria, which could be used to investigate how thymine deficiency affects mycobacterial growth. Effective drugs that reduce dTTP levels in mycobacteria can then be developed as antimycobacterial agents.