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

Shreya Ghosh, Sourabh Samaddar, Prithwiraj Kirtania, Sujoy K. Das Gupta

Bose Institute, Department of Microbiology, Kolkata, West Bengal, India

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. smeg-matis mc2155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes forDinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class IIribonucleotide reductase in M. smegmatis �DRKIN, 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 thatresulted under these circumstances. The selective inhibition observed in the �DRKIN strain was found to be due solely to a re-duced 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 ofdinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with theeffect 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 poly-merases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this proteinhas been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo functionremains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid myco-bacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymeraseDinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing adTTP-deficient state.

Members of the Y family DNA polymerases are capable oftranslesion synthesis (TLS) that allows them to catalyze the

insertion of deoxyribonucleotide triphosphates (dNTPs) opposite po-tentially lethal replication-blocking lesions (1). The ability of thesepolymerases to bypass such lesions helps the cell to survive underDNA-damaging conditions. However, survival comes at a cost.Mutations are introduced more frequently than under normalconditions, as these polymerases function in an error-prone man-ner (2). In Escherichia coli, DinP/DinB (DNA polymerase IV [PolIV]) and UmuC (DNA Pol V) are the two Y family polymerasesthat mediate TLS (2). Mutations in the genes that encode UmuCand a related protein, UmuD, result in a UV-nonmutable (umu)phenotype. In contrast, mutation of dinB, the gene that codes forthe Pol IV enzyme DinB, does not lead to an apparent phenotype,and therefore, the function of this protein remains enigmatic. Theexpression of the E. coli dinB gene, also known as dinP (3), can beinduced through the SOS pathway. Additionally, it can also beinduced in response to general stress through the involvement ofthe alternative � factor �s (RpoS) (4, 5). The increased expressionof dinB (dinP) results in a hypermutable phenotype (3). Accumu-lation of mutations under stress conditions can be beneficial, asthe resulting variants may be more fit to survive under such con-ditions than their predecessors (1, 6). DinB, however, may haveother roles to play that are not directly related to error-pronerepair. It has been found that overexpression of dinB inhibits fork

progression and is lethal (7, 8). Thus, it has been proposed thatDinB possibly acts as a brake for DNA Pol III, thereby slowingdown fork progression (7). Under stress conditions, slowingdown of fork progression may protect E. coli from genome insta-bility. In Mycobacterium tuberculosis, two DinB orthologs arefound, DinB1 (Rv1537) and DinB2 (Rv3056) (9). The corre-sponding E. coli counterparts are DinX and DinB/DinP, respec-tively. In Mycobacterium smegmatis, the MSMEG_3172 andMSMEG_2294 genes encode the DinB1 and DinB2 orthologs, re-spectively. A third ortholog of DinB has been found in M. smeg-matis (DinB3 [MSMEG_6443]), which appears to be more relatedto DinB1 than to DinB2 (10). The M. smegmatis protein encoded

Received 13 August 2015 Accepted 24 October 2015

Accepted manuscript posted online 2 November 2015

Citation Ghosh S, Samaddar S, Kirtania P, Das Gupta SK. 2016. A DinB orthologenables mycobacterial growth under dTTP-limiting conditions induced by theexpression of a mycobacteriophage-derived ribonucleotide reductase gene.J Bacteriol 198:352–362. doi:10.1128/JB.00669-15.

Editor: R. L. Gourse

Address correspondence to Sujoy K. Das Gupta, sujoy@jcbose.ac.in.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00669-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

crossmark

352 jb.asm.org January 2016 Volume 198 Number 2Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

by MSMEG_2294, henceforth referred to only as DinB2, has beencharacterized biochemically, and it has been demonstrated that itcan function as an “unfaithful” DNA polymerase (10–12). How-ever, the in vivo role of DinB2 is unclear, as deletion of the genesencoding DinB2 orthologs did not affect either the spontaneous(9) or DNA damage-induced (13) mutation rate in M. tuberculo-sis. Thus, the M. tuberculosis DinB2 homologs do not appear to beinvolved in error-prone repair, although they are biochemicallycapable of doing so. Error-prone DNA repair was found to bemediated by DnaE2 and not DinB2 in M. tuberculosis (13). Thus,as far as mycobacterial DinB2 is concerned, very little is knownabout how it functions, even though it is expressed at a higher levelthan either DinB1 or DnaE2 (9). The interesting part is that al-though the dinB2 genes are known to be DNA damage inducible inother organisms, they were not found to be so in mycobacteria(14). The general impression derived from research done on my-cobacterial DinB polymerases is that they do not behave like theircounterparts from E. coli or other organisms (9).

The Y family polymerases UmuC and DinB have the intrinsicability to incorporate ribonucleotides into the DNA chain in atemplated manner. However, this ability is controlled by a singleamino acid residue, which is referred to as the “steric gate.” Inwild-type versions of these proteins, the steric gate amino acidresidues are bulky in nature, a result of which is that ribonucle-otides become excluded (15, 16). However, if these bulky residuesare replaced by smaller ones, sugar selectivity becomes relaxed,and the mutants develop the capability of incorporating ribo-nucleotides into DNA efficiently. In the case of DinB of E. coli, ithas been found that the steric gate has a role to play in controllingthe ability of the enzyme to execute TLS over N2-deoxyguanosineadducts (17). Interestingly, in DinB2 of mycobacteria, the stericgate amino acid is a leucine residue (Leu14) and not the bulkyaromatic amino acid phenylalanine (Phe13) that is found in its E.coli counterpart. Therefore, mycobacterial DinB2 is sugar unse-lective, and hence, it is naturally capable of catalyzing the incor-poration of ribonucleotides into DNA (10, 12). Whether thisproperty of DinB2 is beneficial to mycobacteria under special cir-cumstances is an interesting issue that remains to be explored.

One of the consequences of DNA damage is that fork move-ment comes to a halt, as the DNA polymerases, particularly theaccurate ones, cannot replicate past the lesions. Fork movementcan be stalled due to not only DNA damage but also deoxyribo-nucleotide pool imbalances, such as those caused by treatmentwith 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 stalledforks induced by HU (19). Whether the wild-type counterpart canperform the same function is not clear, as deletion of the geneencoding the wild-type protein had no effect. In E. coli, the Pol IVclass DNA polymerase DinB by itself did not play any significantrole 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 possessUmuC, and hence, how these bacteria cope with stalled forks re-mains unresolved.

M. smegmatis strain �DRKIN is a mutant (20) derived frommc2155, a strain used widely to study mycobacterial molecularbiology. The parental strain mc2155 harbors a 56-kb chromo-somal duplication. One copy of this duplicated region is missing

in �DRKIN. All the genes, including dinB2, that are associatedwith the 56-kb region are thus present as two copies in mc2155 butas one copy in �DRKIN. While investigating the function of aclass II ribonucleotide reductase encoded by mycobacteriophageD29 (D29Gp50), it was observed that overexpression of the geneencoding it in �DRKIN resulted in dTTP-limiting conditions,which in turn led to growth arrest. However, the arrested statecould be overcome by increasing the level of DinB2. The resultsindicate that this DNA polymerase can bring about DNA replica-tion in vivo under dTTP-limiting conditions. Thus, for the firsttime, a role has been found for the enigmatic mycobacterial Yfamily DNA polymerase DinB2.

MATERIALS AND METHODSBacterial strains, bacteriophages, and plasmids. E. coli strain XL1-Bluewas used for basic cloning and expression purposes. M. smegmatis strainsmc2155 and �DRKIN (20) were used for expression of genes in mycobac-teria. Mycobacterium bovis BCG was used as a representative of slow-growing mycobacteria. The BCG strain and not the wild type was used soas to minimize biohazards. Mycobacteriophage D29 (21) was obtained asa gift from Ruth McNerney. Overexpression in E. coli XL1-Blue cells wasdone by using the pQE30 vector system (Qiagen). Mycobacterial expres-sion was performed by using either the acetamide-inducible vectorpLAM12 (22) or the tetracycline-inducible vector pMind (23).

Chemicals and reagents. Restriction endonucleases/DNA-modifyingenzymes were obtained from Thermo Scientific or New England BioLabs.Luria-Bertani (LB) broth and Middlebrook 7H9 (MB7H9) broth wereobtained from Himedia and Difco (BD), respectively. Acetamide was pur-chased from Merck, and anhydrotetracycline (ATc) was purchased fromClontech. Anthranilic acid (AA), nicotinic acid (NA), and diammoniumhydrogen citrate (DAHC) were obtained from Fluka (Switzerland). Allother chemicals/reagents for protein purification and analysis were of thehighest 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 inMB7H9 broth supplemented with 0.25% bovine serum albumin (BSA),0.2% glycerol, and 0.05% Tween 80 at 37°C. The antibiotic used was eitherkanamycin (20 or 50 �g/ml), hygromycin (50 or 200 �g/ml), or both ifneeded. Phage was grown to confluence and then extracted by diffusioninto phage dilution buffer (22). After concentration by ultracentrifuga-tion at 1,00,000 � g for 2 h, phage was finally suspended in a minimumamount of phage dilution buffer. Phage DNA was isolated by using aQiagen lambda phage DNA isolation kit. Bacterial genomic DNA wasisolated by using the HiPurA bacterial genomic DNA purification kit (Hi-media, India).

Cloning and expression of recombinant genes. The gene (gene 50)encoding the mycobacteriophage D29 class II ribonucleotide reductaseenzyme (D29Gp50) (24) was amplified from D29 phage DNA and clonedin the mycobacterial expression vector, either pLAM12 or pMind, to gen-erate pSG6 or pSG19, respectively (see Fig. S1 in the supplemental mate-rial). A mutation that alters the catalytically important cysteine residue,C375S (25), was introduced by site-directed mutagenesis (SDM) using acommercially available kit (Stratagene) to generate pSG6A (see Fig. S1 inthe supplemental material). For the construction of an integrative vectorcorresponding to pSG6, a PciI-XbaI fragment encompassing the origin ofreplication (oriM) was deleted from the plasmid, and an integration cas-sette derived from mycobacteriophage L5 was inserted in its place(pSG6B) (see Fig. S2 and S3 in the supplemental material). The dinB2 geneof M. smegmatis (MSMEG_2294) (20) and D29 gene 48, which codes forD29Gp48, a thymidylate synthase (TS) (24), were amplified by using spe-cific primers (see Tables S1 and S2 in the supplemental material) andcloned into the multiple-cloning site (MCS) of the pMind vector to gen-erate pSG16 and pSG17, respectively (see Fig. S1 in the supplementalmaterial). In order to construct a M. smegmatis strain in which only dinB2

DNA Repair by Mycobacterial DinB2

January 2016 Volume 198 Number 2 jb.asm.org 353Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

is duplicated, a HindIII-NheI fragment spanning the dinB2 sequence inpSG16 was cloned into pSG6B to generate an integration vector, pSG18(see Fig. S2 and S4 in the supplemental material). All the PCR-amplifiedgene inserts and mutations were confirmed by DNA sequencing using a3130X genetic analyzer (Applied Biosystems).The recombinant vectorswere introduced into electrocompetent mycobacterial cells by using stan-dard protocols (26), and transformants were selected by using the appro-priate antibiotics, either kanamycin or hygromycin. Integration into thehost genome following transformation was confirmed by monitoring an-tibiotic resistance and also PCR using primers derived from the genomicand phage-derived DNA sequences.

For induction of gene expression in M. smegmatis with acetamide, cellswere grown overnight to an optical density at 600 nm (OD600) of 0.4 andthen induced with 0.2% acetamide for 3 h (27). In the case of the tetracy-cline-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 waspelleted 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 bysonication. To confirm that the target genes were successfully expressed,Western blot analysis was performed with antisera raised in rabbits, es-sentially as reported previously (24).

Growth monitoring experiments. The viability of mycobacterial cellswas monitored by measuring the OD600 of the culture medium spectro-photometrically or by counting the CFU after plating the cells ontoMB7H9 agar. To monitor the effect of target gene overexpression on thegrowth of mycobacterial cells, a saturated culture (OD600 of �3) was usedto inoculate MB7H9 broth in such a way that the initial OD600 became�0.02. The cells were grown overnight (�16 h) in MB7H9 broth undershaking conditions, (200 rpm) to an OD600 of �0.3. The culture thusobtained was divided into two equal parts, one of which was induced andthe other of which was kept uninduced. The time point at which theinducer was added was considered time zero. In particular experimentswhere marker frequencies were compared (see below), the cultures thatwere growing exponentially were diluted from time to time with freshmedium to make certain that the saturation stage was never reached. Thedilutions were performed by discarding a certain volume of the culture,usually one-fourth, and replenishing the culture with fresh medium. Insome cases where the expression of dual genes was needed, the inducerswere added one after the other, allowing a gap of 4 h in between, so that theproduct of the first gene reached the maximum level before the inductionof the second gene. For the isolation of genomic DNA, 2-ml aliquots wererecovered at the desired time points.

Determination of Ori/Ter ratio (marker frequency analysis). Formarker frequency analysis, a real-time PCR (RT-PCR)-based techniquewas used. For this technique, two sets of primers were designed (see TableS1 in the supplemental material), one amplifying the origin (Ori) regionof 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 ex-tracted. RT-PCR was set up by using Power Sybr green PCR master mix(Applied Biosystems), 2 pmol of each primer, and 1 �l of the DNA sample.The reaction was done by using the 7500 Fast real-time PCR system (Ap-plied Biosystems). The Ori/Ter ratio of DNA extracted from cells ali-quoted at a particular time point was calculated from the difference in thethreshold cycle (CT) values obtained by using the respective probes. Thearithmetic difference of 1 unit between the CT(Ori) and CT(Ter) valuestranslates to an Ori/Ter ratio of 2 (2�CT). The Ori/Ter ratio thus obtainedwas normalized to that corresponding to time zero. In some experimentsinvolving the determination of Ori/Ter ratios, the cultures were pre-treated with the antibiotic rifampin (60 �g/ml) (29).

Isolation and analysis of the nucleotide pool. Growth of mycobacte-rial 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 bothuninduced and induced cells were poured into tubes containing formicacid 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 wasin the range of 0.5 to 0.8, corresponding to mid-log phase, in the cases ofboth uninduced and gene 50-induced cultures. The frozen cell sampleswere thawed at 37°C for 30 min and immediately placed on ice, with mildvortexing at intervals for �30 min. Thawed cells were centrifuged at7,000 � g for 10 min, filtered, and diluted with high-performance liquidchromatography (HPLC)-grade water 20-fold. The filtrate was passedthrough a Q-Sepharose Fast Flow column (15-mm diameter) with a bedvolume of 3 ml. The nucleotides were then eluted with 2 bed volumes of 1M ammonium formate (30). The eluate was then dialyzed, using dialysistubing with a 100-Da cutoff (Spectrum), against deionized water over-night. The dialyzed fractions were then frozen and lyophilized.

Nucleotide pool analysis was performed by using matrix-assisted laserdesorption ionization–time of flight (MALDI-TOF) mass spectrometry.For analysis, a matrix solution comprising 45 mM AA, 45 mM NA, and 55mM DAHC in 45% acetonitrile was prepared, and 1 �l of this solution waspipetted onto an Anchor Chip target plate (Bruker Daltonics, Germany)and dried. Subsequently, the lyophilized sample was resuspended inHPLC-grade water, 1 �l of which was pipetted onto the crystallized ma-trix. Analysis was performed by using an Autoflex II MALDI-TOF massspectrometer (Bruker Daltonics, Germany), using the negative-reflectionmode. Laser attenuation was set at 50 to 100 Hz, and 100 shots were usedfor each mass spectrum. The instrument was calibrated by using peptidecalibration standard II (Bruker Daltonics, Germany). Prior to analysis ofthe test sample, commercially acquired dNTPs (Sigma), which werehighly pure, were analyzed individually. The monoisotopic masses de-rived by using the commercial dNTP samples were used as reference stan-dards to identify the peaks corresponding to dNTPs present in the exper-imental sample. Once identified, the peaks were annotated, and theirheights (intensities) were determined. The peak height corresponding toeach dNTP was considered to be a measure of its abundance. The exper-iments were performed in triplicate by inoculating three flasks in parallelto begin with. The heights obtained from the replicates were averaged andexpressed as means � standard deviations (SD).

RESULTSExpression of gene 50 in mycobacteria and its effects. Gene 50 ofmycobacteriophage D29 and related phages encodes a class II ri-bonucleotide reductase (RNRII). In a previous study, this proteinwas biochemically characterized (24). In order to investigate itsfunction, an attempt was made to express this gene in M. smegma-tis strain �DRKIN, in which one copy of a 56-kb duplicationfound in M. smegmatis mc2155 is missing (20). As a result, it has areduced dosage of genes associated with the 56-kb region. Onesuch gene is nrdE, which codes for the host class I ribonucleotidereductase. Since this mutant has only one copy of nrdE, it is there-fore highly sensitive to the well-known class I RNR inhibitor HU(31). To investigate whether the deficiency of RNR activity causedby the loss of one nrdE copy could be compensated for by express-ing the mycobacteriophage D29-derived gene encoding Gp50, aplasmid (pSG6) that expresses this gene under the control of anacetamide-inducible promoter was introduced into the �DRKINstrain. A similar construct, pSG6A, from which a mutant versionof the protein, Gp50(C375S), is synthesized, was also introducedseparately into the same strain. The C375S mutation alters a cru-cial Cys residue (Fig. 1B) that is considered to be necessary forRNR activity (25). Induced expression of the wild-type and mu-tant alleles in either M. smegmatis mc2155, �DRKIN, or both wasconfirmed by Western blotting using antisera against D29Gp50(Fig. 1A). In order to determine whether the expression of gene 50has any effect on the growth rates of bacteria, the OD600s of thecultures at different time points following induction of expressionwere determined. The results indicate that although induced ex-

Ghosh et al.

354 jb.asm.org January 2016 Volume 198 Number 2Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

pression of gene 50 did not have any effect on M. smegmatismc2155 growth, it had an adverse effect in the case of �DRKIN(Fig. 1C and D, respectively). The observed phenomenon was fur-ther confirmed by performing viability assays (Fig. 1E and F, re-spectively). When the gene encoding the C375S mutant ofD29Gp50 was expressed by using plasmid pSG6A, no significantdifference in growth kinetics before and after induction was ob-served (Fig. 1G and H). This observation indicated that the inhib-itory effect produced by D29Gp50 was specifically due to its ribo-nucleotide-reducing function. The same experiment was repeatedwith M. bovis BCG. In this slow-growing mycobacterium, thegenes corresponding to those found within the 56-kb duplicatedregion of M. smegmatis mc2155 are all present in single copies.Overexpression from pSG6 (Fig. 1I) but not pSG6A (Fig. 1J) led togrowth inhibition in M. bovis. The results thus obtained indicatethat the induced expression of gene 50 is growth inhibitory in cellsthat have single copies corresponding to genes associated with the56-kb duplication found in M. smegmatis mc2155.

Marker frequency analysis. Marker frequency analysis (32)was performed by determining the relative Ori/Ter ratios at eachtime point during the growth of M. smegmatis cells in the presenceor absence of inhibitory conditions. The principle behind thisanalysis is that in an actively replicating chromosome, the markerslocated close to the origin replicate at a higher frequency thanthose that are located at a distance. If a circular chromosome is inthe process of being replicated in the theta mode, the frequency atwhich a marker located close to the origin (Ori) is replicatedshould 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 ratiowill be 1. The experiment was performed by using the M. smegma-tis �DRKIN::pSG6B strain, a recombinant which harbors an in-tegrated copy of gene 50 in its genome, the expression of which canbe induced by the addition of acetamide (see Fig. S2 and S3 in thesupplemental material). The results indicated that the Ori/Ter ra-tio increased by 8-fold (Fig. 2B, blue trace) in cells that were grow-

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. smegmatisstrains, either mc2155 or �DRKIN, which were transformed with plasmids, either pSG6 (for synthesis of D29Gp50) or pSG6A [for synthesis of theD29Gp50(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 Lactobacillusleichmannii (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-activeCys 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) Growthpatterns of �DRKIN (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 viablecounts (CFU per milliliter), as indicated. (I and J) Similar experiments were performed by using M. bovis transformed with either pSG6 (I) or pSG6A (J). Thecolor 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 redline and therefore is invisible. Each data point represents the mean � SD of data from three biological replicate experiments performed in parallel. In the figurespresented here, the error bars are often not visible as they are too small compared to the symbols.

DNA Repair by Mycobacterial DinB2

January 2016 Volume 198 Number 2 jb.asm.org 355Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ing in an uninhibited manner (uninduced cells) (Fig. 2A, bluetrace). 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 con-trol, a similar experiment was repeated with M. smegmatis mc2155cells in which growth inhibition was induced with rifampin, awell-known replication initiation inhibitor (29, 33), at a concen-tration of 60 �g/ml (Fig. 2C, red trace). The Ori/Ter ratio of ri-fampin-treated cells did not change over time (Fig. 2D, red trace),although in the case of the untreated ones, an increase of �8-foldwas evident, as expected (blue trace).

The experiments performed here indicate that the Ori/Ter ra-tio reached a value of 2 in cells that were growing in an uninhib-ited manner. The observations thus obtained show that, unlikewhat has been claimed in several recent investigations (34–36),multifork replication occurs in M. smegmatis. Multifork replica-tion is observed in bacteria in such cases where the mass doublingtime is less than the sum of their chromosomal replication and celldivision times, the C and D periods, respectively (37, 38). The Cand D periods usually remain constant, but the mass doublingtime tends to vary with the culture conditions. If the cells growfaster due to better culture conditions, the mass doubling time willbecome shorter than the (C D) period. The differences in theobservations made in this study and those that were reported in

previous single-cell-based investigations (34–36) could be due todifferences in culture conditions and/or experimental strategiesused. In the case of single-cell experiments, growth is achieved onmicroscopic slides, within specialized chambers, whereas here themore traditional method of growing cells in culture flasks andunder highly aerated conditions was used. Overall, the results in-dicate that whereas multiple forks formed in cells growing nor-mally, none developed in cells that had stopped growing due toeither the induction of gene 50 expression or, for that matter,treatment with rifampin.

Reversal of Gp50-induced growth inhibition by coexpres-sion of D29 gene 48. The results presented above indicate thatthe overexpression of mycobacteriophage D29 gene 50 in the�DRKIN strain led to growth inhibition. To understand themechanism by which growth inhibition occurs, it was noted thatribonucleotide reductases can produce only three of the four de-oxyribonucleotides, dATP, dCTP, and dGTP, necessary for DNAsynthesis, while the fourth, dTTP, is synthesized by the action ofthymidylate synthase (TS). Hence, if only ribonucleotide reduc-tase is overproduced, the level of dTTP may become limiting. Invarious investigations, it has been shown that nucleotide pool im-balances could lead to genotoxicity and, eventually, cell death(39). A dTTP-deficient state may also be considered as one ofnucleotide imbalance, and therefore, in such a situation, either

FIG 2 Comparison of Ori/Ter ratios for chromosomal DNA isolated from mycobacterial cells growing either normally (without any inhibition) (blue traces) orsubjected 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 ata 60-�g/ml concentration (C). (B and D) Corresponding Ori/Ter ratio profiles. The arrows in the growth profiles indicate the time points at which the culturesgrowing 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 culturefollowed 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 DNAwas extracted. Ori and Ter regions were PCR amplified by using specific primer pairs in real time, and the corresponding CT values were determined. Thedifference between the CT values was used to calculate the Ori/Ter ratios. The values thus obtained were further normalized with respect to the ones correspond-ing 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, theerror bars are larger than those in panel C, as the individual assays were performed on different days.

Ghosh et al.

356 jb.asm.org January 2016 Volume 198 Number 2Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

growth inhibition or cell death can happen. Moreover, dTTP de-ficiency could specifically lead to what is known as “thyminelessdeath” (TLD), which has been studied extensively in various or-ganisms (40). The genomes of D29 and related phages possessgenes encoding not only RNR (Gp50) but also TS (D29Gp48)(24). It was therefore hypothesized that if D29Gp48 is synthesizedwithin the cell along with Gp50, the disparity in the dTTP levelrelative to the other dNTPs would be corrected, resulting in unin-hibited cell growth. To test this possibility, gene 48 was cloned inthe extrachromosomal tetracycline-inducible plasmid vectorpMind (23) to create the recombinant vector pSG17 (see Fig. S1 inthe supplemental material), which was then introduced into theM. smegmatis mc2155::pSG6B and �DRKIN::pSG6B integrants,both of which express an acetamide-inducible, genomically inte-grated copy of gene 50 (see Fig. S2 and S3 in the supplementalmaterial). The results indicate that, as expected, overexpression ofgene 50 resulted in growth inhibition in �DRKIN::pSG6B (Fig.3A, compare red trace and blue trace) but not in mc2155::pSG6B(Fig. 3C). However, the retarding effect observed in �DRKIN::pSG6B could be reversed by the addition of ATc, which inducesthe expression of gene 48 from pSG17 (Fig. 3B). Hence, it appearsthat growth retardation induced in �DRKIN cells overexpressinggene 50 is due primarily to a decrease of the dTTP level in thesecells relative to the levels of the other dNTPs.

Evidence for dTTP deficiency in cells overexpressing gene 50.In order to confirm that the alteration of the dTTP level is thereason behind the growth-inhibitory phenotype mentionedabove, the nucleotide pool was isolated from �DRKIN cells ex-pressing gene 50 (�DRKIN::pSG6B cells) and examined by usingMALDI-TOF mass spectrometry. The results show that in the caseof uninduced cells, which were growing exponentially, barringdCTP, the peak heights of the remaining dNTPs were comparable(Fig. 4A [representative profile] and B [graphical representationof the mean data from three experiments]). The peak height cor-responding to dCTP was found to be consistently shorter thanthose of the other dNTPs in all the determinations. It could be thatdCTP is indeed less abundant in mycobacterial cells. Alternatively,the observed lower intensity may be due to some limitation of theanalytical 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 uninducedconditions], and compare Fig. 4B and C). However, the maximumchange was observed in the case of dTTP, the level of which wasreduced to such an extent that it was undetectable. The dTTP-deficient state could nevertheless be reversed by prior expressionof the TS gene from plasmid pSG17 (Fig. 4A [pSG17 induced], andcompare Fig. 4C and D). Thus, the results confirm that the rootcause of growth inhibition was the drastic reduction of the dTTPlevel in the affected cells, as proposed above. The MALDI-TOFprofile (Fig. 4A) is a representative one. These experiments wererepeated at least three times, if not more. Since the dNTP profilefor the gene 48 expression set (pSG17 induced) is of crucial im-portance, at least insofar as this study is concerned, the raw datacorresponding to each of the three replicates used to calculate themeans in Fig. 4D are therefore provided in the supplemental ma-terial (see Fig. S5 in the supplemental material).

Gp50-induced lethality is determined by dinB2 copy num-ber. The inhibitory effect of gene 50 overexpression was selectivelyobserved in strain �DRKIN but not in mc2155. �DRKIN isknown to possess only one copy of a 56-kb region that is dupli-cated in mc2155. We therefore hypothesized that a reduced copynumber of genes associated with this segment could be responsi-ble for the observed phenotype in �DRKIN. Since a large numberof genes are present in this region (20), a knowledge-based ap-proach was taken to identify the possible candidate(s). One of thegenes present in the region is dinB2, which encodes the Y familypolymerase DinB2. Considering previous reports (19) that the Yfamily polymerase UmuC can help in overcoming DNA replica-tion defects, we hypothesized that a reduction of the DinB2 level in�DRKIN, caused by a copy number deficiency of its gene (dinB2),could be the determining factor. To test this possibility, the�DRKIN strain was modified to include a second chromosomalcopy of dinB2 (Fig. 5A). The introduction of the second copy wasdone by using a bacteriophage L5-based integrative vector, pSG18(see Fig. S2 and S4 in the supplemental material). The resultingintegrant, �DRKIN::pSG18, which has two chromosomally car-ried copies of dinB2 (dinB2/dinB2), and also �DRKIN, inwhich one copy is missing (dinB2 negative/dinB2), as well asmc2155, which has both copies (dinB2/dinB2), were trans-formed with pSG19 (see Fig. S1 in the supplemental material), a

FIG 3 Effect of D29 gene 48 expression on survival of M. smegmatis strains overexpressing gene 50. M. smegmatis �DRKIN::pSG6B and mc2155::pSG6Bintegrants were further transformed with the extrachromosomal D29 gene 48-expressing vector pSG17. Expression of gene 48 in transformed cells was inducedby using ATc (50 ng/ml) for 4 h prior to the induction of gene 50 expression using acetamide. Growth was monitored spectrophotometrically for bothacetamide-treated (red traces) and untreated (blue traces) cells. Each data point in these experiments represents the mean of data from three biological replicatesperformed in parallel � SD.

DNA Repair by Mycobacterial DinB2

January 2016 Volume 198 Number 2 jb.asm.org 357Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

pMind-derived vector expressing gene 50 under the control of atetracycline-inducible promoter. Following the induction of ex-pression of gene 50, only the growth of the �DRKIN strain (Fig.5C), and not the growth of �DRKIN::pSG18 (Fig. 5D) or mc2155(Fig. 5B), was found to be adversely affected. The results confirmour hypothesis that the decreased dinB2 copy number is the solereason why �DRKIN is highly susceptible to the lethal effects ofgene 50 expression.

Ectopic expression of dinB2 reverses gene 50-induced lethal-ity and restores Ori/Ter ratios. To further test whether DinB2 hasany role to play in the differential susceptibilities of the mutantand the parent strains, its gene was cloned in the extrachromo-somal tetracycline-inducible plasmid vector pMind (23) to createthe recombinant plasmid pSG16 (see Fig. S1 in the supplementalmaterial), which was then introduced into the M. smegmatis

mc2155::pSG6B and �DRKIN::pSG6B integrants, both of whichcarry an integrated copy of gene 50 in their chromosomes. Surviv-ability under various inducible conditions was then determined.The results show that following acetamide-induced expression ofgene 50, growth of �DRKIN::pSG6B came to a halt (Fig. 6C, com-pare red and blue traces), whereas the corresponding mc2155 con-struct continued to grow normally (Fig. 6A). Prior induction ofDinB2 synthesis from pSG16 by the addition of ATc, however,allowed acetamide-treated �DRKIN::pSG6B cells to survive (Fig.6D). Determination of the Ori/Ter ratio of DNA samples isolatedfrom the gene 50 integrants transformed with pSG16, the dinB2-expressing plasmid, indicated that in the absence of any form ofinduction, the ratio increases in a time-dependent manner, as ex-pected (Fig. 6E). Following the induction of gene 50 expressionfrom the �DRKIN::pSG6B integrant using acetamide, no in-

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 isolatedfrom �DRKIN::pSG6B integrant cells transformed with pSG17, the gene 48-expressing plasmid (�DRKIN::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 �DRKIN::pSG6B/pSG17 cells (B), �DRKIN::pSG6B/pSG17 cells induced for gene 50 expression (C), and �DRKIN::pSG6B/pSG17 cells induced for both gene 48 and gene 50 expression (D). The y axis valuesrepresent 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 inthese 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 intotwo 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 givenin the supplemental material (see Fig. S5 in the supplemental material).

Ghosh et al.

358 jb.asm.org January 2016 Volume 198 Number 2Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

crease, rather a decrease, in the Ori/Ter ratio was observed (Fig.6F). The addition of the inducer (ATc) for dinB2 to �DRKIN::pSG6B cells overexpressing gene 50 had no effect (mock experi-ment) on the Ori/Ter ratio, as the corresponding expression plas-mid (pSG16) was not present in these cells (Fig. 6F). The Ori/Terratio of �DRKIN::pSG6B integrants, harboring dinB2-expressingplasmid pSG16, decreased following the selective induction ofgene 50 by using acetamide (Fig. 6G). However, the decline in theratio could be reversed by introducing ATc, a dinB2-specific in-ducer (compare Fig. 6H to G and F). The results indicate that theadverse effects of gene 50 overexpression on growth as well as theOri/Ter ratio can be reversed by increasing the level of DinB2 inthe affected cells. The finding that the DinB2-mediated reversal ofthe lethal phenotypes mentioned above is not due to an elevationof dTTP levels through some indirect mechanism was verified. Itwas found that cells overexpressing dinB2 in addition to gene 50were as deficient of dTTP as those overexpressing gene 50 alone(see Fig. S6 in the supplemental material).

DISCUSSION

In the present study, we report the growth-inhibitory conse-quences of the expression of a mycobacteriophage D29-derivedgene (gene 50) encoding a class II RNR in a particular strain of M.smegmatis known as �DRKIN. In this strain, there is only onecopy of a 56-kb region, compared to two copies found in M. smeg-matis mc2155, the strain from which it was derived. The growth-inhibitory effect is apparently due to alterations in the nucleotidelevels that resulted following the induced expression of gene 50. Inthe altered scenario, the relative level of dATP was found to beincreased, possibly due to the enhanced reduction of ATP to dATPby D29Gp50. However, the levels of dCTP and dGTP were foundto be decreased, although they were still in the detectable range.The most-affected dNTP was dTTP, the level of which decreasedto such an extent that it could not be detected. Coexpression of a

gene encoding a TS, along with gene 50, resulted in the reversal ofgrowth inhibition, confirming that a deficiency of dTTP is theprimary reason for cell death under the given conditions. Osten-sibly, the phenomenon appears to be related to thymineless death(TLD) (40), which has been studied extensively in E. coli (41) andBacillus subtilis (42). TLD is a globally important issue, as it can beexploited for the development of antimicrobial as well as antican-cer drugs (43).

It has been demonstrated using marker frequency determina-tion experiments that gene 50 overexpression in �DRKIN cellsleads to the cessation of DNA replication. A similar effect could beproduced by treatment of cells with rifampin, a transcriptionalinhibitor that can inhibit replication by preventing initiation (29,33, 44). Given the similarity in the phenotypes observed betweengene 50 expression and rifampin treatment, it is tempting to spec-ulate that under dTTP-deficient conditions such as those de-scribed here, replication initiation in mycobacteria is inhibited.However, if dTTP is limiting, the elongation step will also be in-hibited. Thus, in those copies of the genome in which replicationinitiation has already been initiated, no further replication forkprogression is expected to take place following the expression ofgene 50.

To the best of our knowledge, this is the first attempt to usemarker frequency analysis to investigate DNA replication in my-cobacteria. The markers chosen were a DNA sequence derivedfrom the origin region of M. smegmatis (28), the proximal marker,and another from the diametrically opposite end, the distalmarker. The proximal marker was designated Ori and the distalmarker was designated Ter based on the Ori/Ter nomenclatureused in the case of E. coli (45). In the case of mycobacteria, termi-nation of replication is not known in detail, and therefore, theterm Ter is used loosely to describe a site where the bidirectionalforks possibly meet.

FIG 5 Effect of dinB2 copy number duplication on survival of �DRKIN cells expressing gene 50. (A) Schematic diagram of the chromosome (thick line) of the�DRKIN::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 theintegrated copy of plasmid pSG18 (thin line bordered by hatched boxes representing the mycobacteriophage L5 chromosomal left and right attachment sites, attLand 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),�DRKIN (C), and �DRKIN::pSG18 (D) cells in the presence (red) or absence (blue) of ATc, the inducer of gene 50 expression from pSG19. Genetically, thesestrains have either two copies of dinB2 (dinB2/dinB2) or just one copy (dinB2 negative/dinB2). Each data point in these experiments represents the mean ofresults 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.

DNA Repair by Mycobacterial DinB2

January 2016 Volume 198 Number 2 jb.asm.org 359Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

The experiments performed here indicate that the Ori/Ter ra-tio more than doubled (multifork) in cells that were growing in anuninhibited manner. The increase in Ori/Ter ratios in activelygrowing cells is a novel finding. This finding indicates that underconditions such as those mentioned here, multiple forks developduring mycobacterial DNA replication.

The results of the marker frequency analysis also show thatunlike actively growing cells, the Ori/Ter ratio of growth-inhibitedcells remains constant. Our initial expectation was that in retardedcells, Ori/Ter ratios should be higher, as was observed for “dGTP-less” death (45) in E. coli, a phenomenon that may have similari-ties with TLD. TLD and other related phenomena are very com-plex, and the exact mechanisms behind them are not fullyunderstood. However, one consensus that has emerged is thatunder TLD-inducing conditions, origin sequences are lost, and asa result, marker frequency at the Ori region becomes apparentlythe same as that at Ter (40, 46, 47). The constancy of the Ori/Terratio in growth-retarded cells observed here could be due to eithera lack of replication initiation or deletion of origin sequences re-sulting from aberrant initiation. A more detailed high-resolutionmarker frequency analysis may have to be done to come to a spe-cific conclusion. Nevertheless, considering that the dTTP level wasfound to be low in the affected cells and that the region wherereplication initiates is A/T rich (28), it is perhaps not difficult toconclude that dTTP deficiency leads to some kind of a defect inorigin function. The finding that the expression of gene 48, the

TS-encoding gene, leads to recovery is an additional indicationthat such a hypothesis could be correct.

An important question may arise as to why �DRKIN, whichretains one copy of dinB2, is unable to resist the toxic effects ofgene 50 expression. Is gene dosage the only discriminating factor?The precise answer to this question is not known as of now. How-ever, it may be noted that the dosage of genes associated with the56-kb region that is duplicated in M. smegmatis mc2155 appears tohave an important role to play in deciding the phenotypes of thisorganism. For example, as mentioned in Results, it has been dem-onstrated that a lower gene dosage of another 56-kb region-asso-ciated gene, nrdE, encoding a ribonucleotide reductase type I sub-unit, leads to increased susceptibility of the �DRKIN strain to theclastogen hydroxyurea (HU) (31). Therefore, the copy numbersof not only dinB2 but also the other genes present in this locusappear to have a role to play in determining the fate of M. smeg-matis mc2155 under circumstances where their products are nec-essary. Why the single copies of the genes, be it dinB2 or nrdE,present in the �DRKIN strain are incapable of giving protectionwhen needed is also an issue that needs to be addressed. The re-sults presented in this study suggest that it is the copy numberalone that is important. However, it is possible that in a duplicatedcontext, the genes are expressed more efficiently for some un-known reasons.

How the higher level of DinB2 influences the growth proper-ties of �DRKIN cells is another important issue. Recent biochem-

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 �DRKIN::pSG6B (C andD) integrants were transformed with the extrachromosomal dinB2-expressing vector pSG16. The transformed cells were either treated with ATc for dinB2overexpression (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 thecases was monitored spectrophotometrically by determining the OD600. Each data point in these experiments represents the mean of results from three biologicalreplicates performed in parallel � SD. (E to H) Ori/Ter ratios of DNA isolated from �DRKIN::pSG6B cells harboring either no plasmid (F) or pSG16 (E, G, andH). 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 gene50 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 .

Ghosh et al.

360 jb.asm.org January 2016 Volume 198 Number 2Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ical studies have indicated that DinB2, but not the other two my-cobacterial DinBs, has the ability to scavenge dNTPs and alsomisincorporate them (10, 12) under limiting conditions. More-over, DinB2 has low sugar selectivity and thus appears to be nat-urally capable of incorporating ribonucleotides during templatedDNA synthesis (10). The observations made in this study may beexplained by considering that under dTTP-limiting conditionscreated by the overexpression of gene 50, synthesis of DNA at theorigin is hampered (40), which results in the formation of single-stranded gaps. The creation of such gaps could trigger the subse-quent generation of lethal double-stranded DNA breaks. DinB2can help in rectifying the situation by ensuring that such gaps are“patched up” through the incorporation of the ribonucleotideUTP (12). The “ribopatches” may be subsequently removedthrough ribonucleotide excision repair (RER) mechanisms, whichhave been characterized in E. coli (48, 49) but, to the best of ourknowledge, not in mycobacteria.

The ability of a Pol IV, DinB2 being one, to execute TLS de-pends on its ability to compete with the replicative DNA Pol III ina concentration-dependent manner (50). At low concentrations,both polymerases remain associated with the � clamp in an ar-rangement that has been compared to a “tool belt.” Such an ar-rangement allows efficient polymerase “switching” from Pol III toPol IV and back. At higher concentrations, such as those observedfollowing the induction of the SOS pathway, Pol IV displaces PolIII from the clamp and takes over the role of synthesizing DNA ina templated manner. The observation that the lethal effect of gene50 expression in �DRKIN cells can be overcome only in cellshaving a relatively high concentration of DinB2 suggests the in-volvement of the second mechanism, although the first may alsobe operative. By displacing Pol III and/or taking over its functions,at least to a limited extent, DinB2 may aid the survival of cellsunder dTTP-limiting conditions.

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

ACKNOWLEDGMENTS

We thank Ruth McNerney, Valerie Mizrahi, and Brian D. Robertson formycobacteriophage D29, the M. smegmatis �DRKIN strain, and thepMind vector, respectively. The technical assistance given by P. Halderwas of great help.

S.G., S.S., and P.K. acknowledge CSIR, Government of India, for theirfellowship.

REFERENCES1. Nohmi T. 2006. Environmental stress and lesion-bypass DNA poly-

merases. Annu Rev Microbiol 60:231–253. http://dx.doi.org/10.1146/annurev.micro.60.080805.142238.

2. Jarosz DF, Beuning PJ, Cohen SE, Walker GC. 2007. Y-family DNApolymerases in Escherichia coli. Trends Microbiol 15:70 –77. http://dx.doi.org/10.1016/j.tim.2006.12.004.

3. Kim SR, Maenhaut-Michel G, Yamada M, Yamamoto Y, Matsui K,Sofuni T, Nohmi T, Ohmori H. 1997. Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinPresults in strongly enhancing mutagenesis in the absence of any exogenoustreatment to damage DNA. Proc Natl Acad Sci U S A 94:13792–13797.http://dx.doi.org/10.1073/pnas.94.25.13792.

4. Layton JC, Foster PL. 2005. Error-prone DNA polymerase IV is regulatedby the heat shock chaperone GroE in Escherichia coli. J Bacteriol 187:449 –457. http://dx.doi.org/10.1128/JB.187.2.449-457.2005.

5. Storvik KA, Foster PL. 2010. RpoS, the stress response sigma factor,plays a dual role in the regulation of Escherichia coli’s error-proneDNA polymerase IV. J Bacteriol 192:3639 –3644. http://dx.doi.org/10.1128/JB.00358-10.

6. Galhardo RS, Do R, Yamada M, Friedberg EC, Hastings PJ, Nohmi T,Rosenberg SM. 2009. DinB upregulation is the sole role of the SOS re-sponse in stress-induced mutagenesis in Escherichia coli. Genetics 182:55– 68. http://dx.doi.org/10.1534/genetics.109.100735.

7. Uchida K, Furukohri A, Shinozaki Y, Mori T, Ogawara D, Kanaya S,Nohmi T, Maki H, Akiyama M. 2008. Overproduction of Escherichia coliDNA polymerase DinB (Pol IV) inhibits replication fork progression andis lethal. Mol Microbiol 70:608 – 622. http://dx.doi.org/10.1111/j.1365-2958.2008.06423.x.

8. Tan KW, Pham TM, Furukohri A, Maki H, Akiyama MT. 2015. Re-combinase and translesion DNA polymerase decrease the speed of repli-cation fork progression during the DNA damage response in Escherichiacoli cells. Nucleic Acids Res 43:1714 –1725. http://dx.doi.org/10.1093/nar/gkv044.

9. Kana BD, Abrahams GL, Sung N, Warner DF, Gordhan BG, Ma-chowski EE, Tsenova L, Sacchettini JC, Stoker NG, Kaplan G, MizrahiV. 2010. Role of the DinB homologs Rv1537 and Rv3056 in Mycobacte-rium tuberculosis. J Bacteriol 192:2220 –2227. http://dx.doi.org/10.1128/JB.01135-09.

10. Ordonez H, Uson ML, Shuman S. 2014. Characterization of three my-cobacterial DinB (DNA polymerase IV) paralogs highlights DinB2 as nat-urally adept at ribonucleotide incorporation. Nucleic Acids Res 42:11056 –11070. http://dx.doi.org/10.1093/nar/gku752.

11. Sharma A, Nair DT. 2012. MsDpo4 —a DinB homolog from Mycobac-terium smegmatis—is an error-prone DNA polymerase that can promoteG:T and T:G mismatches. J Nucleic Acids 2012:285481. http://dx.doi.org/10.1155/2012/285481.

12. Ordonez H, Shuman S. 2014. Mycobacterium smegmatis DinB2 misin-corporates deoxyribonucleotides and ribonucleotides during templatedsynthesis and lesion bypass. Nucleic Acids Res 42:12722–12734. http://dx.doi.org/10.1093/nar/gku1027.

13. Boshoff HI, Reed MB, Barry CE, III, Mizrahi V. 2003. DnaE2 polymer-ase contributes to in vivo survival and the emergence of drug resistance inMycobacterium tuberculosis. Cell 113:183–193. http://dx.doi.org/10.1016/S0092-8674(03)00270-8.

14. Boshoff HI, Myers TG, Copp BR, McNeil MR, Wilson MA, BarryCE, III. 2004. The transcriptional responses of Mycobacterium tuber-culosis to inhibitors of metabolism: novel insights into drug mecha-nisms of action. J Biol Chem 279:40174 – 40184. http://dx.doi.org/10.1074/jbc.M406796200.

15. Vaisman A, Kuban W, McDonald JP, Karata K, Yang W, Goodman MF,Woodgate R. 2012. Critical amino acids in Escherichia coli UmuC re-sponsible for sugar discrimination and base-substitution fidelity. NucleicAcids Res 40:6144 – 6157. http://dx.doi.org/10.1093/nar/gks233.

16. DeLucia AM, Grindley ND, Joyce CM. 2003. An error-prone family YDNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a‘steric gate’ residue for discrimination against ribonucleotides. NucleicAcids Res 31:4129 – 4137. http://dx.doi.org/10.1093/nar/gkg417.

17. Jarosz DF, Godoy VG, Delaney JC, Essigmann JM, Walker GC. 2006.A single amino acid governs enhanced activity of DinB DNA poly-merases on damaged templates. Nature 439:225–228. http://dx.doi.org/10.1038/nature04318.

18. Davies BW, Kohanski MA, Simmons LA, Winkler JA, Collins JJ, WalkerGC. 2009. Hydroxyurea induces hydroxyl radical-mediated cell death inEscherichia coli. Mol Cell 36:845– 860. http://dx.doi.org/10.1016/j.molcel.2009.11.024.

19. Godoy VG, Jarosz DF, Walker FL, Simmons LA, Walker GC. 2006.Y-family DNA polymerases respond to DNA damage-independent inhi-bition of replication fork progression. EMBO J 25:868 – 879. http://dx.doi.org/10.1038/sj.emboj.7600986.

20. Warner DF, Etienne G, Wang XM, Matsoso LG, Dawes SS, Soetaert K,Stoker NG, Content J, Mizrahi V. 2006. A derivative of Mycobacteriumsmegmatis mc(2)155 that lacks the duplicated chromosomal region. Tu-berculosis 86:438 – 444. http://dx.doi.org/10.1016/j.tube.2005.10.001.

21. Ford ME, Sarkis GJ, Belanger AE, Hendrix RW, Hatfull GF. 1998.Genome structure of mycobacteriophage D29: implications for phage

DNA Repair by Mycobacterial DinB2

January 2016 Volume 198 Number 2 jb.asm.org 361Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

evolution. J Mol Biol 279:143–164. http://dx.doi.org/10.1006/jmbi.1997.1610.

22. van Kessel JC, Marinelli LJ, Hatfull GF. 2008. Recombineering myco-bacteria and their phages. Nat Rev Microbiol 6:851– 857. http://dx.doi.org/10.1038/nrmicro2014.

23. Blokpoel MC, Murphy HN, O’Toole R, Wiles S, Runn ES, Stewart GR,Young DB, Robertson BD. 2005. Tetracycline-inducible gene regulationin mycobacteria. Nucleic Acids Res 33:e22. http://dx.doi.org/10.1093/nar/gni023.

24. Bhattacharya B, Giri N, Mitra M, Gupta SK. 2008. Cloning, character-ization and expression analysis of nucleotide metabolism-related genes ofmycobacteriophage L5. FEMS Microbiol Lett 280:64 –72. http://dx.doi.org/10.1111/j.1574-6968.2007.01047.x.

25. Sintchak MD, Arjara G, Kellogg BA, Stubbe J, Drennan CL. 2002. Thecrystal structure of class II ribonucleotide reductase reveals how an allos-terically regulated monomer mimics a dimer. Nat Struct Biol 9:293–300.http://dx.doi.org/10.1038/nsb774.

26. Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR, Jr. 1990.Isolation and characterization of efficient plasmid transformation mu-tants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919. http://dx.doi.org/10.1111/j.1365-2958.1990.tb02040.x.

27. Triccas JA, Parish T, Britton WJ, Gicquel B. 1998. An inducible expres-sion system permitting the efficient purification of a recombinant antigenfrom Mycobacterium smegmatis. FEMS Microbiol Lett 167:151–156.http://dx.doi.org/10.1111/j.1574-6968.1998.tb13221.x.

28. Qin MH, Madiraju MV, Zachariah S, Rajagopalan M. 1997. Character-ization of the oriC region of Mycobacterium smegmatis. J Bacteriol 179:6311– 6317.

29. Messer W. 1972. Initiation of deoxyribonucleic acid replication inEscherichia coli B-r: chronology of events and transcriptional controlof initiation. J Bacteriol 112:7–12.

30. Buckstein MH, He J, Rubin H. 2008. Characterization of nucleotidepools as a function of physiological state in Escherichia coli. J Bacteriol190:718 –726. http://dx.doi.org/10.1128/JB.01020-07.

31. Mowa MB, Warner DF, Kaplan G, Kana BD, Mizrahi V. 2009.Function and regulation of class I ribonucleotide reductase-encodinggenes in mycobacteria. J Bacteriol 191:985–995. http://dx.doi.org/10.1128/JB.01409-08.

32. Sueoka N, Yoshikawa H. 1965. The chromosome of Bacillus subtilis. I.Theory of marker frequency analysis. Genetics 52:747–757.

33. Nair N, Dziedzic R, Greendyke R, Muniruzzaman S, Rajagopalan M,Madiraju MV. 2009. Synchronous replication initiation in novel Myco-bacterium tuberculosis dnaA cold-sensitive mutants. Mol Microbiol 71:291–304. http://dx.doi.org/10.1111/j.1365-2958.2008.06523.x.

34. Santi I, Dhar N, Bousbaine D, Wakamoto Y, McKinney JD. 2013.Single-cell dynamics of the chromosome replication and cell division cy-cles in mycobacteria. Nat Commun 4:2470. http://dx.doi.org/10.1038/ncomms3470.

35. Santi I, McKinney JD. 2015. Chromosome organization and replisomedynamics in Mycobacterium smegmatis. mBio 6:e01999 –14. http://dx.doi.org/10.1128/mBio.01999-14.

36. Trojanowski D, Ginda K, Pioro M, Holowka J, Skut P, Jakimowicz D,Zakrzewska-Czerwinska J. 2015. Choreography of the Mycobacteriumreplication machinery during the cell cycle. mBio 6:e02125–14. http://dx.doi.org/10.1128/mBio.02125-14.

37. Wang JD, Levin PA. 2009. Metabolism, cell growth and the bacterialcell cycle. Nat Rev Microbiol 7:822– 827. http://dx.doi.org/10.1038/nrmicro2202.

38. Cooper S, Helmstetter CE. 1968. Chromosome replication and the divi-sion cycle of Escherichia coli B/r. J Mol Biol 31:519 –540. http://dx.doi.org/10.1016/0022-2836(68)90425-7.

39. Mathews CK. 2006. DNA precursor metabolism and genomic stability.FASEB J 20:1300 –1314. http://dx.doi.org/10.1096/fj.06-5730rev.

40. Guzman EC, Martin CM. 2015. Thymineless death, at the origin. FrontMicrobiol 6:499. http://dx.doi.org/10.3389/fmicb.2015.00499.

41. Hanawalt PC. 2015. A balanced perspective on unbalanced growth andthymineless death. Front Microbiol 6:504. http://dx.doi.org/10.3389/fmicb.2015.00504.

42. Buick RW, Harris WJ. 1975. Thymineless death in Bacillus subtilis. J GenMicrobiol 88:115–122. http://dx.doi.org/10.1099/00221287-88-1-115.

43. Ahmad SI, Kirk SH, Eisenstark A. 1998. Thymine metabolism andthymineless death in prokaryotes and eukaryotes. Annu Rev Microbiol52:591– 625. http://dx.doi.org/10.1146/annurev.micro.52.1.591.

44. Lark KG. 1972. Evidence for the direct involvement of RNA in the initi-ation of DNA replication in Escherichia coli 15T. J Mol Biol 64:47– 60.http://dx.doi.org/10.1016/0022-2836(72)90320-8.

45. Itsko M, Schaaper RM. 2014. dGTP starvation in Escherichia coli pro-vides new insights into the thymineless-death phenomenon. PLoS Genet10:e1004310. http://dx.doi.org/10.1371/journal.pgen.1004310.

46. Sangurdekar DP, Hamann BL, Smirnov D, Srienc F, Hanawalt PC,Khodursky AB. 2010. Thymineless death is associated with loss of essen-tial genetic information from the replication origin. Mol Microbiol 75:1455–1467. http://dx.doi.org/10.1111/j.1365-2958.2010.07072.x.

47. Kuong KJ, Kuzminov A. 2012. Disintegration of nascent replicationbubbles during thymine starvation triggers RecA- and RecBCD-dependent replication origin destruction. J Biol Chem 287:23958 –23970.http://dx.doi.org/10.1074/jbc.M112.359687.

48. Vaisman A, McDonald JP, Noll S, Huston D, Loeb G, Goodman MF,Woodgate R. 2014. Investigating the mechanisms of ribonucleotide exci-sion repair in Escherichia coli. Mutat Res 761:21–33. http://dx.doi.org/10.1016/j.mrfmmm.2014.01.005.

49. Vaisman A, McDonald JP, Huston D, Kuban W, Liu L, Van HoutenB, Woodgate R. 2013. Removal of misincorporated ribonucleotidesfrom prokaryotic genomes: an unexpected role for nucleotide excisionrepair. PLoS Genet 9:e1003878. http://dx.doi.org/10.1371/journal.pgen.1003878.

50. Kath JE, Jergic S, Heltzel JM, Jacob DT, Dixon NE, Sutton MD, WalkerGC, Loparo JJ. 2014. Polymerase exchange on single DNA moleculesreveals processivity clamp control of translesion synthesis. Proc Natl AcadSci U S A 111:7647–7652. http://dx.doi.org/10.1073/pnas.1321076111.

Ghosh et al.

362 jb.asm.org January 2016 Volume 198 Number 2Journal of Bacteriology

on Decem

ber 16, 2020 by guesthttp://jb.asm

.org/D

ownloaded from