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Department of Medicine, Johns Hopkins University, CRB2, Room 1.08, 1550 Orleans Street, Baltimore, MD 21231-1044, USA
Correspondence
William R. Bishai
wbishai{at}jhmi.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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15 kDa band in M. tuberculosis lysates. Following overexpression of sense and anti-sense constructs in their cognate mycobacterial hosts, whiB2 and whmD transformants displayed a small-colony phenotype, exhibited filamentation, and showed a reduction in viability. These observations reveal that the two proteins are functionally homologous and that their intracellular concentration is critical for septation in mycobacteria. Colonies of M. tuberculosis overexpressing whiB2 were spherical and glossy, suggesting a change in composition of the cell envelope. Filaments of the conditionally complemented M. smegmatis whmD mutant were non-acid-fast, also indicating changes in characteristics of surface lipids. M. smegmatis transformants carrying a whmD–gfp fusion showed a diffuse pattern of fluorescence, consistent with the putative role of WhmD as a regulator. These observations strongly suggest that M. tuberculosis whiB2 is an essential gene and its protein product in all likelihood regulates the expression of genes involved in the cell division cascade.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), and an adaptive response to host immunity which further slows or even stops its growth during infection. This ability to enter a persistent or latent state within humans allows the organism to await immune deterioration of its host for long periods of time. In addition, clinical observations suggest that the organism develops a state of phenotypic tolerance to antimycobacterial drugs during infection, which may be characterized by a reduced rate of cell division (Colangeli et al., 2005). The basis for the prolonged cell division times is unclear, although the limited number of rRNA operons and the metabolic costs of maintaining the complex cell wall characteristic of the genus Mycobacterium are often cited as possible contributing factors (Wheeler & Ratledge, 1994). Of late, significant progress has been made towards an understanding of the temporal and spatial regulation of prokaryotic cell division. A number of proteins in Escherichia coli and Bacillus subtilis have been shown to be essential, to be localized to the division septum and to give filamentous phenotypes when conditionally knocked out (Jacobs & Shapiro, 1999).
Although the step-wise assembly pathway involved in formation of the E. coli cell division complex has been extensively described (Goehring & Beckwith, 2005) this process is just beginning to be understood in members of the mycobacteria. These studies suggest that FtsZ is an essential cell division protein and that M. tuberculosis FtsZ is a target of FtsH protease (Anilkumar et al., 2004, 2001; Dziadek et al., 2002, 2003). Optimal levels of FtsZ are required for cell division, with unregulated expression resulting in lethality (Dziadek et al., 2002). M. tuberculosis ftsZ can functionally substitute for its Mycobacterium smegmatis orthologue, signifying that the FtsZ-catalysed phases in the cell division processes of slow- and fast-growing members of mycobacteria are analogous (Dziadek et al., 2003). In addition, M. tuberculosis ftsZ has been shown to possess multiple promoters, reflecting the requirement to maintain a high basal level of, or to differentially regulate, FtsZ expression during different growth conditions of the pathogen in vivo (Roy & Ajitkumar, 2005). M. tuberculosis FtsZ and the membrane protein FtsW have been shown to interact, in vitro (Datta et al., 2002) and in vivo (Rajagopalan et al., 2005), suggesting that this interaction could serve to anchor FtsZ to the membrane and link septum formation to peptidoglycan synthesis in M. tuberculosis.
Like ftsZ, M. smegmatis whmD is an essential cell division gene. WhmD belongs to the growing WhiB-like family of proteins shown to be involved in the regulation of significant cellular processes such as cell division (Gomez & Bishai, 2000), pathogenesis (Ramakrishnan et al., 2000; Steyn et al., 2002), antibiotic resistance (Morris et al., 2005) and response to oxidative stress (Kim et al., 2005). Each WhiB-like protein contains four invariant cysteine residues and is relatively short (76–139 residues). Members of this family also possess a conserved C-terminal motif comprising two helices and an intervening turn characterized by the 7-residue signature (FYG)-G-(VI)-W-G-G-(LVIM). Although this motif does not constitute a typical HTH motif (Aravind et al., 2005), it is believed to be involved in DNA binding (Davis & Chater, 1992; Soliveri et al., 2000). In a conditionally complemented system, on inducer withdrawal, a 
whmD mutant exhibited irreversible filamentous branched growth with diminished septum formation and aberrant septal placement, while WhmD overexpression resulted in growth retardation and hyperseptation (Gomez & Bishai, 2000). Together these phenotypes indicated a role for this protein in septum formation and cell division. The septal defects were found not to be due to insufficient levels of FtsZ, since FtsZ accumulation did not vary following WhmD withdrawal. WhmD is therefore proposed to play a role in the early stages of mycobacterial cell division, perhaps in FtsZ localization or polymerization or as a regulator of cell division genes other than ftsZ (Gomez & Bishai, 2000). No functional information is available on whiB2, the homologue of whmD in M. tuberculosis, apart from its induction in a nutrient-starvation model of M. tuberculosis (Betts et al., 2002) and in the lungs of infected mice (Dubnau et al., 2005). The two genes share synteny at the genomic level and almost 70 % identity in their protein sequence. Furthermore, whereas the C-termini of WhmD and WhiB2 are nearly identical to that of Streptomyces coelicolor WhiB, the mycobacterial proteins contain a unique 38–48 aa N-terminal extension not found in WhiB. Although whiB2 is annotated as an 89-codon ORF (Cole et al., 1998) based on homology to whiB, the protein is believed to be translated from a start codon 102 nt upstream of that predicted (Gomez & Bishai, 2000). To examine if WhmD and WhiB2 are true homologues, we have carried out a comparative analysis of the two genes at the level of their transcripts, protein products and the phenotypic effects of their perturbation. Our results indicate that the two proteins are functionally equivalent, and strongly suggest that M. tuberculosis whiB2 is an essential gene involved in mycobacterial cell division.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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(F'/endA1 hsdR17 [
] glnV44 thi-1 recA1 gyrA [Nalr] relA1 [lacIZYA–argF]U169 deoR [
80dlac(lacZ)M15]), from Stratagene, was used for cloning purposes. E. coli BL21(DE3) (F– ompT hsdSB [
] gal dcm [DE3]), used for protein expression, was purchased from Novagen. M. smegmatis mc26 1-2c was kindly provided by Dr Bill Jacobs, Albert Einstein College of Medicine, NY, USA, and M. tuberculosis CDC1551 and its genomic DNA were obtained from Colorado State University, CO, USA. Luria–Bertani (LB) broth and LB agar were used to culture E. coli. For culturing M. smegmatis, 7H9 broth and 7H10 agar from Difco were supplemented with albumin dextrose complex (ADC; 5 g BSA, 2 g glucose and 0.85 g NaCl l–1), 0.5 % (v/v) glycerol and 0.05 % Tween 80. For M. tuberculosis, ADC was substituted with Middlebrook OADC (oleic acid/albumin/dextrose/catalase) enrichment (Becton Dickinson). Both E. coli and mycobacteria were grown at 37 °C with shaking at 200 r.p.m. Antibiotics were added when necessary: ampicillin (200 µg ml–1), kanamycin (50 µg ml–1 for E. coli and 15 µg ml–1 for mycobacteria), hygromycin (200 µg ml–1 for E. coli and 50 µg ml–1 for mycobacteria), zeocin (25 µg ml–1 for E. coli and 50 µg ml–1 for mycobacteria) and apramycin (30 µg ml–1).
DNA techniques.
Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (NEB), and Taq polymerase was purchased from Invitrogen. Klenow fragment of DNA polymerase was from NEB. Protocols for DNA manipulations, including plasmid DNA preparation, restriction endonuclease digestion, agarose gel electrophoresis, isolation and ligation of DNA fragments, and E. coli transformation were performed as described by Sambrook et al. (1989). Mycobacterial strains were transformed by electroporation. PCR amplifications were carried out according to the manufacturer's specifications (Bio-Rad). Each of the 30 cycles was carried out at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, followed by a final extension cycle at 72 °C for 10 min. DNA fragments used for cloning and labelling reactions were purified by using the Qiagen gel extraction kit according to the manufacturer's specifications.
Western blotting.
For immunoblotting experiments, M. tuberculosis and M. smegmatis cells were harvested at their exponential phase of growth, lysed by bead beating on a mini bead beater (Biospec Products) and protein was quantified by the Bradford assay (Bradford, 1976). Samples containing 30 µg of total cell lysate proteins were electrophoresed on a 12 % SDS-PAGE system and the proteins transferred to nitrocellulose. WhmD (Gomez & Bishai, 2000) and Gfp antisera (Invitrogen) were used at 1 : 100 and 1 : 5000 dilutions, respectively. Horseradish-peroxidase-conjugated goat anti-rabbit IgG at a 1 : 5000 dilution and chemiluminescent substrate (Amersham) were used to develop the blots.
Mapping the 5' ends of whmD and whiB2 mRNA.
The 5'-RACE (rapid amplification of cDNA ends) system (Frohman, 1993) was used to determine the transcription initiation site with the Invitrogen kit (version 2.0). Total RNA was isolated from M. smegmatis and M. tuberculosis cells harvested at their exponential phase of growth by using the Trizol method according to the supplier's instructions (Invitrogen). The abridged anchor primer (AAP) and abridged universal amplification primers (AUAP) were used in combination with the gene-specific primers. The gene-specific primer whmD RACE gsp1 (5'-ACGTCCAGCTGCCCCCGTCG-3') was used for RT-PCR and nested PCR1 while whmD RACE gsp2 (5'-GGCTCACCGAGCCGAGTAGC-3') was used for the second nested PCR with AUAP. Likewise, whiB2 RACE gsp1 (5'-CGGCCCTCGGGAACCAAAC-3') was used for the RT-PCR and first nested PCR step, while whiB2 RACE gsp2 (5'-ATGCGGTAGCCGATCCGGTA-3') was used for nested PCR 2. The PCR products were subsequently subjected to nucleotide sequencing by using the gene-specific primers whmD RACE gsp2 and whiB2 RACE gsp2.
Construction of lacZ transcriptional fusions and measurement of 
-galactosidase activity.
To generate PwhmD–lacZ, a 187 bp DNA fragment containing the whmD promoter sequence from positions –187 to –1 with respect to the start codon (ATG), was PCR-amplified from M. smegmatis chromosomal DNA using the primers PwhmD5B-F (5'-CTAGTCTAGAGAATTCGCGCCCTGGAGC-3') and PwhmD5B-R (5'-ACATGCATGCATCCCCCGCCTCCTCACT-3'). The transcriptional fusion construct was generated by cloning the amplified fragment in pSD5B at the XbaI and SphI sites. PwhiB2–lacZ was constructed by PCR amplification of a 200 bp fragment from M. tuberculosis genomic DNA using the primers pSD5BwhiB2p-F (5'-CTAGTCTAGATTACGAGATGATATGGAA-3') and pSD5B MtwhiB2p-R (5'-ACATGCATGCGCCTCCGCCTCCTCACTC-3') and cloning the fragment into pSD5B at the XbaI–SphI sites. The positive control fusion, Phsp60–lacZ, was made by amplifying a 385 bp fragment containing the M. bovis hsp60 promoter from the vector pMV261 using the primers pSD5Bhsp60p-F (5'-CTAGTCTAGAAAATCTAGACGGTGACCA-3') and pSD5Bhsp60p-R (5'-ACATGCATGCTGCGAAGTGATTCCTCCG-3') followed by cloning the fragment at the XbaI–SphI sites of pSD5B. All the above constructs and the control plasmid pSD5B were introduced into M. smegmatis by electroporation, and promoter activity was determined by -galactosidase assays (Miller, 1972), using cell lysates of the cultures harvested at their exponential phase of growth. Protein concentrations were determined by the Bradford assay, with BSA as the standard. At least three biological replicates were performed for each sample before calculating the specific activity.
Expression of sense and anti-sense constructs of whmD and whiB2 and examination of their phenotypes.
Sense and anti-sense constructs of whmD and whiB2 were generated by PCR-amplifying the entire ORFs of the two genes from genomic DNA of their cognate hosts in the sense and anti-sense orientations, and cloning the products into the NdeI–SpeI sites of pCK0218 (Manabe et al., 1999). This results in the replacement of the sigF gene in the plasmid with the above products, allowing the regulation of their expression in response to acetamide. The primers used to amplify the two genes were as follows. For pAce whmD: pAcewhmD-F (5'-GGGAATTCCATATGTCTTATGAGAGCGGCGAT-3'), pAcewhmD-R (5'-GGACTAGTCTAGATGATGCCGCGCTT-3'). For pAce anti-whmD: pAce anti-whmD-F (5'-GGGAATTCCATATGCTAGATGATGCCGCGCTT-3'), pAce anti-whmD-R (5'-GGACTAGTATGTCTTATGAGAGCGGC-3'). For pAce whiB2: pAcewhiB2-F (5'-GGGAATTCCATATGTCCTATGAACACCTTCGG-3'), pAcewhiB2-R (5'-GGACTAGTTCAGATGATCCCGCGTTT-3'). For pAce anti-whiB2: pAce anti-whiB2-F (5'-GGGAATTCCATATGTCAGATGATCCCGCGTTT-3'), pAce anti-whiB2-R (5'-GGACTAGTATGTCCTATGAACACCTT-3'). The control plasmid pAce was constructed by digesting pCK0218 with NdeI/SpeI, purifying the vector fragment, endfilling the fragment with the Klenow fragment of DNA polymerase and ligating the vector to itself. To alter the levels of WhmD/WhiB2, the recombinant plasmids described above were transformed into their cognate mycobacterial hosts along with the control plasmid pAce. Transformants were grown to their exponential phase of growth (OD600 1.0), and then grown overnight in the presence or absence of 0.2 % acetamide. For colony phenotypes and viability measurements, appropriate culture dilutions were plated on 7H10 agar medium either containing or lacking the inducer. For induced cultures, it was necessary to maintain a concentration of 0.2 % acetamide on agar plates. To document colony phenotypes, plates were photographed using a Fuji-Finepix A340 digital camera and the images edited using the Adobe Elements 2.0 software package. Percentage viability was calculated by dividing the number of c.f.u. obtained under inducing conditions by that observed in the absence of the inducer. The data represent at least two independent replicates. To observe cellular morphology, cultures were washed and resuspended in PBS, heat fixed on slides and stained with carbolfuchsin for 5 min. After washing off the excess dye with distilled water, the slides were dried and examined at 600x magnification on a Nikon Eclipse E800 microscope under oil. Images were captured using the Nikon digital still camera DXM1200 and edited using the software package ACT-1 ver2. Cell length measurements were made using a sample size of 30.
Real-time RT-PCR analysis.
To quantify transcript levels of whmD and whiB2 in their cognate hosts under conditions where the levels of the genes were being perturbed, RNA was isolated from the acetamide-induced cultures (as described above), treated with RNase-free DNase (Ambion) and subjected to reverse transcription. This was followed by real-time quantitative PCR using SYBR Green Supermix (Bio-Rad). whmD was amplified using the primers whmD RT-F (5'-GTGAGCCATGCACCGCAC-3') and whmD RT-R (5'-CGCTTGGCCTCTCGGGTG-3'). The primers whiB2 RT-F (5'-GCGCCATTCGAGGAACCT-3') and whiB2 RT-R (5'-CAGATGCCGAACCGCTCG-3') were used to amplify whiB2. Both sets of primers amplify 200 nt of the respective gene. The relative fold change of mRNA of the two genes under each of the experimental conditions was measured by normalizing its transcript level to that of sigma factor A (sigA) [amplified by using primers sigA-F (5'-CGATGACGACGAGGAGATCGC-3') and sigA-R (5'-CAGCGCTACCTTGCCGATCTG3')]. Data from three independently derived RNA samples were used to determine mean fold increase in transcript levels.
Acid-fast staining of M. smegmatis 628-53.
Ms 628-53, the conditionally complemented whmD mutant, was cultured in 7H9 broth supplemented with 0.2 % acetamide, grown to an OD600 of 1.0 and washed twice with 7H9 broth, The cells were then resuspended in 7H9 broth, split into two and grown overnight (16 h) in the presence and absence of 0.2 % acetamide. The cultures were washed and resuspended in PBS, and heat-fixed on slides. Staining was carried out with the Difco acid-fast staining kit. Briefly, the smears were first stained with carbolfuchsin for 5 min followed by rinsing off the primary stain with water. The slide was then drained and de-colorized with acid-alcohol until no more stain appeared. Counterstaining was carried out with TB brilliant green for 1 min. After washing off the excess dye with distilled water, the slides were dried and examined at 600x magnification on a Nikon Eclipse E800 microscope under oil. Images were captured using the on-board digital still camera DXM1200 and manipulated using the software package ACT-1 ver2.
Construction of the whmD–gfp fusion and fluorescence microscopy.
To generate pWG, the vector containing the whmD–gfp translational fusion, whmD was first amplified with its promoter region from M. smegmatis genomic DNA using the primers PwhmD27-F (5'-AGCGATATCGAATTCGCGCCCTGGAGC-3') and whmDtag-R (5'-CGGGGTACCGATGATGCCGCGCTTGAG-3'). The gfp gene was then PCR-amplified from the vector pFPV27 (Ramakrishnan et al., 2000) using the primers gfpatg-F (5'-CGGGGTACCTCTAAAGGTGAAGAAT-3') and gfp27-R (5'-ACATGCATGCTTATTTGTACAATTCATC-3'). The whmD fragment digested with EcoRV/KpnI was then ligated to KpnI/SphI-digested gfp amplified as above and EcoRV/SphI-digested pFPV27 in a three-way ligation. The resultant recombinant contained whmD expressed from its own promoter, translationally fused to gfp in the pFPV27 backbone. pFL-whmD was constructed by PCR-amplifying whmD with its promoter region from M. smegmatis chromosomal DNA with the primers PwhmD27-F and whmD27-R (5'-ACATGCATGCCTAGATGATzGCCGCGCTT-3') and cloning the fragment into the EcoRV–SphI sites of pFPV27. pWG, pFLwhmD and the control plasmids pFPV27 and pBEN were transformed into M. smegmatis mc26 1-2c and the transformants cultured to their exponential phase of growth. The cells were then harvested, washed and resuspended in PBS. A drop of this suspension was put on a glass slide under a cover slip, and Gfp fluorescence visualized at 600x magnification on a Nikon Eclipse E800 microscope under oil. Images were captured using the SPOT epi-fluorescence camera and manipulated using the SPOT software package.
M. smegmatis 628-53 complementation assays.
To generate pBP10WG, the whmD–gfp fusion construct was amplified from pWG using the primers pBP10whmD-F (5'-AAAACTGCAGGAATTCGCGCCCTGGAGC-3') and pBZgfp-R (5'-GGACTAGTTTATTTGTA caattcatc-3') and cloned at the PstI–SpeI sites of pBP10 zeo. The plasmid pBP10 zeo whmD was generated by cloning a wild-type copy of M. smegmatis whmD into pBP10 zeo (Raghunand & Bishai, 2006) to serve as a positive control for complementation. For complementation analysis, pBP10 zeo whmD-gfp (WG), pBP10 zeo whmD (W) and the parent plasmid pBP10 zeo (V) were transformed into Ms 628-53 and selected on 0.2 % acetamide-supplemented 7H10 agar plates containing apramycin, hygromycin and zeocin. Acetamide withdrawal was performed as follows: transformants were cultured in 7H9 broth supplemented with 0.2 % acetamide, grown to an OD600 of 1.0, washed twice with 7H9 broth, resuspended in 7H9 broth and grown overnight (16 h) in the absence of acetamide. Following inducer withdrawal, cultures were washed and resuspended in PBS, heat-fixed on slides and stained with carbolfuchsin for 5 min. After washing off the excess dye with distilled water, the slides were dried and examined at 600x or 1000x magnification on a Nikon Eclipse E800 microscope under oil. Images were captured using the on-board digital still camera DXM1200 and edited using the software package ACT-1 ver2. The cell lengths reported represent the mean±SEM of 30 cells.
Sequence analysis.
All sequence alignments were performed on the BCM Search Launcher, Multiple Sequence Alignment package (Baylor College of Medicine) using the ClustalW 1.8 algorithm. The output files were imported into Boxshade 3.21 (http://www.ch.embnet.org) to generate the formatted alignments shown in Figs. 1 and 2. The boundary coordinates of the 5'UTR sequences shown in Fig. 1(c) with reference to their locations upstream to the start codon are as follows: Ms whmD, –187 to –1; and Mt whiB2, –185 to –1.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) seems to support the accuracy of this annotation. The 89 aa putative polypeptide is predicted to have a molecular mass of 9.7 kDa, while that for the 123 aa protein is 13.5 kDa. To address this discrepancy, we carried out immunoblot analysis of M. smegmatis and M. tuberculosis lysates using polyclonal antiserum to WhmD. This antiserum is specific for WhmD despite the presence of other WhiB-like proteins in mycobacteria. The homology within the WhiB paralogues is limited, apart from the conservation of the WhiB signature, so this antibody was not expected to cross-react strongly with any of the other members of the family. As shown in Fig. 1(b), the antibodies specifically react with a protein of approximately 14 kDa in M. smegmatis lysates (lane 1) as documented before (Gomez & Bishai, 2000), and cross-react with a protein of similar size in M. tuberculosis lysates (lane 2). No cross-reactivity was observed in the 9–13 kDa regions where the M. smegmatis orthologues of WhiB1, WhiB3, WhiB6 and WhiB7 would be expected to migrate. These results strongly suggest that translation of WhiB2 originates from a start codon far upstream of that originally predicted, resulting in a protein product 34 aa longer. The use of this site is also corroborated by the presence of a consensus Shine–Dalgarno sequence (AGGAGG), 7 nt upstream of the WhiB2 start codon (Fig. 2c
). We therefore propose that WhiB2 is a 123 aa protein, consistent with the prediction based on sequence alignment of the homologues.
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, the size of the amplicons indicated that the start site lies 200–300 bp upstream of the gsp2 annealing site. Sequencing of the amplified products (Fig. 2b) indicated that whmD and whiB2 transcription initiates from an A residue 134 and 133 nt upstream of their respective start codons. Sequence analysis of the region upstream of the transcription start site revealed the presence of consensus E. coli promoter-like sequences at the –35 and –10 positions (Fig. 2c). The two genes share identical –35 elements, whereas the –10 elements are mismatched by a single base.
The promoters driving the expression of whmD and whiB2 have similar strengths
The extensive conservation in their 5'UTRs and the near-identical promoter elements of whmD and whiB2 led us to postulate that the transcription of the two genes was driven by promoters of comparable strength. To verify this hypothesis, we determined the activity of the two promoters using a -galactosidase-based reporter system. Fragments consisting of 187 and 200 bp of the sequence upstream of the start codons of whmD (PwhmD) and whiB2 (PwhiB2), respectively, were cloned upstream to a promoterless lacZ gene in pSD5B, a mycobacterial promoter-probe vector (Jain et al., 1997). A recombinant plasmid containing a 385 bp fragment from the Mycobacterium bovis hsp60 promoter (Stover et al., 1991) and the empty vector were used as positive and negative controls, respectively. All plasmids were transformed into M. smegmatis and promoter activity was estimated by -galactosidase assays with cell lysates from transformants harvested in their exponential phase of growth. PwhmD (185.53±4.79 nmol min–1 mg–1) and PwhiB2 (110.73±8.16 nmol min–1 mg–1) showed comparable activities, substantially higher than the control plasmid (12.99±0.65 nmol min–1 mg–1). However, the two promoters were considerably weaker than the hsp60 promoter, which showed a -galactosidase activity of 1107.16±39.1 nmol min–1 mg–1. Although the activities of the two promoters were significantly higher than the baseline, they could be categorized as weak promoters, based on the comparison to the activity observed with the M. bovis hsp60 promoter. We surmise, based on the observed promoter strengths, that the two genes are expressed at low levels in their respective hosts. Since most DNA-binding proteins are expressed at low levels in vivo, these observations of low-level reporter gene expression are consistent with the premise that WhmD and WhiB2 are regulatory proteins.
Physiological consequences of perturbing cellular levels of WhmD and WhiB2
whmD has been shown to be an essential gene in M. smegmatis, and lack of the gene product leads to severe defects in septation (Gomez & Bishai, 2000). To determine if whiB2, the orthologue of M. smegmatis whmD, plays a functionally equivalent role in M. tuberculosis, we examined the effects of artificially perturbing whmD and whiB2 levels in their cognate hosts. If the consequences of this perturbation were similar in the two organisms, this would be a pointer to the two genes being operational counterparts. To alter normal cellular levels of the two proteins, whmD and whiB2 were cloned in the sense and anti-sense orientations, under the control of the acetamide-regulatable promoter Pace (Parish et al., 1997) in pAce, a derivative of the plasmid pCK0218 (Manabe et al., 1999) (Fig. 3a). The plasmids were transformed into their cognate mycobacterial hosts and RNA was extracted from these transformants grown under inducing conditions. Quantitative RT-PCR analysis was used to confirm that all the transformants showed the anticipated changes in whmD/whiB2 transcript levels (Fig. 3b, c). The phenotypic effects of altering WhmD/WhiB2 levels were assessed by examining the transformants for colony size, cellular morphology and viability under inducing conditions.
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, panels S+, A+) while control transformants containing the empty plasmid alone in the presence of acetamide induction showed no such defect (Fig. 4a, panel V+). In addition, the M. smegmatis whmD sense and anti-sense transformants also exhibited elongation/filamentation, as assessed by carbolfuchsin staining followed by light microscopy (Fig. 4b, panels S+, A+), while control transformants showed normal cellular morphology (Fig. 4b, panel V+). Cell length measurements showed that transformants containing either the sense or the anti-sense construct were significantly elongated compared to controls (Table 1a). Estimation of viability by comparing c.f.u. of transformants grown under uninduced vs induced conditions revealed that overexpression of either construct led to a decrease in viability of M. smegmatis as compared to controls (Fig. 4c). The loss in viability on overexpression of whmD can be associated with the presence of multiply septate bacteria seen in a whmD-overexpressing strain (Gomez & Bishai, 2000), since such cells are unlikely to contribute to c.f.u. counts. All the observed phenotypes are expected, since whmD is an essential cell division gene, and reduction in WhmD levels is lethal to normal cellular physiology. In keeping with this premise, expression of anti-sense constructs of dnaA, an essential DNA replication gene had similar consequences in M. smegmatis (Greendyke et al., 2002).
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, panels S+, A+ vs V+), displayed significant filamentation (Fig. 5b, panels S+, A+ vs V+, Table 1b) and showed reduced viability as compared to control transformants (Fig. 5c). In M. tuberculosis, anti-sense overexpression of whiB2 resulted in a 90 % loss of viability, strongly suggesting that whiB2 is an essential gene in M. tuberculosis.
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Cell envelope changes associated with alteration of WhiB2 or WhmD concentrations in their cognate hosts
On closer observation of the morphology of M. tuberculosis transformants overexpressing whiB2, we found that these colonies were glossy and spherical, whereas the corresponding controls were rough and flat (Fig. 6a, S+ vs S–). This phenotype closely resembles the appearance of the fbpA mutant of M. smegmatis, where the change in colony morphology was attributed to a decrease in production of ,'-trehalose dimycolate (Nguyen et al., 2005). This raises the possibility that WhiB2 may be associated with regulating the expression of components of the M. tuberculosis cell envelope. However, no such change was observed in transformants expressing the anti-sense construct of whiB2. Curiously, the conditionally complemented M. smegmatis whmD mutant Ms 628-53 (Gomez & Bishai, 2000) was observed to be non-acid-fast under conditions of acetamide withdrawal (Fig. 6b). It is known that the carboxyl and hydroxyl groups of cellular lipids like mycolic acid are essential to the acid-fast reaction of mycobacteria (Harada, 1976), which could imply that in the absence of WhmD there are changes in modification of the surface lipids of M. smegmatis. These two observations seem to point to the involvement of these proteins in cell-envelope-related transactions in their respective hosts.
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) led us to hypothesize that WhmD perhaps plays a structural role in cell division by localizing to developing septa. To determine its subcellular localization, whmD was fused to the gene encoding green fluorescent protein (Gfp), in the vector pFPV27 (Ramakrishnan et al., 2000), with the fusion protein being expressed from the whmD promoter. A construct carrying the full-length whmD gene, and the empty vector, were used as negative controls. The plasmid pBEN (Saviola et al., 2003) expressing gfp from the hsp60 promoter (Stover et al., 1991) served as the positive control for Gfp fluorescence (Fig. 7a) The expression of the whmD–gfp fusion was confirmed by RT-PCR analysis (data not shown) and Western blotting using polyclonal antisera to GFP (Fig. 7b). To assess the functionality of the fusion protein, the whmD–gfp construct was cloned into pBP10 zeo (Raghunand & Bishai, 2006), a derivative of pBP10 (Bachrach et al., 2000), transformed into Ms 628-53, a conditionally complemented whmD mutant (Gomez & Bishai, 2000), and its complementation phenotype evaluated in a filamentation rescue assay (Raghunand & Bishai, 2006). Under non-permissive conditions, Ms 628-53 containing the control plasmid showed extensive filamentation, with a mean cell length of 11.99±0.56 µm. Transformants containing both the whmD–gfp fusion (mean cell length 4.0±0.27 µm) and a WT whmD construct (mean cell length 3.69±0.22 µm) were able to rescue this phenotype (Fig. 7c), establishing that the fusion construct was indeed functional. Upon Gfp fluorescence microscopy, no fluorescence was seen in cells containing the negative control plasmids pFPV27 and pFLwhmD, as expected. Transformants of M. smegmatis expressing the WhmD–Gfp fusion from the plasmid pWG displayed a diffuse pattern of fluorescence, mirroring that observed with pBEN (Fig. 7d). No subcellular localization or distinct foci of fluorescence were detected in cells in any of the several fields examined, which suggests a regulatory role for WhmD in cell division as opposed to a structural role where WhmD would form a part of the septation complex. WhmD and WhiB2 are therefore assumed to influence the process of cell division by regulating the expression of genes directly or indirectly involved in septation. The identity of these genes is currently unknown. In the recent past, defects in several different gene products have been associated with mycobacterial growth and morphology. These include the eukaryotic-like serine/threonine kinases PknA (Chaba et al., 2002; Kang et al., 2005), PknB (Kang et al., 2005) and PknF (Deol et al., 2005) as well as the gene encoding phosphomannose isomerase (Patterson et al., 2003). In addition penicillin-binding proteins, which are septum-specific peptidoglycan synthetic enzymes, are required for cell division, illustrated by the induction of mycobacterial filamentation on treatment with -lactam antibiotics (Mizuguchi et al., 1985). It is conceivable that WhmD/WhiB2 regulate the expression of one or more of the above-mentioned genes, or other annotated cell-division-associated genes. Transcriptional studies to define the WhiB2 regulon in M. tuberculosis may help clarify the role of this protein in cytokinesis.
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; Margalit et al., 2004; White et al., 2002). These molecules have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery. Although significant progress has been made in our understanding of the physiology of M. tuberculosis, no definitive explanations exist for its extremely slow growth rate. Understanding the genetic mechanisms by which M. tuberculosis regulates the process of cell division is imperative to our overall comprehension of the process of latency. Of the 20 annotated cell division genes in the M. tuberculosis genome (Cole et al., 1998), 10 are predicted to be essential and theoretically are targets for therapeutic intervention. The delineation of the mycobacterial cell division cycle could therefore play a vital role in stimulating the development of novel anti-tuberculosis agents. Here we demonstrate that M. tuberculosis WhiB2 is functionally equivalent to its homologue WhmD in M. smegmatis. Our studies strongly suggest that whiB2 is an essential gene and that its protein product is expected to regulate the expression of genes involved in mycobacterial cell division. WhiB2 therefore represents yet another cell division protein which could be a candidate for rational drug design.
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ACKNOWLEDGEMENTS |
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Received 9 February 2006;
revised 6 May 2006;
accepted 24 May 2006.
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