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Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 70 0009, India
Correspondence
Joyoti Basu
joyoti{at}vsnl.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
A supplementary figure showing an alignment of the deduced amino acid sequences of the PBPA orthologues from different mycobacteria is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Ser/Thr/Tyr-specific protein phosphorylation represents one of the fundamental regulatory mechanisms in eukaryotes (Krebs & Fischer, 1989). Genomics has now documented the presence of Ser/Thr protein kinases (STPKs) in prokaryotes as well (Leonard et al., 1998; Madec et al., 2002; Shi et al., 1998). M. tuberculosis has 11 putative STPKs, one phospho-Ser/Thr phosphatase and two Tyr phosphatases (Cole et al., 1998). Eight of the 11 STPKs identified in the M. tuberculosis genome appear to be transmembrane proteins, with a putative extracellular sensor domain and an intracellular kinase domain. Among these, PknA, PknB, PknD, PknE, PknF, PknG and PknH catalyse autophosphorylation (Av-Gay et al., 1999; Chaba et al., 2002; Cowley et al., 2004; Koul et al., 2001; Molle et al., 2003; Ortiz-Lombardia et al., 2003; Peirs et al., 1997), and conserved phosphorylated residues within the activation loop are necessary for activity (Duran et al., 2005). Two recent reports have identified Wag31, the mycobacterial orthologue of Bacillus subtilis DivIVA, as a substrate of PknA (Kang et al., 2005), and GarA as a substrate of PknB (Villarino et al., 2005). PknH has been shown to phosphorylate EmbR, which is a putative transcriptional activator that belongs to the OmpR-like family (Molle et al., 2003), whereas PknF phosphorylates an ABC transporter encoded by Rv1747 (Molle et al., 2004).
The final stages of peptidoglycan synthesis are carried out by penicillin-binding proteins (PBPs), a group of serine acyl transferases involved in cell wall expansion, cell shape maintenance, septum formation and cell division (Goffin & Ghuysen, 1998; Popham & Young, 2003). Höltje (1998) postulated the existence of distinct holoenzyme complexes for cell division and elongation. M. tuberculosis has the ability to shift from a state of active replication to a latent state with minimum replication. It is likely that the regulation of cell wall synthesis and cell division in response to varying environmental conditions is central to this process.
Based on in silico analyses and sequence/motif similarity-based clustering, the ORF Rv0016c encodes a putative PBP, PBPA, which belongs to a cluster termed class B-like I (Goffin & Ghuysen, 2002). PBPA lacks one conserved motif in the N-terminal module. Comparison of the derived amino acid sequences of PBPA from different mycobacterial species shows that this protein is highly conserved among M. tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Mycobacterium avium subsp. paratuberculosis and Mycobacterium smegmatis (see supplementary Fig. S1, available with the online version of this paper). This suggests that the role of this protein is likely to be conserved among mycobacteria. We have focused our present studies on PBPA and its possible regulation by the STPK PknB (encoded by Rv0014c), and PstP (encoded by Rv0018c), a serine/threonine phosphatase (Av-Gay & Everett, 2000) similar to the PP2C family of enzymes. Our results support the idea that signal transduction by serine/threonine kinases and phosphatases provides a novel means of regulating cell septation and cell shape in mycobacteria, and possibly other Gram-positive bacteria that harbour counterparts of these proteins.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Cosmid MTCY10H4 was a generous gift from Stewart Cole, Institut Pasteur, Paris, France. Enzymes used to manipulate DNA were from Roche Applied Sciences. All constructs made by PCR were sequenced to verify their integrity.
Bacterial strains and growth conditions.
Escherichia coli strains were grown in Luria–Bertani (LB) Miller (Difco) medium. Mycobacterial strains were grown in Middlebrook (MB) 7H9 (Difco) or Lemco medium (per litre: 5 g Lemco powder, 5 g NaCl, 10 g Bacto peptone) (Parish et al., 2001) supplemented with 0·05 % Tween 80. Antibiotics were used at the following concentrations: ampicillin, 75 µg ml–1; kanamycin monosulfate, 50 µg ml–1 for E. coli and 25 µg ml–1 for M. smegmatis; and hygromycin B, 100 µg ml–1 for E. coli and 50 µg ml–1 for M. smegmatis.
Genetic manipulations.
pbpA (Rv0016c) was amplified from cosmid MTCY10H4 using the primer pair 5'-TTGGATCCCATATGAACGCCTCTCTGCGCCGA-3'(sense) and 5'-TTGAATTCTCATGGTTCCCCCTGCAG-3'(antisense), and cloned between the asymmetric BamHI and EcoRI sites (underlined) of pK19 to generate pJBA201. After sequencing, the gene was excised with BamHI and EcoRI and cloned between the same sites of pET28a to generate pJBA202.
The gene encoding the cytosolic kinase domain of PknB (M1–V276) [PknB-Kin] was amplified from MTCY10H4 using the primer pair 5'-AAATACATATGACCACCCCTTCCCA-3' (sense) and 5'-TTCTCGAGCACGCGGACCAGGTCGGC-3' (antisense), and cloned between the asymmetric NdeI and XhoI sites (underlined) of pET28a to generate pJBA302. To create a deletion within the catalytic kinase core of PknB, pJBA302 was digested with SalI and a 297 bp fragment encoding V95–D194 was discarded. The larger fragment was gel-purified and religated to generate pJBA303.
To coexpress PBPA and PknB-Kin of M. tuberculosis, both the genes were cloned at the two multiple cloning sites (MCS) of pET Duet-1 (Novagen). Briefly, pbpA with the N-terminal His tag was excised from pJBA202 using NcoI and HindIII and cloned in the MCS1 of pET Duet-1 at the same sites to generate pJBA203. The pknB-kin gene was excised from pJBA302 using NcoI and XhoI and cloned in pBAD mycHisB (Invitrogen) between the same sites to generate pJBA204 encoding pknB-kin carrying a C-terminal myc epitope. This gene was then amplified by PCR using the primers 5'-TTGGATCCAATG ACCACCCCTTCCCA-3'(sense) and 5'-ATGGAATTCTCAGTCGACGGCGCATTCAGATC-3' (antisense). The amplicon was digested with BamHI (underlined) and cloned in the MCS2 of pJBA203 between the BglII and EcoRV sites to generate pJBA205. A similar strategy was employed to generate the plasmid pJBA206 encoding pbpA and myc-pknB-KD. The sequences of all clones were confirmed by DNA sequencing.
Expression and purification of PknB-Kin and PknB-KD.
Transformed cells were grown to an OD600 of 0·6. IPTG was added to a final concentration of 0·1 mM, and growth was continued at 37 °C with shaking for 2 h. Cells were harvested and broken by sonication in 10 mM Tris/HCl, pH 7·5, containing 1 mM MgCl2, 1 µg DNase ml–1, 1 µg leupeptin, 1 ml–1 µg aprotinin ml–1, 1 µg pepstatin ml–1 and 1 mM Pefabloc. The post-sonicate supernatant was loaded on a Ni2+-NTA-agarose column equilibrated with 50 mM sodium phosphate, pH 7·4, containing 0·5 M NaCl (buffer A). After washing the column with 100 mM imidazole in buffer A, PknB-Kin was eluted with 200 mM imidazole in buffer A. PknB-KD was similarly expressed and purified.
Expression and purification of PBPA.
E. coli BL21(DE3)/pJBA202 was grown to an OD600 of 0·6. IPTG was added to a final concentration of 0·1 mM, and growth was continued at 37 °C with shaking for 4 h. Cells after sonication were centrifuged at 5000 g for 5 min, the pellet was discarded and membranes were pelleted from the supernatant by centrifugation at 100 000 g for 1 h. PBPA was extracted by suspension of membranes in 10 mM Tris/HCl (pH 8) containing 1 % (w/v) cetyltrimethylammonium bromide (CTAB) at 4 °C for 40 min. After centrifugation at 100 000 g for 30 min, the supernatant was removed and loaded on to a 1 ml Ni2+-NTA agarose column equilibrated with buffer A. After washing the column with buffer A containing 100 mM imidazole, PBPA was eluted with a gradient of 100–200 mM imidazole/1 % (w/v) CTAB in buffer A. All steps were carried out at 4 °C.
Penicillin-binding assays.
Purified 6xHis-tagged PBPA (His-PBPA) or membranes from cells expressing PBPA were analysed by SDS-PAGE, Coomassie blue staining and fluorography of the gels after labelling of the protein with different concentrations of benzyl[14C]penicillin for 30 min at 37 °C as described by Granier et al. (1994).
In vitro kinase assay.
Purified PknB-Kin (1 µg) was added to a 25 µl reaction volume containing 2 µg purified PBPA (or its derivatives) in kinase buffer (25 mM Tris/HCl, pH 7·5, 5 mM 
-glycerophosphate, 2 mM DTT, 2 mM MnCl2, 0·1 mM sodium orthovanadate). The reaction was started by addition of 1 µCi (37 kBq) [
-32P]ATP (Jonaki, Board of Radiation and Isotope Technology, Hyderabad, India), continued at 25 °C for 30 min and stopped by adding 5 µl 5x Laemmli sample buffer. The samples were boiled and separated by SDS-PAGE. Gels were dried and exposed to Kodak XAR film for autoradiography. In some experiments, proteins separated by SDS-PAGE were electrotransferred and Western blotting was performed using monoclonal anti-phosphothreonine (Sigma), anti-phosphoserine (Sigma) or anti-phosphotyrosine (Cell Signalling Technology) antibody.
Coexpression of PBPA and PknB-Kin and analysis of phosphorylation.
E. coli BL21(DE3) transformed with either pJBA205 or pJBA206 was grown at 37 °C to mid-exponential phase. Induction was started by the addition of IPTG (0·05 mM) and growth was continued for 4 h at 37 °C. Induced samples were denatured with SDS gel denaturing buffer, separated by SDS-PAGE, transferred to PVDF membranes and blotted with anti-His antibody to check the expression of His-PBPA. The membrane was then reprobed with anti-myc antibody to confirm the expression of myc-tagged PknB-Kin or PknB-KD. To analyse the phosphorylation of both PBPA and PknB, proteins were extracted from the induced cells harbouring either pJBA205 or pJBA206 using Cell Lytic BII bacterial extraction reagent (Sigma) according to the manufacturer's recommendations. Extracted proteins were separated by 10 % SDS-PAGE, transferred to PVDF membranes and blotted with anti-phosphothreonine monoclonal antibody in order to detect the phosphorylated threonine residues. Membranes were reprobed with anti-His or anti-myc antibody to confirm the identity of the bands as PBPA and PknB respectively.
Construction of truncated derivatives of PBPA, site-directed mutagenesis, expression and purification.
A 690 bp fragment encoding amino acid residues M1–G229 of PBPA was excised from pJBA202 with BamHI and NotI and cloned between the same sites in pET28a, generating pJBA207. A 1·1 kb fragment encoding amino acid residues L127–P491 of PBPA was excised from pJBA202 using NruI and EcoRI, and cloned between the PvuII and EcoRI sites of pBADHisB (Invitrogen), generating pJBA208. pJBA208 was digested with NcoI and EcoRI and the fragment encoding 6xHis-tagged [L127–P491]PBPA was cloned between the same sites of pET28a to generate pJBA209. To generate truncated constructs, the gene fragments encoding E240–E320, Q316–P491, P346–Q420 and V383–P491 of PBPA were amplified using the sense (s) and antisense (as) primer pairs 5'-ATGGATCCATGGAACAGCTGACTGCGGCG-3' (s) and 5'-ATGAATTCTTCCGCCACTTGCAGCGG-3' (as); 5'-ATGGATCCATGCAAGTGGCGGAATCAACC-3' (s) and 5'-ATGAATTCTCATGGTTCCCCCTGCAG-3'(as); 5'-ATGGATCCATGCCGCTAGCGAACGCAGAA-3' (s) and 5'-ATGAATTCCTGCACGCCGGGGATTGC-3' (as); and 5'-ATGGATCCATGGTCGGATACCAGCAGCGC-3' (s) and 5'-ATGAATTCTCATGGTTCCCCCTGCAG-3' (as) respectively. All amplicons were cloned between the asymmetric BamHI and EcoRI sites of pET28a. The genes encoding [Q316–P491]PBPA harbouring mutations T362A, or T437A, or T362A and T437A, in pET28a, were generated by the principle of overlap extension PCR, giving rise to pJBA216, pJBA217 and pJBA218 respectively. To generate constructs carrying the mutated full-length pbpA genes in pET vectors, pJBA216 or pJBA217 or pJBA218 was digested with NheI (located 1038 bp downstream from the start of the pbpA gene) and EcoRI, and the resulting fragment was first cloned between the NheI and EcoRI sites of pJBA201. The mutated full-length pbpA genes were then excised from these constructs and cloned between the BamHI and EcoRI sites of pET28a. For dual expression in pET-DUET, the mutated genes along with the 6xHis-encoding sequence were PCR-amplified from the pET28a-derived constructs and cloned between the NcoI and EcoRI sites of pJBA205 (replacing the wild-type gene) to generate constructs harbouring the mutated PBPA genes and the gene for PknB-Kin.
The constructs of pbpA (truncations or point mutations) in pET-28a were expressed by induction with 0·1 mM IPTG for 3 h at 37 °C and purified by chromatography on Ni2+-NTA agarose.
Construction of a suicidal delivery vector for inactivation of PBPA in M. smegmatis.
A pbpA deletion mutant of M. smegmatis was constructed employing the allelic replacement method of Parish & Stoker (2000) using p2NIL, the gene-manipulating vector without any mycobacterial origin of replication, and pGOAL19, the marker gene cassette-containing vector. For disruption of pbpA of M. smegmatis, a suicidal delivery vector, pJBA104, was constructed. Briefly, 2083 bp of DNA fragment containing flanking sequences upstream from the start and downstream from the end of the pbpA gene was PCR amplified using the genomic DNA of wild-type M. smegmatis mc2155 as template and the primer pair 5'-TACAACATTTTCGACCACGTG-3' (sense) and 5'-TATGGATCCGGACAGCGTGAC-3' (antisense). The amplicon was cloned in pUC19 between the BamHI (underlined) and SmaI sites to give pJBA101. To create an in-frame deletion construct lacking amino acid residues G47–E222 (including the active-site serine) pJBA101 was digested with SmaI and ScaI, a 525 bp fragment was eliminated and the 4240 bp larger fragment was religated to generate pJBA102. The truncated pbpA gene was excised from pJBA102 and cloned in p2NIL between the PmlI and BamHI sites to generate pJBA103. The final delivery vector, pJBA104, was generated by excising the PacI cassette (hyg, pAg85-lacZ, phsp60-sacB), which confers blue colour, hygromycin resistance and sucrose sensitivity, from the vector pGOAL19 and cloning it into pJBA103 bearing the truncated pbpA allele.
Isolation of the pbpA-inactivated mutant.
pJBA104 was electroporated into electrocompetent cells of M. smegmatis mc2155. The cells after electroporation were revived in 1 ml Lemco broth for 3 h and then plated on Lemco agar supplemented with hygromycin B, kanamycin and X-Gal (50 mg ml–1). Blue colonies were streaked onto Lemco agar without any selection to enhance the recombination process. Cells were then plated onto Lemco agar supplemented with 2 % sucrose and 50 mg X-Gal ml–1. The white colonies were restreaked onto replica plates containing X-Gal and sucrose, either without or with kanamycin and hygromycin. Genomic DNA was prepared from the white, KanS, HygS, SucR colonies generated by double crossover. Genotype was analysed by PCR using genomic DNA from mc2155 or from the KanS, HygS and SucR colonies as template and the primer pair 5'-ATCATGGTGTTGATCGTGCTG-3'(sense) and 5'-TCCTGCAGCGACACGGTGGGT-3' (antisense) which flanked the deletion site.
Complementation of PBPA of M. tuberculosis in the PBPA-KO strain using an integrating vector.
Complementation of PBPA was achieved by constructing the integrating vector pJBA105, which contains a hygromycin-resistance cassette along with a positive-selection L5 integrase cassette. For this, first pbpA was cloned in pOLYG under control of the hsp60 promoter to generate pJBA106. hsp60-pbpA was excised from pJBA106 by digesting with XbaI and HindIII and cloned in pUC19 between the sites for these two enzymes to give pJBA107. A 3·757 kb Hyg-integrase cassette was excised from pUC-HY-INT (Mahenthiralingam et al., 1998) by digesting with HindIII and cloned at the single HindIII site of pJBA107 to generate the integrating vector pJBA105. pJBA105 was electroporated into electrocompetent cells of pbpA-inactivated M. smegmatis in order to complement the mutated pbpA with a single copy of the wild-type pbpA. The presence of pbpA was confirmed by PCR with pbpA-specific primers.
Complementation of PBPA by ectopic expression in the PBPA-KO strain.
Complementation of the pbpA genes in the pbpA-inactivated M. smegmatis was carried out as follows. Briefly, the 6xHis-pbpA gene or its T362A, T437A mutant was cloned in pCKB115 (Choudhuri et al., 2002) harbouring an hsp60 promoter. The genes along with the hsp60 promoter were excised with XbaI and HindIII and cloned between the same sites of pOLYG (Gaora et al., 1997). For ectopic expression of PBPA or its mutant, the respective constructs were electroporated into pbpA-inactivated M. smegmatis (designated PBPA-KO). Expression of PBPA was confirmed by Western blotting of the membranes with anti-His antibody.
Growth curve analysis.
Starter cultures were prepared by inoculating M. smegmatis mc2155 or its derivatives from glycerol stocks into Lemco medium. These starter cultures were used to inoculate liquid media to a theoretical OD600 value of 0·05. Cultures were grown at 37 °C in a shaking incubator and samples were collected at different time intervals, passed five times under sterile conditions through a 26-gauge needle and vortexed. Serial dilutions were plated on Lemco agar plates to determine the number of c.f.u. after incubation at 37 °C.
Electron microscopy.
Exponential-phase cultures of different M. smegmatis strains were harvested, washed three times with 0·15 M cacodylate buffer, treated with 1 % osmium tetroxide for 1 h at room temperature, centrifuged, and the collected cells were fixed with 0·15 M cacodylate buffer containing 2 % glutaraldehyde for 2 h at room temperature. Cells were again collected after centrifugation and fixed overnight with 0·15 M cacodylate buffer containing 1 % osmium tetroxide at 4 °C. The cells were dehydrated using graded ethanol (10 %, 30 %, 50 %, 70 % and 100 %), mounted on microscope stubs and sputter coated on the sputter coater. The samples were then examined under a Leica S440 scanning electron microscope.
Fluorescence microscopy.
Immunostaining was adapted from the method of Harry et al. (1995) with a few modifications. Cells were fixed by incubation for 15 min at room temperature and 45 min on ice in 2·5 % (v/v) paraformaldehyde, 0·04 % (v/v) glutaraldehyde, 30 mM sodium phosphate (pH 7·5). After washing in PBS, the cells were permeabilized by exposing to 2 % toluene for 2 min, and immediately transferred to slides. The slides were washed with PBS, air-dried, dipped in methanol (–20 °C) for 5 min and then in acetone (–20 °C) for 30 s and allowed to dry. After rehydration with PBS, the slides were blocked for 2 h at room temperature with 2 % (w/v) BSA-PBS and incubated for 1 h with appropriate dilution of anti-His antibody in BSA-PBS. The slides were washed extensively with PBS and then incubated with a 1 : 1000 dilution of Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) in BSA-PBS. For staining of DNA, 2 µg 4',6-diamino-2-phenylindole (DAPI) ml–1 or 0·1 µg propidium iodide ml–1 was included with the secondary antibody. When propidium iodide was used to stain DNA, the slides were incubated in 100 µg RNase A ml–1 in PBS to remove RNA from the preparation. Wheat germ agglutinin (WGA)-Alexa Fluor 488 (Molecular Probes) (2 µg ml–1) was used for staining of cell walls (Sizemore et al., 1990). WGA is a lectin that binds to oligomers of N-acetylglucosamine and N-acetylmuramic acid. After extensive washing with PBS, the slides were mounted using 50 % glycerol. In controls for assessing specificity of the secondary antibodies, the incubation with the primary antibodies was omitted.
Protein phosphatase assay.
The catalytic domain of PstP was cloned, expressed and purified as described by Boitel et al. (2003). Dephosphorylation of phosphorylated PBPA or myelin basic protein (MBP) was assayed as described by Boitel et al. (2003) with some modifications. Phosphorylated PBPA and MBP were obtained by performing the kinase reaction using [-32P]ATP and PknB as described above. Dephosphorylation assays were carried out in a 25 µl reaction mixture containing 50 mM HEPES buffer pH 7·5, 0·1 mM EDTA, 1 mM DTT and 5 mM MnCl2. The reaction was started with the addition of purified PstP (100 ng per 25 µl) and incubated for different periods of time at 30 °C. The reaction was terminated by adding SDS gel denaturing buffer and boiling for 10 min. Proteins were separated by SDS-PAGE and the disappearance of radiolabelled bands corresponding to phosphorylated PBPA or MBP was monitored by autoradiography. In separate experiments, dephosphorylation was carried out in the presence of 50 mM NaF.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Purified His-PBPA bound benzyl[14C]penicillin in a concentration-dependent manner, reaching saturation at a penicillin concentration of 10–4 M (Fig. 1b), validating its function as a PBP. Membrane-associated PBPA bound benzyl[14C]penicillin with a similar affinity (Fig. 1b). The membrane-associated PBP and the purified protein therefore appeared to be functionally indistinguishable.
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The suicidal delivery vector pJBA104 (harbouring a truncated M. smegmatis pbpA gene) was used for homologous recombination. As expected in the event of successful inactivation of pbpA, a 250 bp PCR product was obtained in the case of two of the transformants, as opposed to a 750 bp PCR product in the case of wild-type mc2155 (Fig. 2). The PCR products were sequenced to verify the identity of the bands and the deletion within the pbpA gene. The mutated strain will be referred to as PBPA-KO.
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). These findings suggested that PBPA is required for maintenance of total cell mass. The in vitro growth of the mutant strain on Lemco solid medium and on slants was poor compared to that of the wild-type strain.
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). The signal was stronger than that of the integrated His-PBPA. All experiments were subsequently performed with the plasmid-encoded system. PBPA-KO complemented with plasmid-encoded His-PBPA showed a substantial recovery of growth characteristics, approaching that of the wild-type (Fig. 3a). Scanning electron microscopy showed that the complemented cells regained wild-type morphology (Fig. 4), confirming the role of PBPA in maintenance of normal cell growth and morphology, and supporting the view that PBPA of M. tuberculosis and M. smegmatis fulfil similar functions.
Staining of PBPA-KO cells with WGA-Alexa Fluor 488
WGA-Alexa Fluor 488 staining is useful for visualizing cell wall and septa. Fluorescence-labelled WGA binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of Gram-positive bacteria (Sizemore et al., 1990), including M. smegmatis. WGA-Alexa Fluor 488 staining of the PBPA-KO strain failed to detect staining between nucleoids (Fig. 5a–c), indicating that peptidoglycan synthesis had not occurred at the expected position of septa. This view was supported by the observation that the mutant contained a larger average number (4±1) of nucleoids per cell. The majority of the cells of the knockout strain (80 %) showed 4 nucleoids per cell.
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). To demonstrate that the anti-His antibody was reacting specifically with His-PBPA, cells from exponential-phase cultures of the wild-type and PBPA-KO were subjected to the same treatments. No red fluorescence was observed in either of these cells (data not shown). Nucleoids were visualized by staining with DAPI (blue) (Fig. 5f). Staining with WGA-Alexa 488 (Fig. 5e) showed formation of septal peptidoglycan between nucleoids. Overlaying the three images obtained by staining for PBPA (red), for peptidoglycan (green) and for nucleoids (blue) showed that PBPA colocalizes (yellow) with newly formed septal peptidoglycan in the gap between segregated nucleoids (Fig. 5g) in the case of the complemented strain. These observations clearly suggested that PBPA localizes to the septum. To exclude the possibility that the His tag directs PBPA to mid cell, an irrelevant His-tagged protein was expressed in M. smegmatis PBPA-KO. This protein could not be located at mid-cell (data not shown).
Coexpression of PknB and PBPA of M. tuberculosis in E. coli and evidence that PBPA is phosphorylatable by PknB
The interest in PBPA and PknB arises from the fact that they are encoded by neighbouring ORFs, one being a protein likely to be important in cell division and growth, the other an STPK. PknB is a 626 aa transmembrane protein with an intracellular N-terminal kinase domain and an extracellular C-terminal domain. The kinase domain of PknB has been crystallized (Young et al., 2003). Boitel et al. (2003) have demonstrated that the kinase domain of PknB undergoes autophosphorylation, shows a preference for Mn2+ as the divalent cation required for kinase activity, and phosphorylates the model substrate MBP. We attempted to address the question whether PBPA is an endogenous substrate of PknB. The coexpression of His-PBPA and myc-PknB-Kin (or myc-PknB-KD) in E. coli was visualized by Western blotting using anti-His (Fig. 6a) and anti-myc (Fig. 6b) antibodies. Using this dual expression system, we tested whether PBPA is phosphorylatable by PknB. Electrophoretically separated extracts of coexpressed proteins developed positive bands after probing with anti-phosphothreonine antibody when the kinase domain of PknB was coexpressed with PBPA, but not when its kinase-dead counterpart was coexpressed (Fig. 6c). The lack of phosphorylation of PBPA when it was coexpressed with a kinase-inactive mutant of PknB confirmed the specificity of the phosphorylation of PBPA by PknB. No bands developed when anti-phosphoserine antibody was used to detect phosphorylation of PBPA by PknB (data not shown), indicating that phosphorylation occurred specifically on threonine residues.
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, recombinant, purified PknB-Kin (Fig. 6d) was capable of autophosphorylation and also phosphorylated MBP in vitro in the presence of Mn2+ (Fig. 6e). On the other hand, PknB-KD did not undergo autophosphorylation and could not phosphorylate MBP under similar conditions. We next tested whether PBPA was a direct substrate for PknB, by performing an in vitro kinase assay using purified proteins. Phosphorylation of PBPA was observed in the presence of PknB-Kin, but not in the presence of PknB-KD (Fig. 6e).
Determination of the site(s) of PknB-mediated phosphorylation of PBPA
Considering that PknB appeared to phosphorylate PBPA on threonine residues, we attempted to map the site(s) of PknB-mediated phosphorylation of PBPA. Primary sequence analysis revealed the presence of 38 threonine residues in PBPA. The NetPhos 2.0 program available at http://www.cbs.dtu.dk (Blom et al., 1999) was used to map putative phosphorylatable threonine residues. Five putative high-scoring threonine residues at positions 26, 223, 314, 362, 437 were identified as candidate phosphorylatable residues. A number of His-tagged truncated constructs as well as threonine point mutants of PBPA were generated covering the entire PBPA amino acid sequence. The results of in vitro kinase assays using these constructs are summarized in Table 1. The candidate phosphorylatable residues predicted by NetPhos in each construct are indicated. Taking together the results obtained with each construct, T437 and T362 appeared to be the two residues that were phosphorylated by PknB. The results of in vitro phosphorylation of the truncated construct [Q316–P491]PBPA (or its threonine mutants) by PknB-Kin followed by Western blotting with anti-phosphothreonine antibody are depicted in Fig. 6(f). No band was observed in the T362A, T437A double mutant. Bands were obtained using the construct [Q316–P491]PBPA when only one of the two threonine residues at positions T437 or T362 was mutated to alanine, indicating that PknB-mediated phosphorylation occurred at both these residues. Coexpression of PBPA(T362A, T437A) with PknB-Kin in E. coli showed that this mutant was not phosphorylated by PknB (Fig. 6c). Taken together, these results clearly indicated that threonines 362 and 437 of PBPA of M. tuberculosis represent the residues phosphorylatable by PknB.
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). CLUSTAL W analysis of the sequences of PBPA of different mycobacterial species showed that T437 was conserved across slow- and fast-growing species. On the other hand, T362, while being conserved in M. tuberculosis, M. bovis, M. leprae and M. avium subsp. paratuberculosis, was not conserved in M. smegmatis (supplementary Fig. S1). It therefore appeared likely that phosphorylation at T437 could play an essential role in regulating cell division across mycobacterial species. Taking this together with the fact that the T437A mutant failed to complement PBPA-KO, we focused our attention on the effects of complementation of PBPA-KO with PBPA (T437A) on cell morphology and localization of PBPA. When PBPA-KO was complemented with His-pbpA(T437A), the cells remained unaltered in morphology as observed by scanning electron microscopy (Fig. 4). His-PBPA(T437A) failed to localize to the septum when observed by immunofluorescence microscopy of the complemented strain. WGA-Alexa 488 and DAPI staining revealed a lack of septum formation and an increase in the average number of nucleoids per cell, just as observed in the case of PBPA-KO (data not shown).
Dephosphorylation of phosphorylated PBPA by PstP
PstP belongs to the PP2C family of serine/threonine phosphatases, which are characterized by their dependence on the divalent cations Mg2+ or Mn2+ (McCarty & Chory, 2000; Pullen et al., 2004). Given that reversible protein phosphorylation provides a ubiquitous signalling mechanism that defines the response to changing environmental conditions, we asked the question whether PknB and PstP could exert opposing effects on PBPA to coordinately regulate cell division. To address this question, purified PBPA was phosphorylated by PknB using radiolabelled ATP. The phosphorylated protein was then incubated with purified PstP. Phosphorylated MBP was also used as a positive control. The time-dependent disappearance of the label associated with both MBP and PBPA (Fig. 7) confirmed that MBP and PBPA could both be dephosphorylated by PstP. NaF, a known phosphatase inhibitor, inhibited the PstP-mediated dephosphorylation of both these proteins (Fig. 7). This confirmed the specificity of the PstP-mediated dephosphorylation reaction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Durocher et al., 2000) in a gene cluster in M. tuberculosis strengthened the view that these are likely to be involved in coordinated regulation of cell division and/or cell elongation. The genomic region encompassing these genes is conserved in M. leprae (in spite of extensive gene decay in this species), M. avium subsp. paratuberculosis and M. bovis, as well as in fast-growing mycobacteria such as M. smegmatis (Fsihi et al., 1996; Salazar et al., 1996). The conservation of these genes argues in favour of an essential role of the encoded proteins in cell function, possibly related to cell growth. Sassetti et al. (2003) suggested that PknB and PknA are likely to be required for the growth of M. tuberculosis in vitro.
Cell growth and development require the cell wall to have a dynamic structure. PBPs are crucial for the synthesis of peptidoglycan in specific phases of growth or development, underscoring the need to elucidate the functions of the mycobacterial PBPs. pbpA, pknB and pstP are cotranscribed (Kang et al., 2005), raising the possibility that PBPA could be regulated by reversible phosphorylation. Expression of PBPA in E. coli, its purification and analysis of penicillin binding confirmed that PBPA is a bona fide PBP. A mutant of M. smegmatis inactivated in the counterpart of the pbpA gene required nutrient-rich medium for growth and grew more slowly than the wild-type, suggesting that PBPA likely plays an important role during the active phase of growth. This was supported by the observation of Betts et al. (2002) that pbpA is downregulated during nutrient starvation. His-PBPA expressed ectopically in the mutant localized to the septum along with newly synthesized peptidoglycan, implying that it plays a role in cell division. Studies with E. coli and Haemophilus influenzae lend support to the view that cell wall synthesis is executed by two distinct holoenzyme complexes, one involved in elongation and the other in division (Alaedini & Day, 1999; Höltje, 1998). E. coli PBP3 is the classical transpeptidase demonstrated to be essential for peptidoglycan synthesis at the division site (Weiss et al., 1999). However, several PBPs from different bacterial species such as B. subtilis (Scheffers et al., 2004) and E. coli (PBP2) (Den Blaauwen et al., 2003) have been shown to localize to the lateral cell wall as well as to the septum in dividing cells. Although our immunofluorescence studies failed to localize PBPA to the lateral wall, we do not rule out the possibility that the fluorescence signals may have been too weak to be visible at the periphery.
PknB-mediated phosphorylation of PBPA suggested that this modification may modulate the function of PBPA in regulating cell division. T362 and T437 were identified as the residues phosphorylatable by PknB. The preferred substrate motif predicted by Kang et al. (2005) using a proteomic approach was not corroborated by our studies. In agreement with the observations of Kang et al. (2005), PknB phosphorylated PBPA on threonine but not on serine residues. However, the two sites of phosphorylation identified by us did not carry a Q at the +1 position, the preferred amino acid residue predicted by Kang et al. (2005) from peptide library screening data. Although PBPA carries a single TQ motif within the sequence NATMT26QVFT, T26 was not phosphorylated by PknB (Table 1). On the other hand, T437 was predicted as a putative phosphorylation site by NetPhos (Blom et al., 1999). Our studies suggest that the elegant peptide library screening methods used by Kang et al. (2005) do not exclude the possibility of identification of additional substrates of PknB.
T362 was conserved among the slow-growing species of mycobacteria, namely M. leprae, M. bovis, M. tuberculosis, and M. avium subsp. paratuberculosis, but replaced by an alanine in M. smegmatis. On the other hand, T437 was conserved across all species of mycobacteria for which the genomes have been sequenced. The single T437A mutation in PBPA made the protein incapable of complementing the growth and morphological defects of the PBPA-KO strain. The failure of the T437A mutant to localize to the site of division suggested that PknB-mediated phosphorylation targets PBPA to the septum. This appeared likely considering that M. smegmatis PknB was 65 % identical to M. tuberculosis PknB and the kinase domains of the two PBPs shared 86 % identity. We argued that PknB of M. smegmatis could probably exert the same effect on ectopically expressed PBPA of M. tuberculosis as PknB of M. tuberculosis would do to endogenous PBPA. This was supported by our unpublished observations that recombinant PknB (kinase domain) of M. smegmatis phosphorylated recombinant PBPA of M. tuberculosis in vitro.
Boitel et al. (2003) and Chopra et al. (2003) have shown that the catalytic domain of PknB is inactivated by dephosphorylation by PstP, the phosphatase present in the same operon. Our present report documents that PBPA is also a substrate of PstP. It appears possible that dephosphorylation of PBPA by PstP may shuttle it to another location within the cell, leading to the termination of cell division. Identification of PBPA as a substrate of PknB and PstP, together with the fact that inactivation of PBPA leads to impaired growth kinetics and altered morphology of the cells, suggests that this cluster of genes regulates growth and division of mycobacteria and related members of the GC-rich Gram-positive bacteria.
The role of signalling molecules in cell division or in cell growth has remained largely elusive. Reversible phosphorylation-dependent coordinated regulation of cell division and cell fate has been established in B. subtilis. Reversible phosphorylation of anti-sigma factor SpoIIAA (Duncan & Losick, 1993; Duncan et al., 1995, 1996) regulates its binding to 
F, thereby regulating transcription. SpoIIE is also required for normal formation of the asymmetrically positioned septum during B. subtilis sporulation (Lucet et al., 2000). The CckA, PleC and DivJ histidine kinases are dynamically localized to the cell poles of Caulobacter crescentus, each at distinct stages of the cell cycle (Jacobs et al., 1999; Wheeler & Shapiro, 1999). In a recent study, the pole-to-pole oscillation of the signalling molecule DivK has been elegantly demonstrated to be controlled by the kinase DivJ and the phosphatase PleC in C. crescentus (Matroule et al., 2004). Collectively these reports indicate that signal transduction pathways via histidine kinases, serine/threonine kinases and phosphatases influence growth, cell division and segregation in many organisms. Understanding the precise mechanism involved in the localization of phosphorylated PBPA to the septum, as well as the defined physiological signal leading to PknB-mediated PBPA phosphorylation, needs further investigation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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), PBPA-KO (
) and PBPA-KO complemented with pOLYG harbouring the genes of wild-type pbpA of M. tuberculosis (
) or the mutants T362A (
), T437A (
), or T362A, T437A (
). The results are means±SE of three experiments (some of the error bars are smaller than the symbols). (b) Expression of PBPA in the complemented strain. Membranes from PBPA-KO and PBPA-KO complemented with wild-type His-tagged pbpA (PBPA-KO+ pJBA206), and purified PBPA, were separated by SDS-PAGE followed by Western blotting using anti-His-antibody. The arrow indicates the position of His-PBPA.
