TY - JOUR
AU - Ogura, Mitsuo
AB - Abstract Disruptants of genes encoding the ZnuABC high-affinity zinc incorporator and zosA encoding a P-type ATPase for zinc incorporation were identified to show low transformability. The low transformability of the znuB cells was rescued by excess zinc addition and epistatic analysis of the mutation revealed no effect on the expression of comK, which encodes a master regulator for late com operons. We further examined the expression of each late com operon in the znuA mutant and found that the znuA mutation specifically inhibited the expression of comF, but not the other late com operons. The addition of zinc also rescued the low transformability of the zosA cells. In zosA cells, transcription of comK was severely repressed. Using a strain carrying comK driven by a xylose-inducible promoter, we showed that the zosA mutation inhibited the post-transcriptional control of comK. The addition of zinc also rescued the defect of xylose-inducible comK expression in zosA cells, suggesting that post-transcriptional control of comK requires zinc incorporation. Taken together, we propose that the both ZnuABC- and ZosA-mediated zinc incorporation is involved in competence development, although the two zinc transporters are differently implicated in this developmental process. ABC transporter, genetic competence, P-type ATPase, zinc homoeostasis Zinc is an essential metal, but excess zinc is toxic for the cell (1). Zinc serves as a co-factor and/or structural component for various enzymes and regulatory proteins (2, 3). Thus, the intracellular zinc concentration is tightly regulated by zinc transporters and zinc sensors. In the Gram-positive bacterium Bacillus subtilis, zinc importers, ZnuABC and ZosA are known, and the genes encoding the components for ZnuABC are negatively regulated by the zinc-sensor Zur functioning as a DNA-binding protein (4), (Supplementary Fig. S1). This regulatory network constitutes zinc homoeostasis. ZnuA (Formerly YcdH), ZnuB (Formerly YceA) and ZnuC (Formerly YcdI) are a zinc-binding lipoprotein, a membrane permease and an ATP-binding protein, respectively, and are all components of an ABC transporter involved in zinc incorporation (5, 6). The ZnuABC transporter serves as a high-affinity zinc transporter and thus, plays a major role in zinc incorporation at an environmental zinc level <1 µM. ZosA is a P-type ATPase and the zosA gene is negatively regulated by PerR-sensing hydrogen peroxide (7). Thus, ZosA, a peroxide-induced zinc transporter, has been thought to play a role under certain conditions, e.g. a condition of >1 µM of zinc, where the genes for ZnuABC are repressed, but induced under oxidative stress by hydrogen peroxide treatment (7). In the latter condition, activated expression of zosA leads to an enhancement of ZosA-mediated zinc transport, which plays an important role in the protection of the cells from oxidative stress (7). In addition, the probable membrane metal chaperon YciC may serve as the third zinc importer for low affinity to zinc, although there is no direct evidence (8). The yciC gene is directly regulated by Zur (8, 9) and YciC might function in ZnuABC-deficient cells grown under a zinc-limited condition. Bacillus subtilis exhibits genetic competence as an adaptive response to stationary-phase stress. The regulatory cascade leading to competence development in B. subtilis has been well elucidated (10, 11). The pheromone ComX is a cell density signal, and triggers autophosphorylation of ComP, the sensor kinase in the ComP–ComA two-component system, resulting in activation of ComA, and thus expression of the srfA operon. The srfA operon encodes the biosynthetic genes for the biosurfactant surfactin (12). Activation of ComK requires ComS, which is encoded by another Open reading frame (ORF) in srfA. ComS mediates the release of ComK from the MecA-ClpCP-ComK complex, namely proteolysis by ClpCP, leading to the autoactivation of ComK (13, 14). ComK is degraded by ClpCP in the presence of MecA in vitro, and the degradation is inhibited by ComS. ComK autoactivation is important for bistability in both competent and non-competent cells (15, 16). Additional activators, such as DegU and repressors, including CodY, AbrB and Rok, are also involved in ComK activation. ComK is a transcriptional regulatory protein that activates the expression of many genes, including late competence operons encoding the protein components needed for the uptake and processing of foreign DNA, comC, comE, comF and comG (17–20). In this article, we identified gene disruptants for ZnuABC transporter and a zosA mutant with low transformability and found that the addition of excess zinc restored the low transformable phenotype of the both mutants. Furthermore, we confirmed the former report that the lack of ZnuABC perturbs zinc homoeostasis (5). It was observed that the znuA mutant exhibited a lowered expression of comF, one of the late com operons, without affecting comK expression. On the other hand, we showed that in the zosA mutant grown under our moderately zinc-replete conditions used, perturbation of zinc homoeostasis did not occur, based on the monitoring of Zur-regulated ytiA gene expression (21). We also observed that the zosA mutation inhibited the post-transcriptional control of comK. Therefore, it is suggested that ZnuABC and ZosA play different roles in regulation of competence development. Experimental Procedures Media and materials In all of the experiments for competence development, liquid modified competence (MC) medium was used (22). Antibiotic III (Difco. Co) was used for the pre-culture under standard conditions. Colonies were counted on Luria–Bertani agar plates (Difco. Co). The bacterial strains and plasmids used in this study are shown in Table I. Table I. Bacillus subtilis strains used in this study. Strain  Genotype  Reference or source  168  trpC2  Laboratory stock  YKVWd  trpC2 zosA::Emr (zosA-lacZ)  BSORFa (Watabe, K.)  YCDHd  trpC2 znuA::Emr (znuA-lacZ)  BSORFa (Yamane, K.)  YCICd  trpC2 yciC::Emr (yciC-lacZ)  BSORFa (Yamane, K.)  OAM584  trpC2 yciC::Emr (yciC-lacZ:: Tcr)  This study  OAM551  trpC2 znuA::Emr (znuA-lacZ:: Tcr)  This study  OAM552  trpC2 znuA:Pmr  This study  OAM298  trpC2 znuB::Pmr  This study  OAM299  trpC2 znuC::Pmr  This study  OAM553  trpC2 zosA::Emr (zosA-lacZ) Pspac-zosA  This study  OAM554  trpC2 zosA::Pmr  This study  OAM555  trpC2 zosA::PmrznuA::Emr (znuA-lacZ:: Tcr)  This study  OAM585  trpC2 znuA::Emr-TcryciC::Emr  This study  OAM586  trpC2 zosA::PmryciC::Emr (yciC-lacZ)  This study  RIK796  trpC2 amyE::ytiA-lacZ (Tcr)  (21)  OAM558  trpC2 amyE::ytiA-lacZ (Tcr) znuA::Pmr  This study  OAM559  trpC2 amyE::ytiA-lacZ (Tcr) zosA::Pmr  This study  8G33  trpC2 comK-lacZ (Kmr)  (31)  OAM560  trpC2 comK-lacZ (Kmr) zosA::Pmr  This study  13G32  trpC2 comG-gfp (Cmr)  Kuipers, O.P.  OAM581  trpC2 comG-gfp (Cmr) zosA::Pmr  This study  OAM236  trpC2 comG-lacZ (Cmr)  (29)  OAM577  trpC2 comG-lacZ (Cmr) znuA::Emr-Tcr  This study  BD2524  hisA1 leu9 metB5 amyE::Pxyl-comK (Spr) comK (Cmr) comG-lacZ (Kmr)  (30)  OAM566  trpC2 amyE::Pxyl-comK (Spr) comK (Cmr) comG-lacZ (Kmr)  This study  OAM567  trpC2 amyE::Pxyl-comK (Spr) comK (Cmr) comG-lacZ (Kmr) zosA::Pmr  This study  OAM340  trpC2 comF-lacZ (Cmr::Tcr)  (29)  OAM576  trpC2 comF-lacZ (Cmr::Tcr) znuA::Emr-Tcr  This study  OAM344  trpC2 comEA-lacZ (Emr)  (29)  OAM578  trpC2 comEA-lacZ (Emr) znuA::Emr-Tcr  This study  OAM348  trpC2 comC-lacZ (Emr)  (29)  OAM579  trpC2 comC-lacZ (Emr) znuA::Emr-Tcr  This study  Plasmid  Description  Reference or source  pPhl2  Insertion vector, phreomycin resistance  (24)  pPhl-zosA  pPhl2 carrying a part of zosA  This study  pPhl-znuA  pPhl2 carrying a part of znuA  This study  pPhl-znuB  pPhl2 carrying a part of znuB  This study  pPhl-znuC  pPhl2 carrying a part of znuC  This study  pMutinIII  Insertion vecter, ampicillin and erythromycin resistance, lacZI  (25)  pMut-zosA  pMutinIII carrying a part of zosA  This study  pDG148  Multicopy vector carrying kanamycin resistance, Pspac promoter, and Ampicillin resistance  (26)  pDG148-znuA  pDG148 carrying the znuAORF  This study  pDG148-znuB  pDG148 carrying the znuBORF  This study  pDG148-znuC  pDG148 carrying the znuCORF  This study  Strain  Genotype  Reference or source  168  trpC2  Laboratory stock  YKVWd  trpC2 zosA::Emr (zosA-lacZ)  BSORFa (Watabe, K.)  YCDHd  trpC2 znuA::Emr (znuA-lacZ)  BSORFa (Yamane, K.)  YCICd  trpC2 yciC::Emr (yciC-lacZ)  BSORFa (Yamane, K.)  OAM584  trpC2 yciC::Emr (yciC-lacZ:: Tcr)  This study  OAM551  trpC2 znuA::Emr (znuA-lacZ:: Tcr)  This study  OAM552  trpC2 znuA:Pmr  This study  OAM298  trpC2 znuB::Pmr  This study  OAM299  trpC2 znuC::Pmr  This study  OAM553  trpC2 zosA::Emr (zosA-lacZ) Pspac-zosA  This study  OAM554  trpC2 zosA::Pmr  This study  OAM555  trpC2 zosA::PmrznuA::Emr (znuA-lacZ:: Tcr)  This study  OAM585  trpC2 znuA::Emr-TcryciC::Emr  This study  OAM586  trpC2 zosA::PmryciC::Emr (yciC-lacZ)  This study  RIK796  trpC2 amyE::ytiA-lacZ (Tcr)  (21)  OAM558  trpC2 amyE::ytiA-lacZ (Tcr) znuA::Pmr  This study  OAM559  trpC2 amyE::ytiA-lacZ (Tcr) zosA::Pmr  This study  8G33  trpC2 comK-lacZ (Kmr)  (31)  OAM560  trpC2 comK-lacZ (Kmr) zosA::Pmr  This study  13G32  trpC2 comG-gfp (Cmr)  Kuipers, O.P.  OAM581  trpC2 comG-gfp (Cmr) zosA::Pmr  This study  OAM236  trpC2 comG-lacZ (Cmr)  (29)  OAM577  trpC2 comG-lacZ (Cmr) znuA::Emr-Tcr  This study  BD2524  hisA1 leu9 metB5 amyE::Pxyl-comK (Spr) comK (Cmr) comG-lacZ (Kmr)  (30)  OAM566  trpC2 amyE::Pxyl-comK (Spr) comK (Cmr) comG-lacZ (Kmr)  This study  OAM567  trpC2 amyE::Pxyl-comK (Spr) comK (Cmr) comG-lacZ (Kmr) zosA::Pmr  This study  OAM340  trpC2 comF-lacZ (Cmr::Tcr)  (29)  OAM576  trpC2 comF-lacZ (Cmr::Tcr) znuA::Emr-Tcr  This study  OAM344  trpC2 comEA-lacZ (Emr)  (29)  OAM578  trpC2 comEA-lacZ (Emr) znuA::Emr-Tcr  This study  OAM348  trpC2 comC-lacZ (Emr)  (29)  OAM579  trpC2 comC-lacZ (Emr) znuA::Emr-Tcr  This study  Plasmid  Description  Reference or source  pPhl2  Insertion vector, phreomycin resistance  (24)  pPhl-zosA  pPhl2 carrying a part of zosA  This study  pPhl-znuA  pPhl2 carrying a part of znuA  This study  pPhl-znuB  pPhl2 carrying a part of znuB  This study  pPhl-znuC  pPhl2 carrying a part of znuC  This study  pMutinIII  Insertion vecter, ampicillin and erythromycin resistance, lacZI  (25)  pMut-zosA  pMutinIII carrying a part of zosA  This study  pDG148  Multicopy vector carrying kanamycin resistance, Pspac promoter, and Ampicillin resistance  (26)  pDG148-znuA  pDG148 carrying the znuAORF  This study  pDG148-znuB  pDG148 carrying the znuBORF  This study  pDG148-znuC  pDG148 carrying the znuCORF  This study  ahttp://bacillus.genome.jp/ View Large Transformation and β-galactosidase assay Transformation assay was carried out as follows. Overnight culture in Antibiotic medium III was inoculated to MC medium for transformation (5%, v/v). Total DNA containing the appropriate antibiotic resistance gene was added to the 0.1 ml of cell cultures at 2 h after entry into the stationary phase (T2) followed by addition of 2 ml of Antibiotic medium III after 30 min and the cells were further incubated for 2 h. After transformation, the cells were subjected to serial 10-fold dilutions. Each diluted fraction was then plated onto two LB agar plates containing the appropriate antibiotics, and colonies were then counted. The chromosomal antibiotic markers used for the experiments were a Cm resistance marker with an amyE::degU-lacZ fusion (23) and a Tc marker with the amyE::ytiA-lacZ fusion (21). Viable cell numbers were counted by plating the culture onto two LB agar plates containing the appropriate antibiotics after a 10−5 dilution. The typical experiments among those conducted at least two times are shown in the Tables (Tables III–V). The β-galactosidase activities of lacZ fusions were measured as described previously (24). Plasmid construction Synthetic oligonucleotides were commercially prepared by the Tsukuba Oligo Service (Ibaraki, Japan). The plasmids and oligonucleotides used in this study are listed in Tables I and II, respectively. To construct pPhl-zosA and pPhl-znuA, the polymerase chain reaction (PCR) products produced by the oligonucleotide pairs, zosA-E and zosA-Sa and ycdH-Sa and ycdH-Sc, were digested by simultaneous restriction enzyme digestion, using EcoRI and SalII, and SalI and SacI respectively, and cloned into pPhl-2 digested with the same restriction enzyme pairs as used for the PCR product (24). To construct pPhl-znuB and pPhl-znuC, PCR products produced by the oligonucleotide pairs, yceA-Sa and yceA-H and ycdI-E and ycdI-Sa, were digested by the combinations of restriction enzymes, SalII and HindIII, and EcoRI and SalI, respectively, and cloned into pPhl-2 digested with the same restriction enzyme pairs as used for the PCR product. To construct pMut-zosA, a PCR product produced by an oligonucleotide pair, pMut-zosA-H and pMut-zosA-B, was digested by HindIII and BamHI, and cloned into pMutIn-III with the same restriction enzymes (25). To construct pDG148-znuA, pDG148-znuB and pDG148-znuC, PCR products produced by the oligonucleotide pairs, pDG148-ycdH-H and pDG148-ycdH-Sa, pDG148-yceA-H and pDG148-yceA-Sa and pDG148-ycdI-H and pDG148-ycdI-Sa, were digested by HindIII and SalI, and cloned into pDG148 with the same restriction enzymes (26). The sequences of all the cloned PCR products were confirmed. Table II. Oligonucleotides used for this study. Name  Sequence  Product  zosA-E  5′-ATGGAATTCAAGTTATCGTTCAACGCGAC-3′  pPhl-zosA  zosA-Sa  5′-ATGGTCGACGTATGTTTCTAAAGCTCCGC-3′  pPhl-zosA  ycdH-Sa  5′-ATGGTCGACGCCTTTGCTTGCATTAACGA-3′  pPhl-znuA  ycdH-Sc  5′-ATGGAGCTCTTTGTTTGTGATTGCAGCGT-3′  pPhl-znuA  yceA-Sa  5′-ATGGTCGACTTGGAATTCATGCGACG-3′  pPhl-znuB  yceA-H  5′-ATGAAGCTTATCGATGCTCATATTGGCTG-3′  pPhl-znuB  ycdI-E  5′-ATGGAATTCTCTCGTCTCATTGAAAGATA-3′  pPhl-znuC  ycdI-Sa  5′-ATGGTCGACAACGTTTAAACCATTTCCC-3′  pPhl-znuC  pMut-zosA-H  5′-ATCAAGCTTCGTTCTCAATTAGAGAGGAG-3′  pMut-zosA  pMut-zosA-B  5′-ATCGGATCCGAACCAATGGCAGCGAAAAT-3′  pMut-zosA  pDG148-ycdH-H  5′-ATCAAGCTTCTGAAAAGAGGGGATATACGAT-3′  pDG148-znuA  pDG148-ycdH-Sa  5′-ATCGTCGACTTATGATTTAACCAATAGTGAA-3′  pDG148-znuA  pDG148-yceA-H  5′-ATCAAGCTTAGAGAGAGGAGAAAAAGGCG-3′  pDG148-znuB  pDG148-yceA-Sa  5′-ATCGTCGACTTATCGGCTTCTTTTTTTGCGC-3′  pDG148-znuB  pDG148-ycdI-H  5′-ATCAAGCTTGTGAGAAAGGAAGATTAAC-3′  pDG148-znuC  pDG148-ycdI-Sa  5′-ATCGTCGACTAGAAAAGCTCGTCGCAT-3′  pDG148-znuC  Name  Sequence  Product  zosA-E  5′-ATGGAATTCAAGTTATCGTTCAACGCGAC-3′  pPhl-zosA  zosA-Sa  5′-ATGGTCGACGTATGTTTCTAAAGCTCCGC-3′  pPhl-zosA  ycdH-Sa  5′-ATGGTCGACGCCTTTGCTTGCATTAACGA-3′  pPhl-znuA  ycdH-Sc  5′-ATGGAGCTCTTTGTTTGTGATTGCAGCGT-3′  pPhl-znuA  yceA-Sa  5′-ATGGTCGACTTGGAATTCATGCGACG-3′  pPhl-znuB  yceA-H  5′-ATGAAGCTTATCGATGCTCATATTGGCTG-3′  pPhl-znuB  ycdI-E  5′-ATGGAATTCTCTCGTCTCATTGAAAGATA-3′  pPhl-znuC  ycdI-Sa  5′-ATGGTCGACAACGTTTAAACCATTTCCC-3′  pPhl-znuC  pMut-zosA-H  5′-ATCAAGCTTCGTTCTCAATTAGAGAGGAG-3′  pMut-zosA  pMut-zosA-B  5′-ATCGGATCCGAACCAATGGCAGCGAAAAT-3′  pMut-zosA  pDG148-ycdH-H  5′-ATCAAGCTTCTGAAAAGAGGGGATATACGAT-3′  pDG148-znuA  pDG148-ycdH-Sa  5′-ATCGTCGACTTATGATTTAACCAATAGTGAA-3′  pDG148-znuA  pDG148-yceA-H  5′-ATCAAGCTTAGAGAGAGGAGAAAAAGGCG-3′  pDG148-znuB  pDG148-yceA-Sa  5′-ATCGTCGACTTATCGGCTTCTTTTTTTGCGC-3′  pDG148-znuB  pDG148-ycdI-H  5′-ATCAAGCTTGTGAGAAAGGAAGATTAAC-3′  pDG148-znuC  pDG148-ycdI-Sa  5′-ATCGTCGACTAGAAAAGCTCGTCGCAT-3′  pDG148-znuC  View Large Fluorescence microscopy Cells were grown in MC medium and 500 µl of the culture were centrifuged, and 400 µl of the supernatant were aspirated off. The cells were then resuspended in the remaining 100 µl. Portions (2 µl) of each sample were mounted on glass slides treated with 0.1% (w/v) poly-l-lysine (Sigma). Microscopy was performed with an Olympus BX51 phase-contrast and fluorescence microscope with a 100× UplanApo objective. Images were captured using a coolSNAP-hq charge-coupled device camera (Nippon Roper) and Metavue 4.6r8 software (Universal Imaging). Green fluorescent protein (GFP) was visualized using a WIB filter set (Olympus). Image processing was performed with Adobe Photoshop. Results Disruption of the genes encoding the ZnuABC transporter for zinc incorporation caused low transformability In our screening for disruptants with low transformability (27–29), the disruptants of znuA, znuB and znuC displayed reduced transformation frequencies (data not shown). To confirm this, three gene-disruption cassettes were back-crossed and the resultant strains exhibited low transformation frequency (Table III). Next, we carried out a complementation test using a multicopy system, i.e. pDG148-znuA, pDG148-znuB and pDG148-znuC, in which an IPTG-inducible promoter is located upstream of each Shine-Dalgarno (SD) sequence for the genes. In all three cases, the introduction of each plasmid into the znu disruptant resulted in evident recovery of transformation frequency without the addition of IPTG (Table III). This is due to amplification of the leaky expression of Pspac-driven genes. When IPTG was added to the culture of the znu strains with the cognate pDG148-znu plasmid, complementation of the disruption by artificial induction of the corresponding gene was observed, although in the case of znuC, only partial complementation was observed due to an unknown reason. Based on these results, it is concluded that each gene disruption of the znuABC operon causes a low transformation frequency phenotype. Table III. Complementation of low transformability in the mutants by IPTG-driven expression of the corresponding gene. Experiment type/Strain  Relevant genotype  IPTG (1 mM)  Viable cells/ml (× 106)  Transformed cells/ml (× 102)  Frequency (× 10−4)  Relative frequency (%)  Experiment 1                  168  Wild-type  −  131  64  0.49  100      YCDHd  znuA pDG148  −  116  3  0.026  4    znuA pDG-znuA  −  99  14  0.14  22      +  81  86  1.06  163  Experiment 2                  168  Wild-type  −  216  492  2.27  100      OAM298  znuB pDG148  −  64  4  0.063  2.8    znuB pDG-znuB  −  52  71  1.37  60      +  86  162  1.89  83      OAM299  znuC pDG148  −  54  17  0.31  14    znuC pDG-znuC  −  89  125  1.4  62      +  85  100  1.18  52  Experiment3                  168  Wild-type  −  271  274  1.01  100      YKVWd  zosA  −  270  10  0.037  3.7      +  190  9  0.047  4.7      OAM553  zosA Pspac-zosA  −  109  27  0.25  24      +  206  175  0.85  84  Experiment type/Strain  Relevant genotype  IPTG (1 mM)  Viable cells/ml (× 106)  Transformed cells/ml (× 102)  Frequency (× 10−4)  Relative frequency (%)  Experiment 1                  168  Wild-type  −  131  64  0.49  100      YCDHd  znuA pDG148  −  116  3  0.026  4    znuA pDG-znuA  −  99  14  0.14  22      +  81  86  1.06  163  Experiment 2                  168  Wild-type  −  216  492  2.27  100      OAM298  znuB pDG148  −  64  4  0.063  2.8    znuB pDG-znuB  −  52  71  1.37  60      +  86  162  1.89  83      OAM299  znuC pDG148  −  54  17  0.31  14    znuC pDG-znuC  −  89  125  1.4  62      +  85  100  1.18  52  Experiment3                  168  Wild-type  −  271  274  1.01  100      YKVWd  zosA  −  270  10  0.037  3.7      +  190  9  0.047  4.7      OAM553  zosA Pspac-zosA  −  109  27  0.25  24      +  206  175  0.85  84  View Large Zinc rescue test We examined whether the addition of excess zinc to the medium restored low transformability in the znu gene disruptants, because the Znu ABC transporter incorporates zinc from the environment when cells are grown in low levels of zinc concentration <1 µM. The culture for transformation should contain some concentration of zinc, because Antibiotic medium III is composed of natural products, such as yeast extract containing zinc. Thus, we needed to know the surrounding milieu of the cells with respect to zinc in the culture system used in our laboratory. To do so, we adopted a lacZ fusion with ytiA encoding L31-like ribosomal protein as a sensor of the zinc concentration, since ytiA is regulated by the metalloregulator Zur (21). When the cells were grown in zinc-depleted medium, the expression of ytiA was derepressed from Zur-dependent control, and when grown in zinc-replete medium, ytiA expression was repressed. First, we cultured the ytiA-lacZ strain in MC medium using the cells grown on an LB agar plate as the pre-culture, i.e. without the natural products derived from the ingredients of the medium. In this case, the expression of ytiA-lacZ was very high (Fig. 1A). Then, the addition of zinc to the medium decreased ytiA-lacZ expression, and finally 1 µM of zinc completely repressed the expression of ytiA-lacZ. This is consistent with the former report (21). Next, we examined the expression of ytiA-lacZ in our standard conditions using Antibiotic medium III as the medium for pre-culture. We observed the repressed expression of the fusion in wild-type cells (Fig. 1B). Based on the results, it was concluded that the cells grow in milieu containing ∼1 µM of zinc in our standard conditions. The addition of 10 µM zinc resulted in a partial recovery of low transformability in the znuB cells (Table IV). In the mutants for znu genes, severe perturbation of zinc homoeostasis is reported to occur (5), which leads to enhancement of the expression of yciC due to derepression of the genes from Zur. Thus, it is possible that external zinc could be incorporated by YciC in the znuB cells, leading to the recovery of transformation efficiency. To confirm this, we constructed the znuAyciC double mutant. The yciC single mutant did not display significant defect in transformability (Table V). The double mutant exhibited a synergistic decrease in transformability compared with that in the znuA single mutant (Table V). When excess zinc was added to the culture, recovery of low transformation frequencies in the double mutant was scarcely observed (Table V). We note that similar result was obtained in a znuByciC mutant (data not shown). The result indicated that the recovery of low transformability in the znuB cells mediated by overproduced YciC. Taken together, it was shown that perturbation of zinc homoeostasis impaired the full induction of transformation. Fig. 1 View largeDownload slide Expression of ytiA in zinc-replete and -depleted conditions. Cells were grown in MC medium and sampled hourly. The X-axis represents the growth time in hours relative to the end of vegetative growth (T0). (A) The RIK796 cells grown on LB-agar plate were used as pre-culture. ZnCl2 was added with the inoculation of pre-culture. Zinc concentrations; closed circles, 0 µM; open circles, 0.1 µM; triangles, 1 µM. (B) The cells grown in Antibiotic medium III were used as pre-culture. Closed circles, wild-type; open circles, zosA; triangles, znuA. Fig. 1 View largeDownload slide Expression of ytiA in zinc-replete and -depleted conditions. Cells were grown in MC medium and sampled hourly. The X-axis represents the growth time in hours relative to the end of vegetative growth (T0). (A) The RIK796 cells grown on LB-agar plate were used as pre-culture. ZnCl2 was added with the inoculation of pre-culture. Zinc concentrations; closed circles, 0 µM; open circles, 0.1 µM; triangles, 1 µM. (B) The cells grown in Antibiotic medium III were used as pre-culture. Closed circles, wild-type; open circles, zosA; triangles, znuA. Table IV. Transformation frequencies in the various mutants under the condition of addition of zinc ion. Experiment type/Strain  Relevant genotype  ZnCl2 (µM)  Viable cells/ml (× 106)  Transformed cells/ml (× 102)  Frequency (× 10−4)  Relative frequency (%)  Fold (zinc addition)  Experiment 1                    168  Wild-type  0  105  235  2.24  100        10  110  113  1.03  46  ×0.46      OAM298  znuB  0  185  19  0.11  4.7        10  128  100  0.78  35  ×7.4  Experiment 2                    168  Wild-type  10  251  138  0.54  100        OAM552  zosA  0  128  0.6  0.0046  0.9        10  193  51  0.26  49  ×54      OAM555  zosA znuA  0  55  <0.01  <0.0002  <0.04        10  175  15  0.086  16  >×400  Experiment type/Strain  Relevant genotype  ZnCl2 (µM)  Viable cells/ml (× 106)  Transformed cells/ml (× 102)  Frequency (× 10−4)  Relative frequency (%)  Fold (zinc addition)  Experiment 1                    168  Wild-type  0  105  235  2.24  100        10  110  113  1.03  46  ×0.46      OAM298  znuB  0  185  19  0.11  4.7        10  128  100  0.78  35  ×7.4  Experiment 2                    168  Wild-type  10  251  138  0.54  100        OAM552  zosA  0  128  0.6  0.0046  0.9        10  193  51  0.26  49  ×54      OAM555  zosA znuA  0  55  <0.01  <0.0002  <0.04        10  175  15  0.086  16  >×400  View Large Table V. Effect of the yciC mutation on the rescue by zinc addition in the znuA and zosA cells. Experiment type/Strain  Relevant genotype  ZnCl2 (µM)  Viable cells/ml (× 106)  Transformed cells/ml (×102)  Frequency (× 10−4)  Relative frequency (%)  Fold (zinc addition)  Experiment 1                    168  Wild-type  0  119  367  3.08  100        YCICd  yciC  0  161  197  1.16  38    Experiment 2                    168  Wild-type  0  163  680  4.17  100        OAM585  znuA yciC  0  126  0.8  0.006  0.15        10  143  3.5  0.014  0.35  × 2.3  Experiment 3                    168  Wild-type  0  203  484  4.17  100        OAM586  zosA yciC  0  33  1.4  0.042  1.8        10  259  24  0.092  3.9  × 2.2  Experiment type/Strain  Relevant genotype  ZnCl2 (µM)  Viable cells/ml (× 106)  Transformed cells/ml (×102)  Frequency (× 10−4)  Relative frequency (%)  Fold (zinc addition)  Experiment 1                    168  Wild-type  0  119  367  3.08  100        YCICd  yciC  0  161  197  1.16  38    Experiment 2                    168  Wild-type  0  163  680  4.17  100        OAM585  znuA yciC  0  126  0.8  0.006  0.15        10  143  3.5  0.014  0.35  × 2.3  Experiment 3                    168  Wild-type  0  203  484  4.17  100        OAM586  zosA yciC  0  33  1.4  0.042  1.8        10  259  24  0.092  3.9  × 2.2  View Large zosA disruption resulted in low transformability In the screening, we also identified YKVWd with a low transformation frequency (data not shown). Similar to the case of the znu genes, transformation frequency of the back-crossed ykvW strain was assessed and the strain exhibited ∼4% of the transformation efficiency of the control parental strain (Table III). The ykvW gene has been renamed zosA, encoding a zinc-transporting P-type ATPase (7). Next, we examined the possibility that the mutation might be polar to the downstream gene, leading to the low transformability. If so, the addition of IPTG should rescue the low transformability in YKVWd, because in the strain, an IPTG-inducible promoter, Pspac, is located upstream of the ykvY ORF (Fig. 2). The addition of IPTG did not change the low transformability in YKVWd (Table III). Thus, the mutation is not polar to the downstream gene. Furthermore, we carried out a complementation test of the zosA disruption by artificial expression of zosA in the strain OAM553. In the strain OAM553, the addition of IPTG leads to an artificial expression of the entire zosA ORF (Fig. 2). When OAM553 was grown in competence medium without IPTG, the strain exhibited relatively high transformability. This partial complementation is likely due to leaky transcription from the Pspac-driven zosA gene. Complete complementation of the disruption of zosA by Pspac-zosA was observed with the addition of 1 mM isopropyl-beta-D-thiogalactopylanoside (IPTG) (Table III). It is concluded that the zosA disruption caused the low transformability. Fig. 2 View largeDownload slide Chromosomal structures around zosA in YKVWd and OAM553. Both strains were constructed by Campbell-type single cross-over event of the corresponding plasmids. Bent arrow, promoter; Rectangle, complete or partial ORF; Small rectangle with SD, Shine Dalgarno sequence; Closed box, cloned chromosomal region into the plasmids to construct the strain; Numbers, nucleotide positions relative to the transcription start site. Fig. 2 View largeDownload slide Chromosomal structures around zosA in YKVWd and OAM553. Both strains were constructed by Campbell-type single cross-over event of the corresponding plasmids. Bent arrow, promoter; Rectangle, complete or partial ORF; Small rectangle with SD, Shine Dalgarno sequence; Closed box, cloned chromosomal region into the plasmids to construct the strain; Numbers, nucleotide positions relative to the transcription start site. No perturbation of zinc homoeostasis in zosA cells The major zinc incorporation transporter is thought to be ZnuABC under conditions where the zinc concentration is on a submicromolar order (5). In fact, the disruptant of znuA encoding a solute-binding protein for zinc in the ZnuABC transporter exhibited highly derepressed expression of ytiA-lacZ (Fig. 1B). This indicates that perturbation of zinc homoeostasis continues in znuA cells due to the lack of the ZnuABC transporter. In contrast, in the zosA mutant, the expression of ytiA-lacZ did not increase compared with that observed in the wild-type cells, although some decrease was observed due to an unknown reason (Fig. 1B). Thus, it is concluded that there is no perturbation of cellular zinc homoeostasis in the zosA cells. External zinc reversed low transformability in zosA cells To examine whether external zinc reverses low transformability in zosA cells, we added zinc to the medium and performed a transformation test. The addition of 10 µM of zinc increased transformation frequency in the zosA cells from 0.9% to approximately half of that observed in the wild-type cells (Table IV). This rescue might be mediated by the incorporation of zinc through the residual zinc transporter, ZnuABC and/or the putative low-affinity zinc transporter, YciC. To determine which of the two contributes to this rescue, we made two types of double-mutants, znuAzosA and yciCzosA and tested the transformation frequency in the presence or absence of excess zinc. In the znuAzosA strain, a very low transformation frequency was observed (Table IV). This decrease would be caused by a synergistic effect of the two mutations. The observed synergy suggests that the two mutations act on different nodes in the regulatory cascade of competence development. The addition of excess zinc rescued the low transformation frequency in the znuAzosA strain, suggesting that the putative zinc transporter, YciC, which could mediate this rescue of both mutations simultaneously. In the yciCzosA cells, the transformation frequency was not distinguishable from that observed in the zosA single mutant (Table V). When excess zinc was added, transformation frequencies in the double mutant did not evidently change (Table V), strongly suggesting that zinc incorporation is mediated by YciC in the zosA cells and that the ZnuABC-mediated zinc incorporation was not able to rescue low transformability in this double mutant. Based on these results, it is concluded that ZosA-mediated zinc incorporation is required for full induction of competence development. It should be noted that the putative low-affinity zinc transporter YciC would replace the ZosA function with respect to zinc requirement of transformability, taking into account of the zosAyciC cells. This suggested that only low levels of zinc would be required for transformability if zinc homoeostasis is maintained. ZosA-mediated zinc transport is required for comK expression Next, we aimed to identify the target molecular event regulated by the zinc transport mediated by ZosA and ZnuABC. To do this, we performed an epistatic analysis of the mutations using srfA-lacZ and comK-lacZ. The zosA and znuA mutations had essentially no effect on the expression of srfA-lacZ (data not shown). Next, we tested the effect of the mutations on the expression of comK-lacZ. The expression of comK-lacZ severely decreased in the zosA strain but not in the znuA strain (Figs. 3A and 4A). The results suggested that both mutations might affect different points in the competence development regulation, as well as, the rescue of low transformability by zinc addition, the low level expression of comK-lacZ in the zosA cells is almost completely rescued by the addition of 10 µM of zinc (Fig. 3A). In addition, the severe decrease in comK expression in the zosA cells was also observed in a zinc-limited condition in which the expression of the genes for ZnuABC are derepressed, i.e. a condition in which the assay was carried out in a culture using cells grown on an LB agar plate as the pre-culture (data not shown). This again suggested that ZosA defect was not rescued by ZnuABC overproduction. A microscopic analysis using comG-gfp (green fluorescent protein) fusion also confirmed a smaller fraction of competent cells among the zosA cells, due to a decrease in ComK activity, compared to that observed in wild-type cells (Fig. 5). In the competent cells carrying the zosA mutation, weak intensities of ComG-GFP fluorescence were frequently observed, which reflects the relatively low activity of ComK in the competent cells (Fig. 5). This would also contribute low transformability in the zosA cells, due to the low level of the competence machinery compared to the wild-type, competent cells. Fig. 3 View largeDownload slide Epistatic analysis of comK and comG by zosA mutation. Cells were grown in MC medium and sampled hourly. The X-axis represents the growth time in hours relative to the end of vegetative growth (T0). The cells grown in antibiotic medium III were used as pre-culture. ZnCl2 was added with the inoculation of pre-culture. The expression of the indicated fusion above the graph was examined in each mutant. (A) Closed circles, wild-type without zinc; open symbols, zosA. Zinc concentrations: circles, 0 µM; squares, 10 µM; triangles, 30 µM. (B) The media contained 2.5% xylose to induce comK. Left panel. Closed circles, wild-type (comG-lacZ comK amyE-Pxyl-comK) without zinc; open squares, wild-type with 10 µM of zinc; open circles, zosA (OAM567). Right panel. zosA (OAM567). Zinc concentrations; Circles, 0 µM; inverted triangles, 3 µM; squares, 10 µM; triangles, 30 µM. Fig. 3 View largeDownload slide Epistatic analysis of comK and comG by zosA mutation. Cells were grown in MC medium and sampled hourly. The X-axis represents the growth time in hours relative to the end of vegetative growth (T0). The cells grown in antibiotic medium III were used as pre-culture. ZnCl2 was added with the inoculation of pre-culture. The expression of the indicated fusion above the graph was examined in each mutant. (A) Closed circles, wild-type without zinc; open symbols, zosA. Zinc concentrations: circles, 0 µM; squares, 10 µM; triangles, 30 µM. (B) The media contained 2.5% xylose to induce comK. Left panel. Closed circles, wild-type (comG-lacZ comK amyE-Pxyl-comK) without zinc; open squares, wild-type with 10 µM of zinc; open circles, zosA (OAM567). Right panel. zosA (OAM567). Zinc concentrations; Circles, 0 µM; inverted triangles, 3 µM; squares, 10 µM; triangles, 30 µM. Fig. 4 View largeDownload slide Gene expressions of comK and late com in znuA cells. Cells were grown in MC medium and sampled hourly. The cells grown in Antibiotic medium III were used as pre-culture. The X-axis represents the growth time in hours relative to the end of vegetative growth (T0). Closed circles, wild-type without zinc; open circles, znuA. The expression of the indicated fusion above the graph was examined in each mutant. (A) comK-lacZ. (B) comF-lacZ. Reverse triangles, znuA with 3 µM of ZnCl2; triangles, znuA with 10 µM of ZnCl2; squares, znuA with 30 µM of ZnCl2. (C) comC-lacZ. (D) comE-lacZ. (E) comG-lacZ. Fig. 4 View largeDownload slide Gene expressions of comK and late com in znuA cells. Cells were grown in MC medium and sampled hourly. The cells grown in Antibiotic medium III were used as pre-culture. The X-axis represents the growth time in hours relative to the end of vegetative growth (T0). Closed circles, wild-type without zinc; open circles, znuA. The expression of the indicated fusion above the graph was examined in each mutant. (A) comK-lacZ. (B) comF-lacZ. Reverse triangles, znuA with 3 µM of ZnCl2; triangles, znuA with 10 µM of ZnCl2; squares, znuA with 30 µM of ZnCl2. (C) comC-lacZ. (D) comE-lacZ. (E) comG-lacZ. Fig. 5 View largeDownload slide Fluorescence microscopic analysis of zosA cells using ComG-GFP. Cells were grown in MC medium. The cells grown in Antibiotic medium III were used as pre-culture. Samples were harvested at T2. (A) wild-type. (B) zosA. Upper panels, phase contrast micrographs. Lower panels, GFP fluorescence micrographs. The phase contrast and GFP fluorescence images are captured in the same field of vision. Fig. 5 View largeDownload slide Fluorescence microscopic analysis of zosA cells using ComG-GFP. Cells were grown in MC medium. The cells grown in Antibiotic medium III were used as pre-culture. Samples were harvested at T2. (A) wild-type. (B) zosA. Upper panels, phase contrast micrographs. Lower panels, GFP fluorescence micrographs. The phase contrast and GFP fluorescence images are captured in the same field of vision. Zinc is required for post-transcriptional control of comK The regulation of ComK is achieved in two phases (Fig. 6). One is the post-transcriptional control involving the ComK release from the MecA-ClpCP proteolysis system by ComS, and the other is the transcriptional control affected by many transcription factors. To distinguish which of the phases is the target of ZosA-mediated zinc incorporation, we used the xylose-dependent comK transcription system constructed by Hahn et al. (30). In the strain carrying comG-lacZ as an indicator of ComK activity, comK is transcribed by the xylose-dependent promoter alone, independent of comK expression from the transcription factors, including ComK autoactivation. If the effect of the zosA mutation were on the one of the transcription factors, the decrease of comK-lacZ expression would be bypassed in this system. As shown in Fig. 3B, the decreasing effect of the zosA mutation was not bypassed. The addition of zinc resulted in a slight increase in comK-lacZ expression in this system (Fig. 3B, left). Contrary to this, in the zosA mutation background, zinc addition resulted in complete rescue of the decrease in the fusion expression. We note that the extent of the rescue reached a plateau at 10 µM of zinc (Fig. 3B, right). These observations indicate that the effect of the zosA mutation on comK acts on the post-transcriptional control of comK. It should be noted that in the zosA cells, ComK function is inhibited post-transcriptionally, leading to the repression of comK transcription, since comK is within the autoregulatory loop. The notion of post-transcriptional control of comK is reinforced by the observation that an introduction of the mecA mutation, which allows comK expression independently from the ClpCP protease and releasing factor ComS, leads to an enhancement of comK transcription and late com gene expression, bypassing the decreasing effect of the zosA mutation (data not shown). On the other hand, the introduction of the mecA mutation did not bypass a deficiency due to a lack of positive transcription factors required for comK expression, including DegU and ComK (31, 32). Fig. 6 View largeDownload slide Regulation of ComK by zinc transportation: comK proteolytic complex and transcription factors are shown. Dotted box indicates comK autoregulatory loop. Oval, protein; Notched oval, degraded protein; Rectangle, comK ORF; Bent arrow, promoter; T-bar, ComS action resulting in release of ComK from proteolytic complex and inhibitory effect of perturbation of zinc homoeostasis in the cells lacking the complete ZnuABC transporter; Arrow, gene activation; Dotted arrow, indirect gene activation. Fig. 6 View largeDownload slide Regulation of ComK by zinc transportation: comK proteolytic complex and transcription factors are shown. Dotted box indicates comK autoregulatory loop. Oval, protein; Notched oval, degraded protein; Rectangle, comK ORF; Bent arrow, promoter; T-bar, ComS action resulting in release of ComK from proteolytic complex and inhibitory effect of perturbation of zinc homoeostasis in the cells lacking the complete ZnuABC transporter; Arrow, gene activation; Dotted arrow, indirect gene activation. znuA mutation specifically decreased comF expression In znuA cells, the expression of comK is normal, which may indicate that the expression of the late com genes naturally occurs, as in the wild-type cells. Recently, however, it was reported that there is one check-point control by YutB and ComN on comE expression, although the expression of comK is not impaired in either disruptant of yutB or comN (29). Thus, it was worth examining each late com gene transcription event in the znuA cells. We introduced the znuA mutation into the strain carrying the lacZ fusion with each late com operon and examined the β-galactosidase activities. As shown in Fig. 4, the expression of comF-lacZ was greatly decreased in the znuA cells, whereas, the expression of the rest of the late com operon fusions, comC, comE and comG, did not change. Next, we tested whether the addition of zinc would rescue the decreased expression of comF. The addition of 10 µM of zinc to the culture of the znuA cells recovered the expression of comF, which is consistent with the results of the transformation assay. These results clearly indicate that ZnuA-mediated zinc incorporation is specifically required for comF expression. Discussion We report that perturbation of zinc homoeostasis caused by the lack of the Znu ABC transporter for zinc incorporation resulted in low transformability (Fig. 6). This perturbation causes a decrease in comF transcription without affecting comK transcription. The ZnuABC high-affinity zinc incorporation system is found in many bacteria and thus, there are several reports showing that the disruption of znu genes results in drastic changes in certain phenotypes. In Streptococcus pneumoniae, disruption of the genes encoding the ABC transporter for zinc incorporation, Adc, leads to a competence-deficient phenotype, although the molecule targeted by zinc remains unknown (33). Furthermore, in pathogenic Salmonella enterica, disruption of the ZnuABC system results in reduced virulence (34, 35). These patterns of reduced virulence are likely caused by the impaired growth of the Znu system-deficient bacteria in the host cells. In addition, in Streptococcus gordonii, disruption of the repressor of the adc operon, leading to an overproduction of the Adc ABC transporter, resulted in a deficiency in both biofilm-formation and genetic competence (36). This indicates an involvement of this transporter in these two biological processes. It was also determined that ZosA-mediated zinc incorporation is required for the post-transcriptional control of comK (Fig.6). The question of which molecules and processes are required for interaction with zinc remains a subject for future investigation. ClpX has a zinc-binding domain, thus zinc binding influences the activity of the protein (37). It has been reported that ClpX is required for post-transcriptional control of comK (38). Thus, it is possible that ClpX is a target of ZosA-mediated zinc incorporation. This hypothesis, however, has a shortcoming, i.e. the unimpaired expression of srfA, which is positively regulated by the proteolytic activity of ClpXP for Spx, was observed in the zosA cells (39). This means that the zosA mutation may not affect the ClpXP activity. Otherwise, the zinc-dependent activities of ClpX required for activation of srfA and comK might be different. In fact, ClpX has a role in FtsZ assembly independently of ClpP (40) and the regulation of ComK by ClpX may not be dependent on ClpXP-dependent proteolysis (38). It has been known that P-type ATPase for ion uptake functions with secondary ion export due to its structure and mode of action (41). In many cases, what secondary ion is associated with uptake of ion remains unknown. Thus, we could not completely rule out the possibility that the effect of ZosA is dependent on, not zinc transport, but unknown secondary ion transport. It is a very important point that perturbation of zinc homoeostasis did not occur in the zosA mutant, which is based on the observation that the expression of ytiA-lacZ is still repressed. In other words, in zosA cells, the need of proteins requiring zinc to be active would be fulfilled. This situation is consistent with the former observation that the total cellular zinc content in the zosA mutant is similar to that in the wild-type cells, when the cells were grown in the presence of 20 nM of zinc and 20 µM hydrogen peroxide in order to induce zosA expression (7). The nature of the ZosA transporter, which works as a major zinc transporter at a high environmental zinc level, where the expression of the genes coding for ZnuABC and YciC are completely repressed (5), is also compatible with this situation. Since there are many protein ligands for zinc in the cell that results in the cellular free zinc pool being scarce, it is thought to be reasonable that Zur reacts to femtomolar levels of zinc concentration (42). Thus, fluctuation of the intracellular free zinc concentration caused by the zosA mutation might be transient and subtle, leading to ComK inactivation. Since the intracellular zinc pool is scant, subtle and transient increases in intracellular zinc might transmit specific signal to the target. It is very intriguing that the expression of the zosA gene is upshifted by partial derepression from the PerR sensor for hydrogen peroxide in the transition from the growing phase to stationary phase, probably because of the increased generation of hydrogen peroxide due to aerobiosis (7, 43). In other words, the entry into the stationary phase is transmitted to the regulatory cascade for ComK as an increase in cellular hydrogen peroxide, leading to the up-regulation of zosA. ZosA-mediated zinc incorporation is apparently required for post-transcriptional control of comK, while ZnuABC-mediated zinc incorporation specifically activates comF transcription. The differential roles of zinc transporter are common phenomena in eukaryotes (44). For example, 14 zinc importer genes have been identified in human cells, and tissue-specific expression and specific cellular localization are reported (45). Thus, each disruption of the genes encoding zinc transporters shows different phenotypes, such as abnormal morphogenesis of embryo and depletion of thymic pre-T cell in zinc-limiting condition and finally lethality (44). This suggested that in B. subtilis, two zinc transporters might regulate competence development at different timing in the regulatory cascade, because comF activation follows post-transcriptional control of ComK. Taken together, to the best of our knowledge, this is the first report of the differential roles of zinc transporters in the cellular differential process of prokaryotes. Funding Grant-in-aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Conflict of interest None declared. 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Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
TI - ZnuABC and ZosA zinc transporters are differently involved in competence development in Bacillus subtilis
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvr098
DA - 2011-08-03
UR - https://www.deepdyve.com/lp/oxford-university-press/znuabc-and-zosa-zinc-transporters-are-differently-involved-in-Lko66KckXC
SP - 615
EP - 625
VL - 150
IS - 6
DP - DeepDyve
ER -