Nucleic Acids Research, 2001, Vol. 29, No. 18 3804-3813
© 2001 Oxford University Press
DNA microarray analysis of Bacillus subtilis DegU, ComA and PhoP regulons: an approach to comprehensive analysis of B.subtilis two-component regulatory systems
Department of Marine Science, School of Marine Science and Technology, Tokai University, 3-20-1 Orido, Shimizu, Shizuoka 424-8610, Japan and 1Faculty of Engineering, Fukuyama University, Fukuyama 729-0292, Japan
Received June 29, 2001; Revised and Accepted July 31, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have analyzed the regulons of the Bacillus subtilis two-component regulators DegU, ComA and PhoP by using whole genome DNA microarrays. For these experiments we took the strategy that the response regulator genes were cloned downstream of an isopropyl-ß-D-thiogalactopyranoside-inducible promoter on a multicopy plasmid and expressed in disruptants of the cognate sensor kinase genes, degS, comP and phoR, respectively. The feasibility of this experimental design to detect target genes was demonstrated by the following two results. First, expression of lacZ fusions of aprE, srfA and ydhF, the target genes of DegU, ComA and PhoP, respectively, was stimulated in their cognate sensor kinase-deficient mutants upon overproduction of the regulators. Secondly, by microarray analysis most of the known target genes for the regulators were detected and, where unknown genes were found, the regulator dependency of several of them was demonstrated. As the mutants used were deficient in the kinase genes, these results show that target candidates can be detected without signal transduction. Using this experimental design, we identified many genes whose dependency on the regulators for expression had not been known. These results suggest the applicability of the strategy to the comprehensive transcription analysis of the B.subtilis two-component systems.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent advances in genomics have revealed the presence of many two-component regulatory systems in various organisms including prokaryotes, lower eukaryotes and plants, indicating that these signal transduction systems have versatile roles in many cellular functions (1–3). In Bacillus subtilis, 37 sensor kinases and 34 response regulators have been found, and among them 30 kinase–regulator pairs reside consecutively in the chromosome (4,5). The roles of most of the systems, however, are still unknown. It is generally thought that many types of information are processed and appropriate responses are made via these two-component systems so that organisms can adapt to changing environmental conditions (3). A typical two-component system is composed of a sensor kinase and its cognate response regulator. The catalytic part of the kinase phosphorylates its own histidine residue by responding to the input of a signal, and the phosphoryl group is then transferred to a conserved aspartate residue on the cognate response regulator, which acts as a transcription factor in most cases. Many kinases also have a phosphatase activity for the cognate phosphorylated regulator, although some response regulators have an intrinsic phosphatase activity (3,6).
The B.subtilis DegS–DegU two-component system regulates many cellular processes including exoprotease production, competence development and motility (7). Phosphorylated DegU (DegU-P) stimulates transcription of aprE and nprE encoding the major extracellular proteases, and inhibits expression or the activity of an alternative sigma factor SigD (7–9), whereas unphosphorylated DegU enhances the transcription of comK encoding the competence transcription factor by binding to its promoter region (7,10). Therefore, DegU is regarded as a molecular switch controlling the expression of two alternative sets of genes. It has been demonstrated that salt stress affects expression of aprE, sacB (levansucrase) and wapA (wall-associated protein) via DegS–DegU (11,12), but how DegU-P exerts its effect on target gene expression remains unknown.
The ComP–ComA two-component system is activated by cell density signals (13,14). Phosphorylated ComA (ComA-P), the activated form of the regulator, binds to the promoter region of the srfA operon (14) encoding the enzyme complex catalyzing the synthesis of a lipopeptide antibiotic surfactin and also the competence regulatory factor ComS, which lies within and out-of-frame with the srfAB gene (7). In addition, ComA stimulates the expression of degQ, rapA and rapC (13).
The PhoP–PhoR system regulates expression of the Pho regulon that is induced by phosphate starvation (15). The Pho regulon includes the structural genes phoA and phoB for the two major alkaline phosphatases, phoD for alkaline phosphatase–phosphodiesterase, the operons pstSACB1B2, tagAB and tagDEF, and tuaABCDEFGH, which are involved in high-affinity phosphate transport, teichoic acid synthesis and teichuronic synthesis, respectively, and phoPR itself (16–18). Recently the glpQ and ydhF genes were shown to belong to the Pho regulon (16). The consensus sequence for the binding of phosphorylated PhoP (PhoP-P) has been determined (19).
The recently developed DNA microarray technique is a powerful tool for transcriptome analysis of the entire genome, as a large amount of information is obtained at a time, and has been successfully applied to transcriptional analysis of several bacteria including B.subtilis (20–23). With respect to the microarray analysis of the B.subtilis two-component system, studies have been reported for two response regulators, ResD and Spo0A (20,21). In both cases, a global change in gene expression has been observed in strains bearing disruption of resD and spo0A.
To deduce the functional roles of all the B.subtilis two-component systems, knowledge of their target genes obtained by a global method such as microarray analysis will be of great help. For such studies, disruption of the regulator gene followed by microarray analysis is certainly the method of choice. However, this strategy may not be applicable to the cases where expression of the target genes is very low and, therefore, the effect of regulator gene disruption is ambiguous. Furthermore, the fact that the signals inducing most of the two-component systems are unknown makes it impossible to identify target genes by stimulating the cells with the signals. One way to overcome these potential problems would be that the regulator gene product is amplified in the cell, so that expression of the target genes is forced to be enhanced or repressed, and thus, the sensitivity of the microarray analysis may be increased. Following this expectation we applied the amplification method for regulators DegU, ComA and PhoP whose targets are known in some detail. We show in this study that overexpression of the response regulator genes indeed resulted in stimulation of target gene expression in the strains carrying disruption of their cognate sensor kinase genes. Therefore, this method may be potentially applicable to identify possible target genes of two-component regulatory systems.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bacterial strains, plasmids and culture media
All the strains and plasmids used in this study are listed in Table 1. Either Schaeffer’s sporulation medium or Luria–Bertani (LB) medium was used for ß-galactosidase assays and the isolation of total cellular RNA. Escherichia coli cells for DNA manipulation were grown in liquid or agar LB medium. The concentrations of the antibiotics used in this study were described previously (9,24).
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Materials
Synthetic oligonucleotides were commercially prepared by Espec Oligo Service (Ibaraki, Japan). PCR fragments were prepared by PJ2000 (Perkin-Elmer Cetus). Nucleotide sequencing was carried out using a 377 DNA Sequencer and a Dye Terminator Cycle Sequencing Kit (Applied Biosystems).
Plasmid construction
DNA regions encompassing the structural genes for degU, comA and phoP and their SD sequences were amplified by PCR using oligonucleotide pairs DegUF1 and DegUR1, ComAF3 and ComAR1, and PhoPF1 and PhoPR1, respectively (Table 2). The amplified DNA fragments were digested with HindIII and SalI, and then cloned into pDG148 (25) digested with the same restriction enzymes, resulting in plasmids pDG148-degU, pDG148-comA and pDG148-phoP, respectively. The nucleotide sequences in the cloned DNA regions were confirmed by sequence determination of the entire regions.
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Growth condition and RNA isolation
Strains TT7291 and OSM103 carrying pDG148-degU and pDG148-comA, respectively, were grown overnight in LB medium. Two milliliters of the overnight cultures were inoculated into 100 ml of Schaeffer’s medium contained in two 500 ml Erlenmyer flasks, and cells were grown at 37°C to a Klett unit of
50. Isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to one of the flasks at a concentration of 1 mM, and the cells were harvested after 2 h (30 min after the end of log phase). For strain MH5913 carrying pDG148-phoP, LB medium was used for cell growth, as the strain did not grow well in Schaeffer’s medium. Total RNA was isolated from the cells essentially as described previously (22).
Preparation of fluorescently-labeled cDNA
Fluorescently-labeled cDNA probes used for hybridization to microarrays were prepared by a two-step procedure: cDNA for total RNA was aminoallyl-labeled by reverse transcription with specific primers in the presence of aminoallyl-dUTP, followed by fluorescence-labeling of the resultant aminoallylated cDNA with N-hydroxysuccinimide-activated Cy3 or Cy5. The procedure was performed according to the manufacturer’s protocol (Atlas glass fluorescent labeling kit; Clontech) with a slight modification. RNA (50 µg) and a mixture of 4050 primers that were complementary to mRNAs (0.5 pmol each) and used for the preparation of microarrays were mixed with human transferrin receptor (hTFR) mRNA and its complementary hTFR primer (0.5 pmol). hTFR mRNA synthesized in vitro was kindly supplied by Takara Shuzo (Shiga, Japan), and used as a positive control for microarray analysis. To this mixture was added Tris–acetate (pH 8.4, 50 mM final concentration), potassium acetate (75 mM), magnesium acetate (8 mM), dithiothreitol (10 mM), 4 µl of 10x dNTP Mix (dNTP and aminoallyl-dUTP) of the labeling kit, RNaseOUT (40 U; Life Technology, Inc., Rockville, MD) and Thermoscript (30 U; Life Technology, Inc.), and the final volume was made 40 µl. Reverse transcription for aminoallyl-labeling of cDNA was carried out at 60°C for 1 h, and then continued for another 1 h after the addition of 30 U Thermoscript. RNA was digested by the addition of 10 U RNaseH, followed by incubation at 37°C for 5 min. To inactivate Thermoscript, the reaction mixture was incubated for 5 min at 85°C. The Cy3- or Cy5-fluorescently-labeled cDNA was prepared exactly following the protocol supplied by the manufacturer, and was finally dissolved in 12 µl of distilled water. This preparation was stable for several months when stored at –20°C in the dark.
Hybridization and microarray analysis
DNA microarrays were prepared as described previously (22). They contained 4005 genes excluding those for rRNA and tRNA, but did not contain 45 genes including degQ and tuaA due to a problem in amplification of DNA by PCR. The hybridization and microarray analyses were performed as described previously (22) except that the microarrays were washed in 2x SSC and 0.1% SDS after pre-hybridization, and in 0.5x SSC and 0.01% SDS at 48°C for 5 min after hybridization.
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RESULTS |
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Specific expression of target genes by induction of response regulator genes in strains bearing a disruption of the cognate sensor kinase genes
Our strategy to identify the target genes of response regulators was to amplify the regulator proteins and examine the expression levels of the chromosomal genes on a microarray (see Introduction). We chose DegU, ComA and PhoP as the model response regulators for a global analysis of the two-component system in B.subtilis, because a part of their target genes have been identified in each case. We placed the regulator genes degU, comA and phoP under the control of the IPTG-inducible Pspac promoter in pDG148, constructing pDG148-degU, pDG148-comA and pDG148-phoP, respectively (Materials and Methods), and then tested the effect of IPTG-induction on gene expression. To monitor the expression of the target genes of DegU, ComA and PhoP, fusions aprE-lacZ, srfA-lacZ and ydhF-lacZ were used, respectively. Cells were grown to mid-log phase in Schaeffer’s sporulation medium except for the cells carrying pDG148-phoP for which LB medium was used, and expression of the regulator genes was induced (for details see Materials and Methods). As shown in Figure 1, the expression levels of aprE-lacZ, srfA-lacZ and ydhF-lacZ were very low in the degS-, comP- and phoR-deficient mutants, respectively, whereas the addition of IPTG greatly increased the expression levels in those strains. When the same experiments were performed with strains carrying the wild-type kinase genes, no enhancing effect on the target genes was observed (data not shown). These results indicate that overexpression of the regulator genes enhanced the target genes without signal transduction through the sensor kinases and mimicked the signal input that results in phosphorylation and activation of the cognate regulators in the wild-type strain.
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We applied the above experimental strategy to identify possible target genes of the response regulators by microarray analysis. RNAs were isolated from the cells grown with and without IPTG addition, and subjected to cDNA synthesis and the microarray procedures (see Materials and Methods). We took the ratios of >4.5- and
0.25-fold as the criteria of stimulation and inhibition of gene expression by the regulators, respectively. The DNA microarray data are available on the web site: http://www.genome.ad.jp/kegg/expression.
Global analysis of regulons of two-component regulators by DNA microarray
DegU regulon. The results obtained with RNA from TT7291 (
degS) carrying pDG148-degU are shown in Table 3. The aprE, nprE and ispA genes have been shown to be the targets of phosphorylated DegU (7), and in good agreement with this the transcription levels of these genes were found to be 12.7-, 9.5- and 8.3-fold higher in the IPTG-induced cells, respectively. Transcription of the nprB gene encoding an exoprotease was stimulated as expected (7). Table 3 also shows that expression of many genes/operons whose relationship to DegU had been unknown was either stimulated or decreased by overproduced DegU. In fact DegU affected the expression of 2.8% of the B.subtilis genes (116 genes) based on our criteria. In order to test whether the genes found in this experiment are indeed under DegU regulation, we examined expression of several genes by lacZ fusion or northern analysis in CU741 (degU+) and its degU-knockout strain. The expression of bpr-lacZ, yukL-lacZ and ycdA-lacZ was found to be decreased in degU strains as shown in Figure 2. It should be noted that the former dhbF (fold ratio, 4.5), yukL and yukM are now in the large dhbF gene and form a gene cluster of siderophore synthesis with the upstream dhbA, C, E and B genes (26,27). Furthermore, the expression level of dhbA was much lower in the degU mutant as shown by northern analysis (Fig. 3A). Among the genes that we tested for DegU dependency, we could not see much difference in expression for the genes ywfD, yvdA, yraJ and yitN, because ß-galactosidase activities in degU+ and degU strains were too low or too close to each other. It seems that the lacZ fusion assay is less sensitive than the microarray assay and also has its limitation possibly due to the difference in stability of natural and fusion-gene mRNAs.
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The other up-regulated genes whose functions are already known include those for energy production (atpB, E, F, H, A, G, D, D and C; 28), polyketide biosynthesis (pksP, M, L, K and G; 29), cell wall biosysnthesis (murD, mraY; 30), pyruvate dehydrogenase (pdhA; 31) and translation (frr and tsf; 5). In contrast to the DegU regulation described so far, murD transcription was found to be increased by degU deficiency as shown by northern analysis (Fig. 3B), apparently indicating that murD is regulated by DegU in both positive and negative ways. In addition, 39 genes whose functions are unknown were found to be positively regulated by DegU (Table 3).
We found that overproduction of DegU resulted in reduced expression of the genes for histidine degradation (hutM, G, I, U and H; 32), the PTS system for lichenan (licB and licC; 33), fatty acid metabolism (acdA; 5), chemotaxis-motility (hag, fliS, mcpA and C; 34), a response regulator aspartate phosphatase (rapA; 13) and fermentation (acoC, B and A; 35), and those located within the 5'-portion of the large fla-che operon (flgE, fliJ, I, H, G, F, E, flgC and B; 34). It is interesting to note that the transcription of the minor exoprotease gene, epr (36), was repressed, in contrast to the positive regulation of other protease gene expression by DegU. In addition, transcription of 20 unknown genes was found to be repressed by DegU (Table 3).
The previous and the current experimental results with aprE, nprE, ispA, bpr, yukL, ycdA and dhbA, together with the DegU effect on the expression of the SigD-driven chemotaxis-motility genes, support the validity of our experimental approach to identify target candidates of DegU.
ComA regulon. Results of a microarray analysis obtained by overexpression of comA in a comPA disruptant are shown in Table 4. The transcription of the srfA operon genes, srfAA, AB, AC and AD, which are known to be directly regulated by ComA-P (14), was greatly increased (7.7–43-fold). Expression of rapA has been reported to be regulated by ComA-P (13), and indeed we found the gene overexpressed in our analysis. ComA-P also regulates degQ and rapC (13), but they are not listed in Table 4, as the degQ DNA had not been spotted on the microarray grid (see Materials and Methods) and the fold expression of rapC (2.8-fold) did not meet our criterion (>4.5-fold expression level).
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The expression of rapF was increased as shown by the microarray analysis, and we confirmed the result by using a rapF–lacZ fusion in comA cells (Fig. 2D). The rapF gene encodes a putative phosphatase gene for an unknown response regulator, which suggests that the target response regulator of RapF is under the control of ComA. A further microarray analysis of rapF would extend the network of ComA regulation. As expected from a computational search for ComA binding sites (13), the expression of pel encoding pectate lyase (37) was increased by overproduced ComA. These results together with those of the known genes described above show again that most of the target genes of ComA can be detected by our strategy.
The genes sacV (a transcriptional regulator of the levansucrase gene sacB; 38) and sunA (sublancin 168 lantibiotic antimicrobial precursor peptide; 5) were identified as possible new members of the ComA regulon. We note that the genes yddI through sacV are overexpressed, although the extents of enhancement were variable. They are arranged on the B.subtilis map in the order of sacV, ydcO, P, Q, R, S, T, yddA, B, C, D, F, G, H and I (5). The fold ratios for yddE and yddJ that reside in or at the terminus of this group of genes were 3.9 and 3.6, respectively. These results suggest that the genes constitute a large operon. We note that yqaT, sunA-yopZ-yoqL and the genes yddI through sacV are constituents of the skin element, phage SPß and prophage2, respectively (5).
PhoP regulon. For the microarray analysis of phoP expression, cells were grown in LB medium, i.e. a condition in which no phosphate starvation was exerted. Overexpression of phoP in phoPR cells stimulated the known target genes phoA, phoB, ydhF, phoD, tuaB, C, D, E, F, G, H, pstS, A, C, B1, B2, glpQ and phoR (Table 5), indicating that overproduced PhoP in the absence of PhoR can stimulate PhoP target genes without phosphate starvation. The expression of the yycP, glnQ and yjdB genes was stimulated by overproduced PhoP. We tried to confirm PhoP dependency for the two genes yycP and yjdB, but the cells carrying a yycP-lacZ (YYCPd) or yjdB-lacZ (BFS436) fusion showed very low levels of ß-galactosidase activity, which precluded an accurate estimation of expression. However, the strains gave blue colonies on low-phosphate medium plates, whereas the colonies of their phoP mutants (OAM143 and OAM144) exhibited no blue color, indicating that the expression of the genes is PhoP-dependent (data not shown). Furthermore, it was found that the expression of yycP was low-phosphate-inducible, whereas no such specificity was observed for yjdB (data not shown).
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Although it has been reported that tagAB, tagDEF and resABCDE are repressed by PhoP (17,18), we could not confirm this under the condition we adopted in this study.
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DISCUSSION |
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The lacZ fusion experiments (Fig. 1) show that overproduction of the response regulators DegU, ComA and PhoP stimulate the expression of the genes aprE, srfA and ydhF, respectively, in their cognate sensor gene disruptants, whereas there was no such stimulation in the wild-type strains (data not shown). These results provided the basis of our experimental strategy to identify possible targets of two-component regulatory systems, and indeed we identified many genes that were proved to be under the regulation of DegU, ComA and PhoP. Several explanations could be envisaged for the successful expression of the target genes in the sensor gene disruptants but not in the wild-type cells. It may be possible that without stimuli the sensor protein would serve as a phosphatase for its overproduced cognate response regulator. The E.coli sensor kinase KdpD is thought to be activated by a physiological signal acting to inhibit the phosphatase activity intrinsic to the sensor protein (39). In the B.subtilis DesK–DesR system, the overproduced DesR regulator stimulates target gene expression in the absence of the DesK kinase, and it was suggested that DesK works as a phosphatase of phosphorylated DesR unless a stimulus (temperature shift down) comes into the cell (40). The other explanation would be that an overproduced response regulator is inhibited by its cognate sensor kinase. It has been demonstrated that the E.coli UhpA response regulator is inhibited by its cognate sensor kinase UhpB in the absence of stimulation by UhpC possibly through binding and sequestration of UhpA by inactive UhpB (41). Regardless of the precise mechanism underlining these phenomena, overproduction of regulators in the absence of the cognate sensor kinases results in ‘constitutive’ expression of the target genes and eliminates the need for physiological signal input. This is important for studying two-component regulatory systems, for most of which the nature of the inducing signal is unknown.
The experimental results that overproduction of DegU, ComA and PhoP in cognate sensor disruption mutants enhanced the expression of the target genes indicate that the overproduced response regulators behave like phosphorylated regulators. Two explanations could be conceivable. First, the response regulators would be phosphorylated by a non-partner kinase or low molecular-weight phosphate donors, for example, acetyl phosphate (42), leading to activation of the target gene expression. In fact, ComA can be phosphorylated by acetyl phosphate in vitro (43). Secondly, an elevated concentration of the response regulator in the cell might result in multimer formation of the response regulator, which is proposed to activate some response regulators (6,44,45). To unravel the mechanism underlying this phenomenon, in-depth analysis is needed.
Expression of the genes for degradative enzyme synthesis and competence development has been shown to be positively but differentially regulated by the DegU response regulator: expression of the former genes is stimulated by phosphorylated DegU, while that of the latter genes is by unphosphorylated DegU (7). Likewise, it may be conceivable that a certain gene is negatively regulated by unphosphorylated DegU, whereas it is positively regulated by phosphorylation of DegU. Based on the assumption that overproduction of DegU mimics the phosphorylated form of DegU as described above, the putative atp and murD-mraY operons are likely to belong to this category, as their expression was stimulated by both multicopy degU (Table 3) and degU disruption (Fig. 3 and M.Ogura and T.Tanaka, unpublished result). On the other hand, expression of dhbA was stimulated by multicopy degU (Table 3), and inhibited in a degU disruptant as revealed by northern analysis (Fig. 3), suggesting that dhbA is positively regulated by DegU-P like aprE.
Several genes were found to be grouped in the DegU regulon, although they were not rigorously proved by lacZ fusion or northern analysis. They include the putative atpIBEFHAGDC and murE-murD-mraY operons and the genes relating to polyketide synthesis (pksG through pksR). However, not all the constituents of these operons were detected by the microarray analysis. For example, atpI and murE in the atp and murE-murD-mraY operons, respectively, and pksH, I, J, N, P and R for the probable pksGHIJKLMNPR operon were not included. One reason for this is due to our rigorous criterion (expression ratios of >4.5-fold). In fact, induction ratios (fold) of these genes were as follows: atpI, 2.8; murE, 4.2; pksH, 2.4; I, 4.6; J, 1.7; N, 2.3; P, 4.7; R, 1.4. It is interesting to note that, in addition to degradative enzyme synthesis and competence development, DegU may participate in cell wall synthesis, energy production, siderophore formation, protein translation and antibiotic synthesis, although implications of these observations are not clear at present.
In this microarray analysis, we could not detect genes whose expression was expected to be high in the DegU-overproducing cells; for example, the amyE gene belonging to the DegU regulon (7). This is probably because expression of the amyE gene is very low in strain CU741 used in this study for an unknown reason (M.Ogura and T.Tanaka, unpublished results). Others include sacB (7), sacX (46), xynD (7) and bglS (7). One possible reason is that these four genes are also under the regulation of another gene(s); for example, sacB expression requires the addition of sucrose in medium.
Elevation of the phosphorylation level of DegU has been shown to inhibit motility function through the inhibition of either transcription of sigD (8), which resides in the large fla-che operon (34), or the function of SigD (9). In concert with this, transcription of the hag and fliS genes that belong to the SigD regulon was inhibited (Table 3). The inhibition of SigD may block the transcription of the entire fla-che operon (47). This agrees with the observation that transcription of the genes related to chemotaxis and motility was repressed (Table 3).
It was found that transcription of rapA was repressed by overproduction of DegU. We note that transcription of the other rap genes was also slightly repressed: rapB, 0.39-fold; rapE, 0.34-fold; rapF, 0.27-fold; rapC, 0.27-fold.
In competence development through the ComP–ComA regulatory system, the extracellular ComX factor triggers ComP-dependent phosphorylation of ComA (13). Our microarray analysis revealed the target genes for ComA in a strain lacking the entire comQXPA region (48). Although the known target genes such as the srfA operon genes and rapA were identified in this global analysis of ComA, we failed to find the known genes, rapC (13) and rapE (49). This is due to low induction ratios of rapC (2.8-fold) and a low basal transcription level of rapE whose induction ratio was 8.0-fold.
In the microarray analysis of PhoP, we detected most of the genes reported to be under Pho-P regulation. However, transcription of the tagAB and tagDEF was not affected by overproduction of PhoP, although these operons are known to be repressed by Pho-P (16,17). This is probably because the tag operons are not repressed under phosphate-replete conditions (17). Although not detected in the current microarray analysis, the resABCDE and ykoL genes are under positive regulation of PhoPR. It has been shown that the addition of glutamate or growth in Schaeffer’s sporulation medium supplemented with glucose greatly reduces the PhoPR dependency of these genes (18,50), respectively. As we used LB medium for this experiment, it is likely that the failure to detect the genes is due to a nutritional effect in the medium used. The tuaA gene whose expression is also expected to be stimulated was not spotted on the microarray plate.
The B.subtilis genome sequencing revealed that this organism possesses 37 sensor kinases and 34 response regulators of the two-component regulatory systems (4,5). Among them, nearly two-thirds remain to be characterized. Although we could not detect several genes by this system probably due to the reasons described above, the current approach will allow us to detect most of the target candidates of many two-component regulatory systems functionally uncharacterized. In these cases too, quantitative analyses such as lacZ fusion or northern experiments on the candidate genes detected may be necessary. We note that most of the kinase-regulator genes reside in the B.subtilis chromosome in pairs (5), so that we can mimic an unknown signal(s) to cause phosphorylation of an uncharacterized regulator by overexpression of the regulator gene and simultaneous disruption of the neighboring cognate sensor gene. We have already applied this strategy to analysis of uncharacterized B.subtilis two-component systems and successfully identified putative target genes for many regulators (K.Kobayashi, M.Ogura, H.Yamaguchi, K.-I.Yoshida, N.Ogasawara, T.Tanaka and Y.Fujita, unpublished observation). Obviously the essential two-component system, yycF–yycG, cannot be applied to this experimental approach (51,52).
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ACKNOWLEDGEMENTS |
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We wish to thank H. Kitoh, W. Shimizu, T. Takahashi, Y. Nakaura and S. Tojo for their assistance. We also thank F. M. Hulett, P. Zuber, M. M. Nakano and K. Ochi for strains, K. Asai for a plasmid, and all the members of EU and Japan Consortia of Bacillus subtilis functional genomics for the pMUTIN-based mutants. This work was supported by Grant-in-aids for Scientific Research on Priority Areas (C) ‘Genome Biology’ and a Grant-in-aid for Scientific Research (B) and (C) from the Ministry of Education, Science and Sports and Culture of Japan.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +81 543 34 0411; Fax: +81 543 34 9834; Email: teruot{at}scc.u-tokai.ac.jp The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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REFERENCES |
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1 Loomis,W.F., Kuspa,A. and Shaulsky,G. (1998) Two-component signal transduction systems in eukaryotic microorganisms. Curr. Opin. Microbiol., 1, 643–648.[ISI][Medline]
2 Mizuno,T. (1997) Comparison of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli. DNA Res., 4, 161–168.[Abstract]
3 Stock,J.B., Ninfa,A.J. and Stock,A.M. (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol. Rev., 53, 450–490.
4 Fabret,C., Feher,V.A. and Hoch,J.A. (1999) Two-component signal transduction in Bacillus subtilis: How one organism sees its world. J. Bacteriol., 181, 1975–1983.
5 Kunst,F., Ogasawara,N., Moszner,I., Albertini,A.M., Alloni,G., Azevedo,V., Bertero,M.G., Besssieres,P., Bolotin,A., Borchert,S. et al. (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature, 390, 249–256.[Medline]
6 Egger,L.A., Park,H. and Inouye,M. (1997) Signal transduction via the histidyl-aspartyl phosphorelay. Genes Cells, 2, 167–184.[Abstract]
7 Msadek,T., Kunst,F. and Rapoport,G. (1995) A signal transduction network in Bacillus subtilis includes the DegS/DegU and ComP/ComA two-component systems. In Hoch,J.A. and Silhavy,T.J. (eds), Two-Component Signal Transduction. American Society for Microbiology, Washington, DC, pp. 447–471.
8 Tokunaga,T., Rashid,M.H., Kuroda,A. and Sekiguchi,J. (1994) Effect of degS–degU mutations on the expression of sigD, encoding an alternative sigma factor and autolysin operon of Bacillus subtilis. J. Bacteriol., 176, 5177–5180.
9 Ogura,M. and Tanaka,T. (1996) Transcription of Bacillus subtilis degR is 
D-dependent and suppressed by multicopy proB through D. J. Bacteriol., 178, 216–222.
10 Hamoen,L.W., Van Werkhoven,A.F., Venema,G. and Dubnau,D. (2000) The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis. Proc. Natl Acad. Sci. USA, 97, 9246–9251.
11 Kunst,F. and Rapoport,G. (1995) Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J. Bacteriol., 177, 2403–2407.
12 Dartois,V., Debarbouille,M., Kunst,F. and Rapoport,G. (1998) Characterization of a novel member of the DegS–DegU regulon affected by salt stress in Bacillus subtilis. J. Bacteriol., 180, 1855–1861.
13 Lazazzera,B.A., Palmer,T., Quisel,J. and Grossman,A.D. (1999) Cell density control of gene expression and development in Bacillus subtilis. In Dunny,G.M. and Winans,S.C. (eds), Cell–Cell Signaling in Bacteria. American Society for Microbiology, Washington, DC, pp. 27–46.
14 Tortosa,P. and Dubnau,D. (1999) Competence for transformation: a matter of taste. Curr. Opin. Microbiol., 2, 588–592.[ISI][Medline]
15 Hulett,F.M. (1996) The signal-transduction network for Pho regulation in Bacillus subtilis. Mol. Microbiol., 19, 933–939.[ISI][Medline]
16 Antelmann,H., Scharf,C. and Hecker,M. (2000) Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis. J. Bacteriol., 182, 4478–4490.
17 Liu,W., Eder,S. and Hulett,F.M. (1998) Analysis of Bacillus subtilis tagAB and tagDEF expression during phosphate starvation identifies a repressor role for PhoP-P. J. Bacteriol., 180, 753–758.
18 Birkey,S.M., Liu,W., Zhang,X., Duggan,M.F. and Hulett,F.M. (1998) Pho signal transduction network reveals direct transcriptional regulation of one two-component system by another two-component regulator: Bacillus subtilis PhoP directly regulates production of ResD. Mol. Microbiol., 30, 943–953.[ISI][Medline]
19 Liu,W., Qi,Y. and Hulett,F.M. (1998) Sites internal to the coding regions of phoA and pstS bind PhoP and are required for full promoter activity. Mol. Microbiol., 28, 119–130.[ISI][Medline]
20 Fawcett,P., Eichenberger,P., Losick,R. and Youngman,P. (2000) The transcriptional profile of early to middle sporulation in Bacillus subtilis. Proc. Natl Acad. Sci. USA, 97, 8063–8068.
21 Ye,R.W., Tao,W., Bedzyk,L., Young,T., Chen,M. and Li,L. (2000) Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions. J. Bacteriol., 182, 4458–4465.
22 Yoshida,K.-I., Kobayashi,K., Miwa,Y., Kang,C.-M., Matsunaga,M., Yamaguchi,H., Tojo,S., Yamamoto,M., Nishi,R., Ogasawara,N. et al. (2001) Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis. Nucleic Acids Res., 29, 683–692.
23 Moreno,M.S., Schneider,B.L., Maile,R.R., Weyler,W. and Saier,M.H. (2001) Catabolite repression mediated by CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Mol. Microbiol., 39, 1366–1381.[ISI][Medline]
24 Ogura,M., Liu,L., Lacelle,M., Nakano,M.M. and Zuber,P. (1999) Mutational analysis of ComS: evidence for the interaction of ComS and MecA in the regulation of competence development in Bacillus subtilis. Mol. Microbiol., 32, 799–812.[ISI][Medline]
25 Stragier,P., Bonamy,C. and Karmazyn-Campelli,C. (1988) Processing of a sporulation sigma factor in Bacillus subtilis: how morphological structure could control gene expression. Cell, 52, 697–704.[ISI][Medline]
26 May,J.J., Wendrich,T.M. and Marahiel,M.A. (2001) The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J. Biol. Chem., 276, 7209–7217.
27 Rowland,B.M., Grossman,T.H., Osburne,M.S. and Taber,H.W. (1996) Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes. Gene, 178, 119–123.[ISI][Medline]
28 Santana,M., Ionescu,M.S., Vertes,A., Longin,R., Kunst,F., Danchin,A. and Glaser,P. (1994) Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants. J. Bacteriol., 176, 6802–6811.
29 Scotti,C., Piatti,M., Cuzzoni,A., Perani,P., Tognoni,A., Grandi,G., Galizzi,A. and Albertini,A.M. (1993) A Bacillus subtilis large ORF coding for a polypeptide highly similar to polyketide synthases. Gene, 130, 65–71.[ISI][Medline]
30 Daniel,R.A. and Errington,J. (1993) DNA sequence of the murE-murD region of Bacillus subtilis 168. J. Gen. Microbiol., 139, 361–370.[Medline]
31 Hemila,H., Palva,A., Paulin,L., Arvidson,S. and Palva,I. (1990) Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase. J. Bacteriol., 172, 5052–5063.
32 Oda,M., Sugishita,A. and Furukawa,K. (1988) Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon. J. Bacteriol., 170, 3199–3205.
33 Tobisch,S., Glaser,P., Kruger,S. and Hecker,M. (1997) Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis. J. Bacteriol., 179, 496–506.
34 Ordal,G.W., Marquez-Magana,L. and Chamberlin,M.J. (1993) Motility and chemotaxis. In Sonenshein,A.L., Hoch,J.A. and Losick,R. (eds), Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics. American Society for Microbiology, Washington, DC, pp. 765–784.
35 Huang,M., Oppermann-Sanio,F.B. and Steinbuchel,A. (1999) Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway. J. Bacteriol., 181, 3837–3841.
36 Sloma,A., Ally,A., Ally,D. and Pero,J. (1988) Gene encoding a minor extracellular protease in Bacillus subtilis. J. Bacteriol., 170, 5557–5563.
37 Nasser,W., Awade,A.C., Reverchon,S. and Robert-Baudouy,J. (1993) Pectate lyase from Bacillus subtilis: molecular characterization of the gene and properties of the cloned enzyme. FEBS Lett., 335, 319–327.[ISI][Medline]
38 Martin,I., Debarbouille,M., Klier,A. and Rapoport,G. (1987) Identification of a new locus, sacV, involved in the regulation of levansucrase synthesis in Bacillus subtilis. FEMS Microbiol. Lett., 44, 39–43.
39 Brandon,L., Dorus,S., Epstein,W., Altendorf,K. and Jung,K. (2000) Modulation of KdpD phosphatase implicated in the physiological expression of the kdp ATPase of Escherichia coli. Mol. Microbiol., 38, 1086–1092.[ISI][Medline]
40 Aguilar,P.S., Hernandez-Arriaga,A.M., Cybulski,L.E., Erazo,A.C. and de Mendoza,D. (2001) Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis. EMBO J., 20, 1681–1691.[ISI][Medline]
41 Wright,J.S., Olekhnovich,I.N., Touchie,G. and Kadner,R.J. (2000) The histidine kinase domain of UhpB inhibits UhpA action at the Escherichia coli uhpT promoter. J. Bacteriol., 182, 6279–6286.
42 Lukat,G.S., McCleary,W.R., Stock,A.M. and Stock,J.B. (1992) Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc. Natl Acad. Sci. USA, 89, 718–722.
43 Roggiani,M. and Dubnau,D. (1993) ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. J. Bacteriol., 175, 3182–3187.
44 Fielder,U. and Weiss,V. (1995) A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules. EMBO J., 14, 3696–3705.[ISI][Medline]
45 Webber,C.A. and Kadner,R.J. (1997) Involvement of the amino-terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli. Mol. Microbiol., 24, 1039–1048.[ISI][Medline]
46 Crutz,A.M. and Steinmetz,M. (1992) Transcription of the Bacillus subtilis sacX and sacY genes, encoding regulators of sucrose metabolism, is both inducible by sucrose and controlled by the DegS–DegU signaling system. J. Bacteriol., 174, 6087–6095.
47 West,J.T., Estacio,W. and Marquez-Magana,L. (2000) Relative roles of the fla/che PA, PD-3 and PsigD promoters in regulating motility and sigD expression in Bacillus subtilis. J. Bacteriol., 182, 4841–4848.
48 Nakano,M.M. and Zuber,P. (1989) Cloning and characterization of srfB, a regulatory gene involved in surfactin production and competence in Bacillus subtilis. J. Bacteriol., 171, 5347–5353.
49 Jiang,M., Grau,R. and Perego,M. (2000) Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis. J. Bacteriol., 182, 303–310.
50 Robichon,D., Arnaud,M., Gardan,R., Pragai,Z., O’Reilly,M., Rapoport,G. and Debarbouille,M. (2000) Expression of a new operon from Bacillus subtilis, ykzB-ykoL, under the control of the TnrA and PhoP-phoR global regulators. J. Bacteriol., 182, 1227–1231.
51 Fabret,C. and Hoch,J.A. (1998) A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J. Bacteriol., 180, 6375–6383.
52 Fukuchi,K., Kasahara,Y., Asai,K., Kobayashi,K., Moriya,S. and Ogasawara,N. (2000) The essential two-component regulatory system encoded by yycF and yycG modulates expression of the ftsAZ operon in Bacillus subtilis. Microbiology, 146, 1573–1583.
53 Mukai,K., Kawata-Mukai,M. and Tanaka,T. (1992) Stabilization of phosphorylated Bacillus subtilis DegU by DegR. J. Bacteriol., 174, 7954–7962.
54 Nakano,M.M., Xia,L.A. and Zuber,P. (1991) Transcription initiation region of the srfA operon, which is controlled by the comP–comA signal transduction system in Bacillus subtilis. J. Bacteriol., 173, 5487–5493.
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