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FEMS Microbiology Letters 195 (2001) 179^183 www.fems-microbiology.org Involvement of acetyl phosphate in the in vivo activation of the response regulator ComA in Bacillus subtilis Seong-Bin Kim a;c , Byung-Sik Shin a , Soo-Keun Choi a , Chi-Kyung Kim c , Seung-Hwan Park b; * a b Laboratory of Microbial and Bioprecess Engineering, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon 305-600, South Korea Genome Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon 305-600, South Korea c Department of Microbiology, Chungbuk National University, Cheong-ju 361-763, South Korea Received 21 September 2000; received in revised form 20 December 2000; accepted 31 December 2000 Abstract Development of genetic competence in Bacillus subtilis is regulated by ComP^ComA, a two-component signal transduction system. The response regulator ComA is primarily activated by ComP, a histidine kinase that mediates response to nutrient conditions and cell density, and the activated ComA is required for transcription of the srf operon, which is essential for the development of genetic competence and surfactin production. In this study we suggested that the ComA could also be activated by a small molecule phospho-donor, acetyl phosphate. Examination of srfA-lacZ expression indicated that a significant amount of srfA expression still occurs in the comP mutant during growth in a sporulation medium containing excess glucose. Analysis of a comP and pta mutant suggests that srfA activation seen in the comP mutant is dependent on the expression of pta, which encodes phosphotransacetylase (Pta). As Pta is responsible for the catalysis for conversion of acetyl coenzyme A to acetyl phosphate, we conclude that the expression of srfA seen in the comP mutant is mainly due to the activation of ComA by acetyl phosphate. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Two-component signal transduction; ComA; Acetyl phosphate ; srfA-lacZ ; Bacillus subtilis 1. Introduction Bacillus subtilis has natural competence, generally de¢ned as a physiological state that permits a bacterial cell to bind and take up high-molecular mass DNA [1]. Early regulatory steps in competence development are activated by two extracellular factors, ComX pheromone and competence stimulation factor (CSF), which accumulate in culture media as cells grow to high density. These two factors were known to deliver signals from outside by two di¡erent sensing pathways and converge to activate expression of srfA [2]. By signaling of ComX pheromone, ComP histidine kinase autophosphorylates on a histidine residue. In vivo, the phosphate from ComPVP is transferred to the ComA response regulator. Then, the phosphorylated ComAVP binds to and activates the transcrip- * Corresponding author. Tel. : +82 (42) 860-4410; Fax: +82 (42) 860-4594; E-mail: [email protected] tion of srfA [3], and resultantly the competence regulatory gene comS, which lies within and out-of-frame with the second gene of the fourth amino acid-activating domain of the srfA operon is also transcribed [4]. Interestingly, however, Solomon et al. reported that comP mutants still could activate the ComA response regulatory protein, and this indicates that there must be an additional source of phosphate for activation of ComA in the cell [2]. Despite the drastic decrease of srfA transcription by the comP mutation, the accumulation of L-galactosidase speci¢c activity from srfA-lacZ in a comP mutant remains V5% of that in the wild-type. Recent studies on Escherichia coli have shown that the major secondary source of phosphoryl groups is a small molecule phosphate donor, acetyl phosphate [5^7]. Several response regulatory proteins, such as CheY [5], PhoB [6], and OmpR [7], have been shown to be phosphorylated in vitro when incubated with acetyl phosphate. It was also reported that partially puri¢ed ComA protein, prepared from E. coli cells overexpressing ComA, could be phosphorylated in vitro by 0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 0 8 - 8 FEMSLE 9782 7-2-01 180 S.-B. Kim et al. / FEMS Microbiology Letters 195 (2001) 179^183 incubation with acetyl phosphate [8]. Genetic studies have indicated that acetyl phosphate functions as the principle phospho-donor in E. coli mutant strains that are de¢cient in the cognate histidine kinase [9]. In B. subtilis, however, there is no evidence that acetyl phosphate is a phosphodonor in the two-component signal transduction system in vivo. In this study, we present evidence that acetyl phosphate is a phospho-donor that can activate the ComA response regulator in B. subtilis, and that this activation is dependent on the regulation of pta that encodes a phosphotransacetylase (Pta). Glucose and fructose, which can activate pta, stimulated the activation of ComA, but pyruvate did not induce this activation. 2. Materials and methods 2.1. Bacterial strains and media The bacterial strains used in this study are described in Table 1. E. coli DH5K was used for plasmid construction. All are derived from the parent strain JH642. Strains were constructed by transformation with plasmid and chromosomal DNA. LB medium was routinely used for cultivating E. coli and B. subtilis. Difco sporulation medium (DSM) was used for growth and maintenance of B. subtilis. Competent cells were obtained in Spizizens medium as described by Albano et al. [10]. For carbon source, pyruvate and fructose were added to sterile DSM to yield 1% ¢nal concentration. 2.2. L-Galactosidase assay The inoculum for the L-galactosidase assay was prepared by growing cells on a DSM agar plate at 30³C overnight with appropriate antibiotics. The cells were then harvested by washing the plate surface with 1.0 ml of prewarmed DSM, and liquid medium was inoculated to an initial absorbance level at 600 nm of about 0.02. Cultures were grown at 37³C and 300 rpm, and growth was monitored by measuring A600 . The assays were performed with toluenized cells, as described by Nicholson and Set- low [11]. The speci¢c activity was expressed as Miller units [12]. 2.3. Plasmid and strain constructions Plasmid pDG1728 contains a sequence homologous to the amyE locus and carries a promoterless copy of lacZ [13]. The srfA promoter region from 3291 to +140 relative to the srfA transcription start site, with £anking EcoRI and BamHI restriction sites, was cloned into the lacZ fusion vector, pDG1728, and the fusion was recombined into the chromosome by double-crossover, selecting for spectinomycin resistance. To construct a comP mutant, a DNA fragment of about 2.2 kb containing the comP front region was ampli¢ed by PCR and then cloned into pGEM-7Zf (+) (Promega), giving pComP-F. Then, the cat gene was ligated into ClaI and SpeI restriction sites of pComP-F, giving pComP(F)-cm. Finally, the fragment of comP back region was cloned by PCR. An approximately 1.6-kb PCR fragment restricted with ClaI and EcoRI was cloned into pComP(F)-cm, giving prComP. This plasmid was integrated into the chromosome by means of double-crossover. To construct a comA mutant, a DNA fragment of about 2.0 kb containing the comA gene was ampli¢ed by PCR and then cloned into pGEM-7Zf (+), giving pComA. A deletion^insertion mutation of the comA gene was constructed by replacing the 0.5-kb ClaI^SpeI fragment of pComA with a 1.1-kb ClaI^SpeI fragment of the cat gene, cloned from pDG1661 by PCR. The resulting plasmid, prComA, was integrated into the chromosome by means of a doublecrossover. 2.4. Site-directed mutagenesis of comA To substitute an alanine (A) for the aspartic acid (D) residue at position 55 on the comA sequence, an Altered sites0 II in vitro Mutagenesis system (Promega) was used. Mutagenesis was performed following the instructions of the manufacturer. This mutation (D55A in the comA sequence) was con¢rmed by DNA sequencing. Mutations were con¢rmed by Southern hybridization. Table 1 Bacterial strains used in this study Strain Genotype JH642 BSK9901 BSK9902 BSK9903 BSK9904 BSK9905 BSK9906 BSK9907 trpC2 trpC2 trpC2 trpC2 trpC2 trpC2 trpC2 trpC2 pheA1 pheA1 pheA1 pheA1 pheA1 pheA1 pheA1 pheA1 Source amyE: :(srfA-lacZ spc) vcomP: :cat amyE: :(srfA-lacZ spc) vcomA: :cat amyE: :(srfA-lacZ spc) vpta: :erm amyE: :(srfA-lacZ spc) vccpA: :erm amyE : :(srfA-lacZ spc) vcomP: :cat vpta: :erm amyE: :(srfA-lacZ spc) vcomP: :cat vccpA: :erm amyE: :(srfA-lacZ spc) FEMSLE 9782 7-2-01 BGSC This work This work This work This work This work This work This work S.-B. Kim et al. / FEMS Microbiology Letters 195 (2001) 179^183 181 from srfA-lacZ grown in DSM-G did not decrease even 3 h after the end of the exponential phase (T3 ). In order to analyze the regulation of srfA expression in cells grown in medium containing a high concentration of glucose, the e¡ect of the comP mutation on the expression of srfA was examined. As expected, expression of srfAlacZ was greatly reduced in the comP mutant grown in DSM (Fig. 1B), which was seen previously [4]. However, the expression of srfA was largely restored in the comP mutant by the addition of glucose to DSM (Fig. 1B). These results suggest that the residual srfA expression in the comP mutant seen in DSM-G is largely dependent on the presence of glucose in the medium. The expression of srfA in the comP mutant was increased much more in this experiment than in the previous report of Solomon et al. [4]. There was no detectable expression of srfA-lacZ from a comA null mutant grown in either DSM or DSM-G (Fig. 1B). It has been reported that the unphosphorylated form of ComA protein can bind to a DNA fragment containing the srfA promoter in vitro, although the binding a¤nity is enhanced when ComA is phosphorylated [8]. As the conserved aspartate at position 55 of ComA is suggested to be the phosphorylation site of ComA [15], we constructed a comA mutant with alanine in place of the conserved aspartate (comAD55A ) to determine whether unphosphorylated ComA could stimulate the expression of srfA. The speci¢c activity of L-galactosidase from srfA-lacZ in the comAD55A mutant was very low in both DSM and DSM- Fig. 1. Expression of srfA-lacZ in wild-type (A) and a comP mutant (B) in the presence and absence of glucose in DSM. Samples were taken for determination of L-galactosidase speci¢c activity at 30-min or 1-h. intervals. (A) Expression of srfA in wild-type is constantly maintained in the stationary phase of growth under the presence of glucose. b, L-galactosidase activity in DSM ; F, L-galactosidase activity in DSM-G. (B) Expression of srfA in comP mutant is activated in the presence of glucose. R, L-galactosidase activity in DSM; 8, L-galactosidase activity in , L-galactosidase activity of comA mutant in DSM; S, LDSM-G; galactosidase activity of comA mutant in DSM-G. Arrows indicate the onset of stationary phase (T0 ). 3. Results and discussion 3.1. E¡ect of glucose on the srfA expression The expression of srfA, required for activation of ComK, increases as cells enter the stationary phase, and this activation of srfA transcription decreases at the lategrowth phase [14]. However, the addition of a high concentration of glucose to the culture medium resulted in constitutive expression of srfA during the late-growth phase (Fig. 1A). The L-galactosidase speci¢c activity Fig. 2. Model for the role of acetyl phosphate in the regulation of srfA expression and competence in B. subtilis. The expression of srfA through these pathways is reached in the end of exponential phase. Upon entry into stationary phase, CSF reaches 50^100 nM, concentrations that stimulate sporulation and inhibit ComA-dependent gene expression. But, the expression of srfA in the presence of glucose or fructose is maintained. Results presented in this report indicate that acetyl phosphate may be involved in the expression of srfA in the stationary phase in which much acetate is produced through acetyl phosphate by AckA and unknown pathways. FEMSLE 9782 7-2-01 182 S.-B. Kim et al. / FEMS Microbiology Letters 195 (2001) 179^183 Table 2 E¡ect of carbon source on the expression of srfA-lacZ in various B. subtilis mutants Medium DSM DSM-G DSM-F DSM-P L-Galactosidase speci¢c activity-Miller units (T0 /T3 ) Genotype BSK 9902 (comP) BSK 9904 (pta) BSK 9905 (ccpA) BSK 9901 (wild-type) BSK 9906 (comP+pta) 3.3/2.5 203/187 237/217 27/12 215/64 214/95 241/98 255/86 190/69 197/133 227/144 224/67 188/43 235/218 277/206 210/56 3.4/2.6 57/36 58/47 4.0/4.5 Values represent means of at least two independent experiments. T0 is the onset of the stationary phase and T3 is 3 h after T0 . Glucose, fructose and pyruvate were added to ¢nal 1% in DSM medium. G, similar to that in the comA null mutant (data not shown), indicating that the unphosphorylated form of ComA does not appear to have a role in the regulation of srfA. All of these results indicate that the majority of srfA expression during growth in DSM-G, in particular at late-growth phase, does not depend on the presence of ComP, and that there must be an additional source of phosphate for activation of ComA in these cells. 3.2. E¡ect of pta regulation on the ComA activation To examine if acetyl phosphate could be another phosphate donor in the activation of ComA, we analyzed the e¡ect of pta regulation on the expression of srfA. In a previous study, we showed that the expression of pta, which encodes a Pta responsible for conversion of acetyl coenzyme A to acetyl phosphate, is activated by the addition of excess glucose or fructose, and that this activation is dependent on CcpA [16,17]. As the expression of pta depends on the presence of glucose in the medium, it is possible that expression of srfA observed in DSM-G may be due to induction of pta expression by the presence of excess glucose. As shown in Table 2, in the comP mutant, the expression of srfA was greatly increased by the addition of glucose or fructose. In contrast, the expression of srfA in the pta mutant was almost not e¡ected by the presence of glucose or fructose. We also observed that the pattern of srfA expression in the pta mutant was almost the same as that in the ccpA mutant. Because the expression of pta requires CcpA [17,18], the expression of srfA in the ccpA mutant should be similar to that in the pta mutant. The dependence of srfA expression on the presence of sugar also could be observed in the wildtype : the addition of glucose or fructose increased the expression of srfA in the wild-type strain, especially during the late-growth phase (T3 ). All of these results indicate that the induction of srfA expression seen in the presence of glucose or fructose is critically dependent on the regulation of the pta gene, suggesting possible involvement of acetyl phosphate in the activation of ComA (Fig. 2). It is interesting to note that the addition of pyruvate did not greatly induce the expression of srfA in the comP mutant. Although pyruvate could be considered a good source for production of acetyl phosphate, we observed that the expression of the pta gene is not induced by the addition of 1% pyruvate in DSM (data not shown). In B. subtilis, CcpA-dependent catabolite repression requires the seryl-phosphorylated form of both the Hpr and Crh proteins; and two glycolytic metabolites, fructose-1,6,-diphosphate (FDP) and glucose-6-phosphate (G6P), have been proposed as e¡ectors of CcpA [19,20]. As the presence of pyruvate did not e¡ect the expression of srfA, it is possible that activation of pta by CcpA also requires either FDP or G6P as a coactivator. We also measured expression of srfA in the comP-pta double mutant. Although the expression of srfA was signi¢cantly lower than that in either single mutant, there was still detectable expression of srfA in the double mutant in the presence of glucose or fructose (Table 2). In B. subtilis, acetate is mainly produced via the Ack^Pta pathway during growth with excess carbohydrate. However, a signi¢cant amount of acetate is still produced in the pta mutant, indicating that there may be another pathway responsible for acetate production [18]. Thus it is possible that the expression of srfA seen in the comP-pta double mutant may be due to acetyl phosphate produced from acetate by reverse reaction of AckA. As the Pta^AckA pathway is the only route for production of acetyl phosphate, examination of a comP-pta-ackA triple mutant should be done to prove this hypothesis. It is also possible that residual expression in the comP-pta double mutant may have resulted from activation of ComA by another histidine kinase, although there is no available evidence in B. subtilis to support this speculation. References [1] Dubnau, D. (1989) The competence regulon of Bacillus subtilis. In: Regulation of Prokaryotic Development (Smith, I., Slepecky, R. and Setlow, P., Eds.), pp. 147^166. American Society for Microbiology, Washington, DC. [2] Solomon, J.M., Magnuson, R., Srivastava, A. and Grossman, A.D. (1995) Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis. Genes Dev. 9, 547^ 558. [3] Nakano, M.M. and Zuber, P. (1991) The primary role of comA in the establishment of the competent state in Bacillus subtilis is to activate the expression of srfA. J. Bacteriol. 173, 7269^7274. [4] Hamoen, L.W., Eshuis, H. and Jongbloed, J. (1995) A small gene, FEMSLE 9782 7-2-01 S.-B. Kim et al. / FEMS Microbiology Letters 195 (2001) 179^183 [5] [6] [7] [8] [9] [10] [11] [12] designated comS, located within the coding region of the fourth amino acid-activating domain of srfA, is required for competence development in Bacillus subtilis. Mol. Microbiol. 15, 55^63. 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. Hiratsu, K., Nakata, A., Shinagawa, H. and Makino, K. (1995) Autophosphorylation and activation of transcriptional activator PhoB of Escherichia coli by acetyl phosphate in vitro. Gene 161, 7^10. McCleary, W.R. and Stock, J.B. (1994) Acetyl phosphate and the activation of two-component response regulators. J. Biol. Chem. 269, 31567^31572. 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. Kim, S.K., Wilmes-Riesenberg, M.R. and Wanner, B.L. (1996) Involvement of the sensor kinase EnvZ in the in vivo activation of the response-regulator PhoB by acetyl phosphate. Mol. Microbiol. 22, 135^147. Albano, M., Hahn, J. and Dubnau, D. (1987) Expression of competence genes in Bacillus subtilis. J. Bacteriol. 169, 3110^3117. Nicholson, W.L. and Setlow, P. (1990) Sporulation, germination and out growth. In: Molecular Biological Methods for Bacillus (Harwood, C.R. and Cutting, S.M., Eds.), pp. 391^450. John Wiley and Sons, New York. Miller, J. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 183 [13] Guèˆrout-Fleury, A., Frandsen, N. and Stragier, P. (1996) Plasmids for ectopic integration in Bacillus subtilis. Gene 180, 57^61. [14] Nakano, M.M., Magnuson, R., Myers, A., Curry, J., Grossman, A.D. and Zuber, P. (1991) srfA is an operon required for surfactin production, competence development, and e¤cient sporulation in Bacillus subtilis. J. Bacteriol. 173, 1770^1778. [15] Weinrauch, Y., Penchev, R., Dubnau, E., Smith, I. and Dubnau, D. (1990) A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes Dev. 4, 860^872. [16] Grundy, F.J., Turinsky, A.J. and Henkin, T.M. (1994) Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA. J. Bacteriol. 176, 4527^4533. [17] Shin, B.S., Choi, S.K. and Park, S.H. (1999) Regulation of the Bacillus subtilis phosphotransacetylase gene. J. Biochem. 126, 333^339. [18] Presecan-siedel, E., Galinier, A., Longin, R., Deutscher, J., Danchin, A., Glaser, P. and Mortin-Verstraete, I. (1999) Catabolite regulation of the pta gene as part of carbon £ow pathways in Bacillus subtilis. J. Bacteriol. 181, 6889^6897. [19] Fujita, Y., Miwa, Y., Galinier, A. and Deutscher, J. (1995) Speci¢c recognition of the Bacillus subtilis gnt. Cis-acting catabolite-responsive element by a protein complex formed between CcpA and serylphosphorylated Hpr. Mol. Microbiol. 171, 953^960. [20] Deutscher, J., Ku«ster, E., Bergstedt, U., Charrier, V. and Hillen, W. (1995) Protein kinase-dependent Hpr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria. Mol. Microbiol. 15, 1049^1053. FEMSLE 9782 7-2-01