CodY Is Required for Nutritional Repression of Bacillus subtilis Genetic Competence

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1 JOURNAL OF BACTERIOLOGY, Oct. 1996, p Vol. 178, No /96/$ Copyright 1996, American Society for Microbiology CodY Is Required for Nutritional Repression of Bacillus subtilis Genetic Competence PASCALE SERROR AND ABRAHAM L. SONENSHEIN* Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts Received 13 May 1996/Accepted 14 August 1996 The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids. This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfa and comk and was relieved in strains carrying a null mutation in the cody gene. DNase I footprinting experiments showed that purified CodY binds directly to the srfa and comk promoter regions. When Bacillus subtilis cells reach stationary phase, they induce several adaptive responses. Among these is the acquisition of genetic competence, a physiological state that enables cells to take up exogenous DNA. The acquisition of competence requires the expression of at least 20 late com genes (7), whose transcription is regulated by a variety of nutritional, growth stage-, and cell type-specific mechanisms. These regulatory mechanisms involve the products of at least 15 additional genes (13). In the primary regulatory cascade, two extracellular signal peptides, competence-stimulating factor (39) and ComX (21), are detected by the products of the oligopeptide permease (opp) operon (29, 32) and comp, respectively, and as a result, the transcription factor ComA becomes phosphorylated. Phosphorylated ComA acts as a positive regulator for the srfa operon (14, 26, 27, 45), one of whose products, ComS, is needed for expression of comk (5, 6, 18). ComK, in turn, activates transcription of the late com genes (e.g., comg [1, 16, 42, 44]). The ComK activation pathway is counterbalanced by the MecA/MecB system, which inhibits ComK activity and can prevent the acquisition of competence under inappropriate environmental conditions (10, 19, 20, 24). During exponential growth in minimal medium, competence is repressed by the addition of Casamino acids (9), an effect that appears to be exerted before srfa expression in the regulatory cascade (15). Such a repressive effect of amino acid mixtures has been described for other operons (2), including hut, the histidine utilization operon (3), dpp (37), which encodes a dipeptide transport system (23), and gsia (25), which codes for a regulatory protein phosphatase (28). The product of the cody gene has been shown to be required for amino acid repression of the dpp operon (36, 38), and a cody mutation partially relieves amino acid control of hut (11, 38) and gsia (35). Moreover, purified CodY binds to a part of the dpp promoter region within which mutations relieve amino acid repression (34). Since competence is also repressed by mixtures of amino acids, we tested whether the cody gene has any role in this repression. We show that CodY is required for the effect of Casamino Acids on competence and that CodY interacts with the srfa and comk promoter regions. MATERIALS AND METHODS Bacterial strains. All B. subtilis strains used in this study (Table 1) were derived from FJS107 (36), a derivative of JH642 (30). Media. S7 minimal medium for competence assays and -galactosidase assays contained S7 minimal salts (46) supplemented with 1% glucose, 0.1% glutamate, and amino acids (50 g/ml) as needed. For S7CAA, S7 medium was further supplemented with 0.1% Casamino Acids. DS medium has been described previously (12). DNA manipulations. Chromosomal and plasmid DNAs were purified as described previously (12). The srfa and comk promoter regions were cloned after amplification by PCR with oligonucleotides based on published sequences (27, 43). pps53 contains DNA from positions 126 to 137 of the srfa promoter region, cloned between the HindIII and BamHI sites of pjpm1 (25). pps54 carries DNA from positions 151 to 114 of the comk promoter region, cloned between the HindIII and BamHI sites of pjpm1. Genetic transformation. Genetic competence was quantitated as described by others (21) except that the selected marker was erm (resistance to 0.5 g of erythromycin per ml and 12.5 g of lincomycin per ml). For strain construction, cells were made competent by the method of Dubnau and Davidoff-Abelson (8) and transformed with plasmid or chromosomal DNA. Transformants were selected on DS plates containing the following antibiotics at the indicated concentrations (in micrograms per milliliter): neomycin, 5; chloramphenicol, 5; spectinomycin, 100; tetracycline, 10; erythromycin, 0.5 to 0.7; and lincomycin, Galactosidase assays. -Galactosidase-specific activity was measured as described previously (4) and is presented as (A 420 per minute per milliliter of culture per A 600 ) 1,000. A 420 indicates the A 420 of o-nitrophenol, the product of cleavage of the -galactosidase substrate, o-nitrophenyl- -D-galactoside; A 600 refers to the A 600 of the culture at the time of sampling. DNase I footprinting experiments. DNA templates used for DNase I footprinting were prepared by PCR using T7 and T3 primers (Stratagene, Inc.). For any given template preparation, one of the primers was labelled with T4 polynucleotide kinase and [ - 32 P]ATP (33). Plasmids pps53 and pps54 were used as templates for amplifying the srfa and comk promoter regions, respectively. Each labelled PCR product was fractionated on a 4% polyacrylamide gel and eluted as described previously (33). Footprinting experiments were performed as described previously (34), with CodY expressed in Escherichia coli from a phage T7 promoter and purified by heparin-agarose chromatography (34). The sequencing reactions that accompanied each experiment were performed with radiolabelled T3 and T7 primers for the template and nontemplate strands, respectively. * Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA Phone: (617) Fax: (617) Electronic mail address: ASONENSH@OPAL.TUFTS.EDU. Present address: Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy en Josas Cedex, France. RESULTS Repression of competence by Casamino acids requires the product of the cody gene. To check whether CodY is involved in the repression of competence by Casamino Acids, we measured the transformation efficiencies of isogenic cody (FJS107) and cody mutant (PS37) strains during growth in competence medium (S7 minimal salts). In this medium, cells 5910

2 VOL. 178, 1996 REPRESSION OF GENETIC COMPETENCE BY CodY 5911 TABLE 1. B. subtilis strains used in this study Strain Genotype Source or reference BD1991 hisb2 leu-8 metb5 amye::( comk -lacz cat) 15 BD1626 hisa1 leua8 metb5 coma124::ptv21 cat 13 FJS107 trpc2 36 JMS107 trpc2 phea1 amye::( comg -lacz neo) 21 JMS374 trpc2 phea1 amye::( srfa -lacz neo) 39 LAB2295 srfa::cat sfp 0 P. Zuber PS29 trpc2 unku::spc a 38 PS37 trpc2 unku::spc cody 38 PS112 trpc2 abrb::cat::tet thrc::( dpp -cat erm) Laboratory strain PS123 trpc2 amye::( comg -lacz neo) FJS107 DNA JMS107 PS124 trpc2 amye::( comg -lacz neo) unku::spc cody PS37 DNA JMS107 PS145 trpc2 amye::( srfa -lacz neo) FJS107 DNA JMS374 PS146 trpc2 amye::( srfa -lacz neo) unku::spc cody PS37 DNA JMS374 PS172 trpc2 amye::( srfa -lacz neo) coma124::ptv21 cat FJS107 DNA BD1626 PS173 trpc2 amye::( srfa -lacz neo) coma124::ptv21 cat unku::spc cody PS37 DNA BD1626 PS205 trpc2 amye::( comk -lacz cat) FJS107 DNA BD1991 PS221 trpc2 amye::( comk -lacz cat::tet) PS205 pps28 b PS232 trpc2 amye::( comg -lacz neo) unku::spc PS123 DNA PS29 PS274 trpc2 amye::( comg -lacz neo) srfa::cat PS123 DNA LAB2295 PS275 trpc2 amye::( comg -lacz neo) srfa::cat unku::spc cody PS124 DNA LAB2295 PS276 trpc2 amye::( comk -lacz cat::tet) unku::spc PS29 DNA PS221 PS278 trpc2 amye::( comk -lacz cat::tet) cody PS37 DNA PS221 PS279 trpc2 amye::( comk -lacz cat::tet) srfa::cat unku::spc PS276 DNA LAB2295 PS281 trpc2 amye::( comk -lacz cat::tet) srfa::cat unku::spc cody PS278 DNA LAB2295 a unku is a locus with an unknown function located upstream of the cod operon (36). b pps28 is a derivative of pcm::tc (40). become maximally competent during late exponential growth phase, coincident with the accumulation of extracellular pheromones (21). Figure 1 shows that the maximal transformation efficiency of the wild-type strain was reduced 10-fold when the FIG. 1. Development of competence in strains FJS107 (cody ) (circles) and PS37 ( cody) (squares) during growth in S7 competence medium (as defined in Materials and Methods) in the absence (open symbols) or presence (closed symbols) of 0.1% Casamino Acids (S7CAA medium). Samples were taken for determination of culture density (A 600 [x axis]) and transformation frequency (y axis). The maximum transformation frequency obtained ( for strain PS37 in S7CAA medium) was defined as 1. Transformation frequency was the number of Ery r transformants per viable cell, after exposure to chromosomal DNA ( 1.5 g/ml) of strain PS112. medium was supplemented with Casamino Acids (i.e., in S7CAA medium) and that this reduction was not observed in the cody mutant strain. The transformation efficiencies of the two strains were similar during growth in the absence of Casamino acids. To see whether the effect of the Casamino Acids on transformation efficiency reflects the altered expression of competence genes, the expression of a late com gene was monitored. Maximal comg-lacz expression was reduced sevenfold in the wild-type strain in the presence of Casamino Acids (Fig. 2A). A cody mutation relieved this repression (Fig. 2C). Since the cody mutant strain also carries a spc gene insertion as a selectable marker upstream of the cod operon, we tested whether this insertion contributed to the relief of repression by Casamino Acids. In fact, the spc insertion had no effect on comg expression (Fig. 2B). Note that the timing of comg-lacz expression was different in the two media, reflecting the fact that in S7CAA medium the cells reach stationary phase at a density higher than that in S7 medium. srfa and comk are targets of repression by Casamino Acids. To determine the point(s) in the competence-regulatory pathway at which Casamino Acids and CodY act, we examined expression from several early competence gene promoters fused to lacz in cody and cody strains. The expression of opp-lacz did not show any clear response to either Casamino Acids or to CodY (data not shown). The expression of srfa-lacz (Fig. 3A), on the other hand, was repressed by Casamino Acids, and this effect was relieved by a cody mutation. Since srfa expression depends on positive regulation by phosphorylated ComA, we wondered whether the effect of the Casamino Acids was occurring directly at the srfa level or at the level of ComA synthesis or activity. Figure 3B shows the expression of a srfa-lacz fusion in coma and coma cody strains. Even though the activity of the fusion was severely reduced because of the coma mutation, the residual activity of the srfa promoter was still regulated by Casamino

3 5912 SERROR AND SONENSHEIN J. BACTERIOL. FIG. 2. Expression of comg-lacz during growth in S7 competence medium (open symbols) and in S7CAA medium (closed symbols). (A) Strain PS123 (unk cody ); (B) strain PS232 (unk::spc cody ) (circles) and strain PS123 (squares); (C) strain PS124 (unk::spc cody). x axis, A 600. Acids. In the cody mutant, the residual srfa expression was increased and insensitive to the presence of Casamino Acids in the medium. Assuming that ComA-independent expression of srfa reflects intrinsic promoter activity, we conclude that the srfa promoter is a direct target of CodY-mediated amino acid repression. The increased level of expression in the cody mutant could indicate that CodY has some activity even in the absence of Casamino Acids. To see whether srfa is the only competence-related target of CodY and Casamino Acids, we assayed the activity of a comglacz fusion in srfa and srfa cody strains (Fig. 4A). As expected, comg expression was strongly reduced by the srfa mutation but, surprisingly, was still subject to CodY-dependent repression mediated by Casamino Acids. Since ComK synthesis is dependent on srfa expression and is required for comg transcription (42), we tested the expression of a comk-lacz fusion in srfa and srfa cody strains (Fig. 4B). The residual comk expression was still repressed by Casamino Acids in the cody strain but not in a cody mutant strain. Assuming that srfa-independent expression of comk is a measure of promoter activity, we conclude that comk is a second target for control by CodY. We cannot exclude the possibility that comg is also a target of CodY. Interaction of CodY with the srfa and comk promoter regions. Since in vivo studies showed that CodY mediates repression of srfa and comk by Casamino Acids, we tested whether these promoters are direct targets for CodY binding. The electrophoretic mobilities of DNA fragments containing the srfa or comk promoter regions were reduced following incubation with CodY (data not shown), suggesting specific binding. To confirm that CodY recognizes specific sites near the srfa and comk promoters and to identify these binding sites, DNase I footprinting studies using DNA fragments carrying the srfa or comk promoter regions were carried out. As shown in Fig. 5 and summarized in Fig. 6, a distinct region was protected on both the template and nontemplate strands of the srfa and comk promoter regions. The srfa promoter region was protected from positions 38 to 45 on the template strand and from positions 32 to 49 on the nontemplate strand. The comk-protected region extended from positions 80 to 16 on the template strand and from positions 77 to 13 on the nontemplate strand. In both cases, the protected regions overlap with the likely RNA polymerase binding site. DISCUSSION Two major signals for induction of genetic competence have previously been identified and extensively studied. First, two pheromones that activate comk transcription through srfa (21, 39) allow a population of cells to monitor its density before inducing competence. Second, the MecA/MecB system inhibits ComK activity (16, 19) and must be bypassed, in response to unknown physiological signals, in order for cells to induce late com gene transcription. CodY mediates a third signal, which is likely to be nutritional. We have shown that the presence of Casamino Acids reduces competence as much as 10-fold, in agreement with results in the literature (9, 47), and that disruption of cody abolishes the inhibitory effect of Casamino Acids. The effect of CodY can be attributed to its role in inhibiting transcription from the srfa and comk promoters in the presence of Casamino Acids, since we have shown directly that CodY binds to the srfa and comk promoter regions. No consensus sequence can be deduced from the CodYprotected regions at the srfa and comk promoters studied here and the dpp promoter analyzed previously (34). This might be either because target recognition is not stringent or because CodY recognizes a three-dimensional DNA structure rather than a linear sequence. In fact, all three promoter regions are A T rich and might adopt a common topology. A fourth regulatory mechanism for the control of ComK expression involves AbrB (16), a negative regulator of many stationary-phase genes (41). AbrB is known to bind to the comk promoter region (22) and does so at a site just upstream

4 VOL. 178, 1996 REPRESSION OF GENETIC COMPETENCE BY CodY 5913 FIG. 3. (A) Expression of srfa-lacz during growth in S7 competence medium (open symbols) and in S7CAA medium (closed symbols). Data for strains PS145 (cody ) (circles) and PS146 ( cody) (squares) are shown. (B) Residual expression of srfa-lacz in a coma mutant strain during growth in S7 competence medium (open symbols) and in S7CAA medium (closed symbols). Data for strains PS172 (cody ) (circles) and PS173 ( cody) (squares) are shown. from the CodY binding site (35). This suggests rather complex regulation of ComK synthesis and activity, with two negative regulators of transcription (CodY and AbrB) and an inhibitor of ComK function (MecA/MecB) in effect competing against two positive regulators, ComS and ComK. Since overproduction of ComK is toxic (16), it is not surprising that the cell has evolved multiple, independent, and partially redundant mechanisms for controlling ComK synthesis and activity. While meca or cody single mutants grow well, a meca cody double mutant was essentially nonviable (35). Overexpression of ComK is the lethal factor, since a meca cody comk triple mutant grew well (35). The lethality of ComK may also explain why CodY represses comk in two ways, both directly and by repressing srfa. The specific mechanism by which CodY acts at the comk promoter is unknown. Since ComK is a positive autoregulator (44) and since the ComK and CodY binding sites in the comk promoter region overlap (17), CodY could inhibit transcription by competing with either ComK or RNA polymerase for binding to the comk promoter. Since the CodY binding site in the srfa promoter region overlaps with the presumed RNA polymerase binding site but not with the ComA binding sites (31), CodY probably inhibits srfa expression by blocking RNA polymerase activity. This suggestion fits with the fact that srfa expression is repressed by CodY in the presence of Casamino Acids whether or not ComA is active. We predict that CodY will prove to regulate a number of stationary-phase genes and suspect that its affinity toward its targets is modulated by an intracellular effector molecule. How the signal is derived from Casamino Acids is unknown. In an opp mutant, however, CodY-dependent repression by Casamino Acids was lost (35), suggesting that an extracellular signal is sensed or transported by the opp system. It will now be important to identify the intracellular signal and to understand how it triggers the activity of CodY. FIG. 4. (A) Residual expression of comg-lacz in a srfa mutant strain during growth in S7 competence medium (open symbols) and in S7CAA medium (closed symbols). Data for strains PS274 (cody ) (circles) and PS275 ( cody) (squares) are shown. (B) Residual expression of comk-lacz in a srfa mutant strain during growth in S7 competence medium (open symbols) and in S7CAA medium (closed symbols). Data for strains PS279 (cody ) (circles) and PS281 ( cody) (squares) are shown.

5 5914 SERROR AND SONENSHEIN J. BACTERIOL. Downloaded from FIG. 5. DNase I footprinting analysis of CodY binding to srfa (A) and comk (B) promoter regions. The left and right sections of each panel show results obtained with the labelled nontemplate and template strands, respectively. (A) Lane 1, no protein; lane 2, 1.8 M CodY protein. (B) Lane 1, no protein, lanes 2 and 3, 0.9 and 1.8 M CodY protein, respectively. Sanger sequencing reactions of the appropriate DNA strand were used as size markers. (Lanes C were read after longer exposures of the gel.) The 10 and 35 hexamers of the promoters are marked, and the protected regions are bracketed. on September 29, 2018 by guest FIG. 6. Summary of footprinting results. The sequences of the srfa (A) and comk (B) promoter regions are shown, with the DNase I protected areas marked by brackets. Coordinates are relative to the transcription initiation sites of srfa (26) and comk (43).

6 VOL. 178, 1996 REPRESSION OF GENETIC COMPETENCE BY CodY 5915 ACKNOWLEDGMENTS We thank B. Belitsky, D. Dubnau, S. Fisher, A. Grossman, L. Hamoen, S. Jin, M. Marahiel, J. Solomon, and P. Zuber for helpful discussions, sharing unpublished results, and donating strains and A. Grossman for suggesting the use of S7 medium to test the effect of Casamino Acids. We also thank B. Belitsky and A. Grossman for critical reading of the manuscript. This work was supported by a research grant from the National Institutes of Health (U.S. Public Health Service) to A.L.S. (GM42219). P.S. was supported by the Institut National de la Recherche Agronomique (France) and a travel fellowship from NATO. REFERENCES 1. Albano, M., J. Hahn, and D. Dubnau Expression of competence genes in Bacillus subtilis. J. Bacteriol. 169: Atkinson, M. R., and S. H. Fisher Identification of genes and gene products whose expression is activated during nitrogen-limited growth in Bacillus subtilis. J. 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