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The CcpA protein is a key central regulator of carbon metabolism in Bacillus subtilis and other gram-positive organisms (7). CcpA is a member of the LacI/GalR family of transcriptional regulatory proteins and binds to conserved cre sites in the promoter regions of its target genes (8, 9, 11, 24). The response to glucose is mediated at least in part by the HPr/Crh signaling pathway (1, 3, 10, 16). While the role of CcpA as a repressor of genes involved in utilization of secondary carbon sources is well established, CcpA is also required for activation of carbon excretion pathways, including those for production of acetate, acetoin, and glycogen, during growth in glucose (5, 15, 18; C. Moran, personal communication).

The ackA, pta, and glg genes all contain cre sites upstream of the promoter which are required for transcriptional activation. However, no cre site was found in the alsSD operon, which encodes acetolactate synthase and acetolactate decarboxylase, enzymes involved in the biosynthesis of acetoin (17, 18). The alsR gene, which encodes a LysR family transcriptional regulator, is transcribed divergently from alsSD and has been proposed to act as an activator of alsSD transcription. Acetate has been implicated as the effector controlling AlsR-dependent activation, since addition of exogenous acetate increased alsSD transcription during vegetative growth (17, 18). This suggested the possibility that the effect of CcpA on alsSD was indirect, perhaps mediated via acetate accumulation due to dependence of the ackA/pta pathway on CcpA.

Isolation of ccpA mutants defective in activation of alsS transcription. An insertion of Tn917lac into the alsS gene was isolated in a search for genes induced during growth in glucose (21). ccpA mutants defective in activation of alsS transcription were then identified. Multiple independent pools of ethyl methanesulfonate-mutagenized SP phage carrying the intact ccpA gene as well as a selectable cat gene were introduced into strain ZB449ccpTn2 containing the ccpA::spc null allele and the alsS::Tn917lac insertion. Transductants (4,000) were patched onto tryptose blood agar base (TBAB) plates (Difco) containing X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside; 40 µg/ml) and 1% glucose, and 17 white colonies were identified. To eliminate mutations in ccpA that resulted in a loss of function, repression of -amylase production during growth on glucose was tested by monitoring starch hydrolysis (8). Three independently derived isolates retained normal glucose repression of amyE. Two of the ccpA mutants, designated CM48RC and CM286PL, exhibited very low alsS-lacZ activity and wild-type colony morphology, while the third, designated CM18AV, exhibited no detectable alsS-lacZ activity on plates and increased colony size during growth on TBAB medium containing glucose.

Effect of ccpA mutations on alsS-lacZ expression. Expression of alsS-lacZ was quantitated during growth in TSS medium (2) with 1% Casamino Acids in the presence or absence of glucose (1%) (Table 1). A derivative of ZB449ccpTn2 containing the wild-type ccpA locus on an SP prophage was constructed as a control. Expression was very low in all strains in the absence of glucose and was induced 45-fold during growth in glucose in the strain containing wild-type ccpA. No induction was observed in strain ZB449ccpTn2 containing the null allele of ccpA, while a small increase in activity was detected in strains carrying ccpA genes with point mutations (ccpA point mutants). This is consistent with the phenotypes observed during the mutant screening, although under those conditions it appeared that CM18AV exhibited the most severe phenotype. The phenotype of the ccpA point mutants therefore resembles that of the ccpA null mutant in having nearly complete loss of alsS expression. Similar effects on alsS-lacZ expression were observed in other growth media, including NSM (19) and Luria-Bertani (LB) medium (13; data not shown).

Effect of ccpA mutations on ackA-lacZ expression. The SP::ccpA phage from the wild-type and mutant strains was transferred into strain BR151MAccp::spc, and an ackA-lacZ transcriptional fusion (5) was introduced into the resulting strains. Cells were grown in TSS medium with 1% Casamino Acids with or without glucose (1%), and -galactosidase activity was measured (Table 2). While the expression of ackA during growth in the absence of glucose was reduced somewhat in the strains containing ccpA with point mutations compared to that of the strain containing the wild-type allele of ccpA, expression during growth in glucose was unaffected. The ccpA null allele resulted in both reduction of basal expression and loss of glucose induction, as previously reported (5). It therefore appears that the ccpA point mutations separate the functions of CcpA in transcriptional activation of ackA and alsS. This result also makes it unlikely that CcpA mediates its effect on alsS expression only through its effect on ackA, since in these mutants ackA expression was unaffected during growth in glucose.

Effect of ccpA mutations on repression of acsA-lacZ expression. The ability of the CcpA variants to repress acsA, another known target of CcpA (4, 6), was examined, since perturbations in levels of acetyl-coenzyme A (CoA) synthetase, which converts acetate to acetyl-CoA, might affect alsS expression. As shown in Table 3, each of the mutant alleles resulted in significant repression of acsA-lacZ expression during growth in glucose, while the ccpA null mutant exhibited total loss of repression, as previously reported (4). The efficiency of repression of acsA-lacZ was somewhat reduced in the ccpA point mutants relative to that in strains carrying the wild-type allele of ccpA, suggesting that although the mutant variants of CcpA are able to bind DNA, their affinity for the acsA cre site may be lower than that of wild-type CcpA. It is also possible that the reduced repression is due to an effect on other factors involved in acsA regulation.

Acetate production in ccpA mutant strains. Acetate has been implicated as the effector for induction of alsS transcription by its activator AlsR (17, 18). Although ackA expression was unimpaired in the ccpA point mutants during growth in glucose, it remained possible that acetate production was reduced because of other effects on carbon metabolism. In addition, the partial derepression of acsA could result in a reduction of acetate accumulation. Acetate concentrations in the culture supernatants were therefore directly measured (Table 4). The ccpA::spc null allele resulted in a twofold drop in acetate levels during growth in glucose, compared to the acetate level for the wild-type strain; the residual acetate production is presumably due to the basal level of ackA transcription in the absence of CcpA-dependent activation (22). The ccpA point mutants CPC18AV and CPC286PL exhibited a small decrease in acetate accumulation, while CPC48RC produced wild-type levels of acetate. It therefore appears that a reduction in acetate accumulation is unlikely to be responsible for the effect of these mutants on alsS expression.

Effect of addition of acetate on alsS-lacZ expression. Addition of acetate has been shown to activate alsS transcription during exponential growth in LB medium (17). Since the accumulation of acetate was partially reduced in the ccpA mutants, the effect of addition of exogenous acetate was tested. Cultures were grown in LB medium containing 1% glucose in the presence or absence of potassium acetate (50 mM). The addition of acetate at either pH 7.0 (Fig. 1) or pH 6.0 (data not shown) activated transcription of alsS-lacZ during the exponential-growth phase in the control strain containing the wild-type ccpA. Acetate addition conferred a small increase in alsS-lacZ expression in the ccpA point mutants, but had no effect on the ccpA::spc null mutant. These results indicate that addition of acetate is not sufficient to restore normal alsS expression in the ccpA mutants, although it clearly influences alsS transcription. These results support the hypothesis that CcpA plays a role in alsS regulation in addition to its role in activating ackA expression.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Effect of addition of acetate on alsS-lacZ expression in the ccpA mutants. All strains contained the Tn2 alsS::Tn917lac insertion (21). Cultures were grown in LB medium (13) containing 1% glucose in the presence (filled symbols) or absence (open symbols) of potassium acetate (50 mM) (pH 7.0). Arrow, time of entry of cultures into stationary phase. -Galactosidase activities are expressed in Miller units (13). (A) ZB449Tn2CWT (ccpA wild-type; circles) and ZB449ccpTn2 (ccpA::spc; squares). (B) CM18AV (circles), CM48RC (squares), and CM286PL (triangles). Cultures were grown concurrently and are presented in two panels for clarity.

Effects on the pH of the culture supernatant. Growth of B. subtilis in the presence of glucose results in a reduction in the pH of the culture medium. The pH generally reaches its lowest point at the end of the exponential phase, which correlates with the time of activation of alsSD transcription. Careful buffering of the medium to pH 7.0 eliminated acetoin production (data not shown), suggesting that a reduction in pH may be required for alsS transcriptional activation, possibly because of effects on acetate transport. The pH of the culture medium was therefore measured during growth in NSM with or without glucose (1%) (Fig. 2). All ccpA mutants exhibited a drop in pH levels during growth in glucose. The ccpA null mutant displayed a defect in both the timing and magnitude of the pH decrease, while the CM18AV mutant exhibited a phenotype intermediate between that of the null mutant and the wild-type strain; the other point mutants exhibited patterns similar to that of the wild-type strain, although the pH increased more rapidly in stationary phase. The modest effects of the ccpA point mutants on the pH profiles make it unlikely that this plays a major role in the major defect in alsS transcription.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   pH profiles of cultures of ccpA mutants. Cultures were grown in NSM broth (19) in the presence (filled symbols) or absence (open symbols) of glucose (1%). Arrow, time of entry of the cultures into stationary phase. BR151MACWT (ccpA wild type), circles; CPC18AV, squares with solid lines; CPC48RC, triangles with solid lines; CPC286PL, squares with dashed lines; BR151MAccp::spc (ccpA null), triangles with dashed lines.

Effect of ccpA mutations on growth. A null mutation in ccpA results in a defect in growth in minimal media with glucose as the sole carbon source (7, 25); this is due at least in part to derepression of nitrogen metabolism genes, in particular rocG, encoding glutamate dehydrogenase (B. R. Belitsky and A. L. Sonenshein, personal communication). The effect of the ccpA point mutations on growth was therefore examined. While the null mutant exhibited no growth on agar plates under the conditions tested, the point mutants all exhibited some growth on glucose minimal medium; however, the growth was clearly defective in comparison to that of the wild-type strain (data not shown). The point mutations therefore are likely to confer a partial defect in regulation of nitrogen metabolism. Growth rates in TSS medium with 1% Casamino Acids with or without glucose were also determined. The ccpA::spc null allele resulted in a significant decrease in growth rate in the presence of glucose, relative to the growth rate of the wild-type strain, presumably reflecting the major role of CcpA in the control of carbon metabolism; in contrast, the point mutants exhibited growth rates indistinguishable from that of the wild-type parent strain (data not shown).

Summary. These studies indicate that certain ccpA point mutations can separate CcpA's function of activating the acetoin biosynthesis pathway from its other functions in the cell. Since neither ackA transcription nor acetate accumulation was affected during growth in glucose and since the defect in alsSD transcription could not be bypassed by addition of exogenous acetate, the effect of CcpA on alsSD transcription must involve factors other than acetate as an effector. The mechanism of alsSD transcriptional activation by CcpA remains to be determined.