INTRODUCTION

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Carbon catabolite repression (CCR) of many genes in Bacillus subtilis is caused by the availability of glucose or other rapidly metabolized carbon sources during growth. The regulatory process involves several proteins including HPr, Crh, HPr kinase, and CcpA. The HPr protein, encoded by the ptsH gene, functions in CCR as well as in the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Crh (catabolite repression HPr) is a protein homologous to HPr that functions in CCR but not in the PTS (13, 29). After glucose is taken up via the PTS, glycolytic intermediates such as fructose-1,6-bisphosphate activate HPr kinase (14, 19, 25). Then, HPr kinase phosphorylates HPr (14, 36) and Crh (13, 14) at a serine residue (Ser46) in an ATP-dependent manner. The seryl-phosphorylated proteins act as corepressors (11, 20, 24) by forming a complex (6) with the trans-acting CcpA (catabolite control protein A), a member of the LacI/GalR family of regulatory proteins (17). The complex binds to a consensus DNA sequence, the so-called cre site (catabolite-responsive element) (18), located upstream of the target gene, where it may act either as a repressor or as an activator of transcription (18).

The key role of CcpA in carbon regulation in B. subtilis has been demonstrated by inactivating the ccpA gene, which resulted in relief of glucose catabolite repression (11, 17) or the abolition of gene activation (34, 40). Mutating Ser46 of HPr resulted in relief from CCR of gnt (7, 31), in partial relief of lev (13) and xynB (12), and no relief of hut (47) and idh (13). In the last two cases, complete relief was observed in a ptsH1 crh double mutant, in which HPr is mutated and the crh gene is disrupted. This suggests an active role for both P-Ser46-HPr and P-Ser46-Crh in CCR, but the individual roles of the two proteins remain obscure.

Uptake of citrate in B. subtilis is strongly enhanced in the presence of divalent metal ions (2). At least two secondary transport proteins, the paralogues CitM and CitH, encoded by open reading frames yflO and yxiQ, respectively (26), mediate citrate uptake in B. subtilis (3). The transporters were expressed in E. coli and functionally characterized (3). CitM turned out to be the transporter that is likely to be responsible for enhanced uptake in the presence of divalent metal ions: CitM transports citrate in a complex with Mg²⁺ and several other divalent metal ions (2; B. P. Krom, J. B. Warner, W. N. Konings, and J. S. Lolkema, submitted for publication).

The structural gene coding for CitM is organized in an operon-like structure (Fig. 1), including citM and a second gene, yflN (26), the function of which is not known. Upstream of citM are reading frames citS and citT, which code for a two-component system (9). The CitS-CitT two-component system is essential for the transcription of the citM-yflN operon. The putative CitT target sequence is believed to be located in the region between nucleotides 62 and 113, upstream of the citM transcriptional start point (H. Yamamoto, M. Murata, and J. Sekiguchi, Abstr. 10th Int. Conf. Bacilli, p. 71, 1999). In addition, just in front of the citM gene lies a sequence that matches the consensus sequence of a cre site (41, 46). The functionality of the cre site has been demonstrated in vivo (32). The location of the citM gene on the chromosome of B. subtilis suggests that expression of CitM might be under the control of the metabolic state of the cell.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Genetic organization of the citM gene on the B. subtilis chromosome. Arrows, direction of transcription; loops, transcription termination sites. citS and citT code for the two-component signal transduction system, citM codes for the secondary transporter of the Mg2+-citrate complex, and yflP and yflN code for unknown proteins. The genes are drawn to scale. Below is shown the alignment of the cre site located in the CitM promoter region and the consensus sequence as described previously (48). The citM cre site is centered at 24 bp relative to the start codon of the citM gene. Symbols for nucleotides in the consensus sequence: W, A or T; R, A or G; Y, C or T; N, A, G, C, or T.

Induction by citrate and inhibition by glucose of citrate uptake in B. subtilis have been described already 3 decades ago (42). In this study we report on the regulation of synthesis of the Mg²⁺-citrate transporter of B. subtilis, encoded by the citM gene. Transcription of citM is strictly dependent on the presence of citrate in the growth medium and is under the control of CCR. Repression was observed in media containing several carbohydrates but also nonsugars. Experiments with wild-type and CCR mutant strains revealed the involvement of the different CCR components.

	MATERIALS AND METHODS

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Bacterial strains, media, and growth conditions. The B. subtilis strains used in this study are listed in Table 1. B. subtilis was grown in C medium (1) in which ferric ammonium citrate was omitted. The C medium was supplemented with 10 mM trisodium citrate (CC medium) or 25 mM myo-inositol (CI medium) or 25 mM myo-inositol and 8 g of potassium glutamate/liter (CIE medium), or 6 g of sodium succinate and 8 g of potassium glutamate/liter (CSE medium). When strain QB5407 was grown in CI medium, 10 mM potassium glutamate was added as nitrogen source (10). Glucose or trisodium citrate was sometimes added at a concentration of 10 mM. Auxotrophic requirements were added at 20-µg/ml final concentration. When appropriate, antibiotics were added at concentrations of 100 µg/ml for spectinomycin (strains QB7097 and QB5407) and 5 µg/ml for kanamycin (QB7102) and chloramphenicol (the PcitM-lacZ fusion strains).

Construction of PcitM-lacZ fusions. Vector pJM116 (5) contains the promoterless spoVG-lacZ gene between two fragments of the B. subtilis amyE gene and carries the cat gene from pC194 (4). The integration vector pCM160 was constructed by cloning an 819-bp-long PCR fragment of the citM promoter region, PcitM, into the multiple cloning site of pJM116. The citM promoter region including the cre site was amplified by PCR using a forward primer (5'-CTCCAAGGAATTCCAGACGGTTGCATTGCC-3') that introduced an EcoRI site (boldface) and a backward primer (5'-AAGCCTAAGGATCCTAACACATCCATTCCC-3') that introduced a BamHI site (boldface). Both pJM116 and the PcitM PCR fragment were digested with BamHI and EcoRI and ligated to yield pCM160. The vector was constructed in Escherichia coli DH5 grown in Luria-Bertani (LB) medium (30) at 37°C. Transformants were selected by including 50 µg of ampicillin/ml in LB agar plates. The construct was checked by restriction and DNA sequence analyses.

Transport assay. Cells from 20-ml cultures were resuspended in 50 mM PIPES, pH 6.5, to yield an OD₆₆₀ of 10 and stored on ice until use. Transport activity was determined by the rapid-filtration method (27). Briefly, cells were diluted 10-fold in 50 mM PIPES, pH 6.5-1 mM NiCl₂ and incubated for 5 min at 30°C. At time zero, 1 µl of [1,5-¹⁴C]citrate (114 mCi/mmol) was added to 99 µl of cell suspension, yielding a final concentration of 4.4 µM citrate. Uptake was stopped by the addition of 2 ml of ice-cold 0.1 M LiCl solution, immediately followed by filtration through a 0.45-µm-pore-size nitrocellulose filter. The filters were washed once with the same LiCl solution. The filters were submerged in scintillation fluid, and the retained radioactivity was counted in a liquid scintillation counter. Samples were taken at time points between 0 and 10 min. Uptake rates were determined from the linear initial part of each uptake curve.

-Galactosidase assay. One milliliter of cell culture at an OD₆₆₀ between 0.3 and 0.6 (exponential growth phase) was harvested by centrifugation for 5 min in an Eppendorf table top centrifuge operated at 14,000 rpm. Cell extracts were obtained by lysozyme treatment, and -galactosidase activities were determined using o-nitrophenyl--D-galactopyranoside as the substrate, as described previously (30). -Galactosidase activities of PcitM-lacZ integrants were corrected for -galactosidase activity of B. subtilis 168 transformed with pJM116, which amounted to 0.4 to 2.5 Miller units. Strain CM004 contains the PcitM-lacZ fusion integrated in the chromosome of strain SA003, which already contains another lacZ fusion (Table 1). Under the growth conditions used in our experiments the -galactosidase activity of strain SA003 was the same as that observed for strain 168 transformed with plasmid pJM116. Consequently, the -galactosidase activity of strain CM004 reflects citM promoter activity.

	RESULTS

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Induction and glucose catabolite repression of citM expression. B. subtilis contains two known transporters for citrate, CitH and CitM. CitM is responsible for citrate-induced citrate uptake activity and transports citrate in complex with Mg²⁺ and other divalent metal ions (2, 3). To study the expression of CitM, the uptake activity was measured using the Ni²⁺-citrate complex as the substrate, which is highly specific for CitM (Krom et al., submitted). Ni²⁺ was chosen rather than Mg²⁺ because of the higher stability of the Ni²⁺-citrate complex, which assures that all citrate is in the complexed state (log K_A is 5.4 and 3.4 for Ni²⁺ and Mg²⁺, respectively [28]).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Ni2+-citrate uptake and citM promoter activity. Uptake of [1,5-14C]citrate in whole cells of B. subtilis 168 grown in CSE medium without further additions (, CSE), with 10 mM citrate (, CSEC), and with 10 mM citrate plus 10 mM glucose (, CSECG). (Inset) -Galactosidase activity (in Miller units [MU]) of B. subtilis CM002 carrying the lacZ gene under the control of the citM promoter grown in the same media.

Ni²⁺-citrate uptake and citM promoter activity in different growth media. The repressive effects of various growth media on Ni²⁺-citrate uptake and citM promoter activity were determined by growing B. subtilis in citrate minimal medium supplemented with different carbon sources. Highest uptake and promoter activities were observed when cells were grown in C medium with citrate as the sole carbon and energy source (Fig. 3). Supplementing the growth medium with the carbohydrate glucose, glycerol, or inositol resulted in dramatic loss of both activities. The repressive effect was not restricted to sugars, because, though less prominent, clear decreases in both Ni²⁺-citrate uptake activity and citM promoter activity were observed with cells grown in medium containing succinate and glutamate. The similar activities of cells grown in inositol and inositol-glutamate media suggest that the latter repression is caused by succinate. In all the growth media tested, there was a fair correlation between CitM transport activity and citM promoter activity, with the relatively low promoter activity in the glycerol medium as the exception. The differences between the two activities most likely represent other regulatory factors acting after the transcription of the gene or differences in the metabolic state of the cells (see Discussion).

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Effect of different growth substrates on transport and promoter activities. B. subtilis strains 168 and CM002 were grown in C minimal medium with 10 mM citrate in the presence of no further additions (none), glucose (Gluc), glycerol (Gly), inositol (I), inositol and glutamate (IE), and succinate and glutamate (SE). Open bars, initial rates of uptake of [1,5-14C]citrate in the presence of 1 mM NiCl2 by whole cells of B. subtilis 168; solid bars, -galactosidase activity of B. subtilis CM002 grown in the different media in Miller units (MU).

Roles of HPr, Crh, and CcpA in repression of citM expression in CI medium. B. subtilis mutants SA003 (genotype ptsH1), QB7097 (crh), and QB5407 (ccpA) are defective in HPr, Crh, and CcpA, respectively, components involved in CCR in B. subtilis (see the introduction) (Table 1). Mutant QB7102 is a double mutant defective in the two homologous proteins HPr and Crh. The PcitM-lacZ fusion was integrated into the chromosome of each of these mutants to measure the promoter activity in the mutant background under inducing and repressing conditions.

View larger version (34K): [in this window] [in a new window]   FIG. 4.   citM promoter activity of CCR mutants grown in CI medium. -Galactosidase activities of B. subtilis strains CM002 (wt), CM004 (HPr), CM006 (Crh), CM008 (HPr/Crh), and CM010 (CcpA) grown in CI medium supplemented with citrate (black bars) and citrate plus glucose (gray bars) are shown. All strains carry the PcitM-lacZ fusion integrated in the amyE locus. (Inset) enlarged part of the graph.

Roles of HPr, Crh, and CcpA in repression of citM expression in CSE and CIE media. Upon growth of cells in the CSE and CIE media, the main features of promoter activity and Ni²⁺-citrate uptake activity of the mutant and wild-type strains were similar to those observed in CI medium (Tables 3 and 4). Importantly, in the succinate-glutamate medium (CSE medium) both transport and promoter activities were four- to fivefold higher in the ccpA mutant and ptsH1 crh double mutant than in the wild-type strain, indicating that the medium components (succinate and/or glutamate) repressed citM expression via the CCR system. Noteworthy is that in CSE medium the double mutant and the ccpA mutant gave comparable -galactosidase and transport activities, indicating that repression by succinate and/or glutamate is completely mediated via HPr and/or Crh. In the two single mutants, both transport activity and promoter activity were stimulated by a factor of two relative to those of the wild-type strain, indicating that both HPr and Crh play a role in succinate and/or glutamate repression but that they cannot completely take over each other's roles as was observed in repression by inositol. Additional repression by glucose in the wild-type and mutant strains was much stronger in CSE medium than in CI medium, except for the ccpA mutant, where repression was completely relieved.

Growth defects of the ptsH1, crh, and ccpA mutant strains. A (partly) functional CCR system was necessary for normal growth on inositol. Both the ptsH1 crh double mutant and the ccpA mutant grew poorly in CI and CIE minimal media, with growth rates of 0.07 to 0.11 h¹ compared to 0.32 (CI medium) and 0.69 h¹ (CIE medium) for the wild-type strain. Especially long lag phases were observed, and the addition of glucose, citrate, or glutamate (44) had no significant effect on the growth rate.

	DISCUSSION

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In B. subtilis the uptake of citrate is mediated by at least two known homologous secondary transport proteins (3). One of them, termed CitM, is a proton motive force-driven transporter that mediates the transport of citrate complexed to Mg²⁺ and several other divalent metal ions in symport with two protons (3). CitM is believed to be the predominant citrate transporter under physiological conditions (2, 3). The conditions under which CitM is expressed were the topic of our research. The expression of citM was monitored at the transcriptional level by measuring the citM promoter activity and at the protein level by measuring Ni²⁺-citrate uptake activity in whole cells. Our results indicate that citM expression is under strict control of the medium composition. CitM is an inducible protein that is only expressed when citrate is present in the growth medium, which is sensed by the CitS-CitT two-component system, whose coding sequence is located upstream of citM (9; Yamamoto et al., Abstr. 10th Int. Conf. Bacilli). The expression was highest when citrate was the only carbon and energy source in the medium. The carbohydrates glucose, glycerol, and inositol are preferred over citrate, resulting in higher growth rates and a strongly repressed expression of citM. Remarkably, the expression of citM was also repressed during growth on the nonsugars succinate and glutamate, albeit to a lesser extent. In B. subtilis, repression by growth substrates other than glucose has been reported for inositol dehydrogenase, which was repressed by glycerol and mannitol (7), for the hut operon, that was found to be repressed by amino acids (43) and for the first three enzymes of the Krebs cycle were found to be repressed by glutamate and glutamine (8, 33).

Transcription of the structural gene is the first step in the biosynthetic pathway of a protein. The amount of a membrane protein such as CitM that ends up in the cytoplasmic membrane depends on the rate of transcription of the gene (measured by the promoter activity) and on other factors such as messenger stability, efficiency of insertion into the membrane, and protein stability. The activity of the ensemble of protein molecules in the membrane, which is the relevant parameter for the cell, may further depend on regulation of the activity of the individual protein molecules by global cellular factors such as pH and redox potential and by more-specific effectors. Most importantly, for secondary transporters, transport activity depends on the energy status of the cell. Transport activity catalyzed by CitM is driven by the electrochemical proton gradient that is maintained across the cytoplasmic membrane by the cellular energy metabolism (3). In this study, a reasonable correlation between citM promoter activity and Ni²⁺-citrate uptake activity in the different strains and under different growth conditions was observed; this is somewhat surprising considering the above discussion. Apparently, in the media tested, the uptake of citrate complexed to divalent metal ions in B. subtilis is mainly regulated at the level of transcription of the citM gene.

The promoter region of citM contains a cre sequence centered 24 bp upstream of the citM start codon. The 14-bp DNA sequence with dyad symmetry deviates very little from the consensus sequence (41, 46) (Fig. 1), which suggested that CitM is likely to be subject to carbon catabolite repression. It has been suggested that A- and T-rich regions intensify the interactions of catabolite control protein A (CcpA) with the cre site flanking the citM cre sequence (46). Recently, it was demonstrated that the citM cre site is active in vivo (32). The strong relief of citM repression in all media tested in a defective-CcpA mutant indicates that repression by not only glucose but also inositol and succinate and/or glutamate in the wild-type strain is mediated by the binding of CcpA to the cre site located in the citM promoter region.

In CCR, the binding of CcpA to the cre site is induced by complex formation of CcpA and the phosphorylated forms of HPr and Crh. Repression of citM expression in CI medium was less relieved in the ptsHI crh double mutant than in the ccpA mutant. Since in the double mutant the CcpA molecule is present, this may indicate an affinity of uncomplexed CcpA for the cre site or promotion of binding by factors other than HPr and Crh (15, 23). Complete relief of repression in the double mutant when grown in CSE medium suggests that in CI medium other metabolic intermediates promote CcpA binding to the cre site.

The specific roles of HPr and Crh in CCR are not clear. HPr is one of the general proteins in the phosphoenolpyruvate-dependent PTS that transports sugars into the cell with concomitant phosphorylation. HPr is a phosphocarrier intermediate that is phosphorylated at a histidine residue (His15) by phosphoenolpyruvate in a reaction catalyzed by enzyme I (EI). It donates its phosphoryl group to a sugar-specific enzyme or enzyme domain termed IIA. In CCR, HPr is phosphorylated by ATP at a serine residue (Ser46), a reaction catalyzed by HPr kinase. It has been suggested that the functions of HPr in the PTS and CCR interact when transcription of a gene is repressed by a PTS sugar, for instance, glucose (35). The Crh protein is not operational in the PTS (29), simply because, at the position corresponding to the phosphorylation site (His15) in HPr, a glutamine residue is found in Crh. The CCR phosphorylation site (Ser46) is present, and it has been shown that Crh is phosphorylated by HPr kinase in vitro (13, 14). In a number of studies, it has been observed that HPr and Crh can take over each other's role in the CCR function (34, 46). Similarly, in this study, repression of expression of citM by inositol in the ptsH1 and crh single mutants was similar to that observed in the wild-type strain, while repression was alleviated in the ptsH1 crh double mutant (Fig. 4). It should be noted that inositol is believed to be taken up by the cell by a secondary transporter (encoded by iolF) (45) and, therefore, that there is no turnover of the PTS during growth on inositol. For a PTS sugar, it may be anticipated that the CCR system discriminates between HPr and Crh (35). In agreement, repression of citM by glucose is more effective in the crh mutant than in the HPr mutant, suggesting that repression by HPr is potentiated during turnover of the PTS. Discrimination between HPr and Crh in the repression of citM in medium containing the nonsugars succinate and glutamate (CSE medium) was also observed in this study. Repression was relieved twofold in both single mutants, suggesting that HPr could only partly take over repression exerted by Crh and vice versa. The mechanism by which succinate and/or glutamate metabolism affects the degree of phosphorylation of HPr and Crh is completely unknown.

The addition of glucose to CI medium resulted in an additional threefold repression of citM expression. The addition of glutamate to CI medium had no effect on citM expression. Remarkably, when both glucose and glutamate were added to CI medium, the additional repression was about 15-fold and of the same order of magnitude of the glucose repression in CSE medium, which also contains glutamate. This suggests that the presence of glutamate in the medium makes repression by glucose much stronger. A similar synergistic repression has been observed for the citB gene of B. subtilis, coding for the tricarboxylic acid (TCA) cycle enzyme aconitase. Expression of the citB gene in minimal medium containing glucose or glycerol was fully repressed when a source of 2-ketoglutarate, such as glutamine or glutamate, was present (33, 37, 39). Repression of citB is mediated by a novel regulator termed CcpC (22). A PcitM-lacZ fusion integrated in the amyE locus of B. subtilis CJB8 containing an inactivated ccpC gene resulted in the same -galactosidase activities when grown on CSE medium in the presence and absence of glucose as those observed for the wild-type strain CM002 (not shown). This strongly suggests that CcpC is not involved in the regulation of CitM expression and that synergistic repression is not restricted to regulation by CcpC.

The ccpA mutant has been reported to be unable to grow on glucose minimal medium with ammonium as the nitrogen source (44). The observation was explained by the inability of the mutant to utilize ammonium as a single source of nitrogen because of the absence of the key enzyme of ammonium assimilation, glutamate synthase (10). Addition of TCA cycle intermediates, such as citrate and glutamate, could restore growth on minimal medium containing glucose and ammonium (44), but not on that containing arabinose (38). In this study, the effect of inositol was as observed with arabinose, i.e., growth in CI and CIE medium was severely slowed down even when citrate or glutamate or both were present. The same was true for the ptsH1 crh double mutant grown in minimal medium in the presence of inositol, whereas the ptsH1 and crh single mutants showed wild-type growth rates under these conditions. Similar growth defects have been described by Zalieckas et al. (47).