INTRODUCTION

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During growth in the presence of rapidly metabolized carbon sources, carbon catabolite repression (CCR) inhibits the utilization of carbon compounds which support low rates of growth. Transcription of the Bacillus subtilis histidine utilization operon (hut) is regulated in response to carbon availability by the global regulatory proteins CodY (33) and CcpA (17). The CodY repressor protein exerts a low level of CCR at the hutO_A site (previously called hutO_CR1) located immediately downstream of the hut promoter (9). Although the metabolic signal regulating CodY activity is not known, CodY regulates dpp and hut expression in response to growth rate (9). The highest levels of CodY-dependent repression occur in cells growing rapidly in media containing amino acids. In cells growing with preferred carbon and nitrogen sources (but no amino acids), only a low level of CodY-mediated repression occurs. Since limitation of growth by carbon availability almost completely abolishes CodY-dependent regulation, CodY mediates a low level of CCR at the dpp and hut promoters (9).

CCR of hut expression is mediated primarily by CcpA-dependent repression at the hut carbon repression element (cre) (previously called hutO_CR2) centered 209.5 nucleotides downstream of the hut transcriptional start site (28, 40). The in vitro binding of CcpA to cre sites is stimulated by seryl-phosphorylated HPr [HPr(ser-P)] (13, 16, 18, 22), a phosphocarrier component of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) (29). HPr can be phosphorylated at two residues, His-15 by phosphoenolpyruvate-dependent PTS enzyme I and Ser-46 by the ATP-dependent HprK kinase (15, 30, 31). The ptsH1 mutation replaces the Ser-46 residue of HPr with alanine and results in the production of a mutant form of HPr that cannot be phosphorylated by HprK (6, 15, 30, 31). CCR of acsA (43), bglPH (19), gnt (6, 22), iol (15), lev (21), and xyl (5) expression is partially relieved in ptsH1 mutants. The Crh protein, a homolog of HPr, can also be phosphorylated by the HprK kinase and is involved in CCR (14). In a ptsH1 crh double mutant, carbon regulation of acsA, ackA, iol, lev, and -xylosidase expression is abrogated (14, 36, 43). Although it has not yet been demonstrated in vitro, the seryl-phosphorylated form of Crh, Crh(ser-P), has been proposed to also function as a corepressor for CcpA (14, 15). CcpB, a homolog of CcpA, has also been reported to control CCR of gnt expression during growth with low levels of aeration and during growth on solid medium (3).

HPr(ser-P) and Crh(ser-P) may not be the only CcpA corepressors. The observation that glucose-6-phosphate (Glc-6-P) enhances the in vitro binding of the Bacillus megaterium CcpA protein to the B. megaterium xyl cre site and of the B. subtilis CcpA protein to the downstream cre site in the B. subtilis gnt operon raises the possibility that Glc-6-P serves as a corepressor for CcpA binding to cre sequences in gram-positive bacteria (16, 22). This hypothesis is supported by the finding that inactivation of the gene encoding glucose kinase partially relieves CCR in Staphylococcus xylosus (37) and B. megaterium (34). Since glucose kinase catalyzes the conversion of glucose (transported by PTS-independent systems) to Glc-6-P, smaller intracellular pools of Glc-6-P may be present in glucose kinase mutants than in wild-type cells during growth with glucose. An additional cofactor, NADP, has been shown to stimulate the binding of the CcpA-HPr(ser-P) complex to the amyE cre sequence and increase the ability of CcpA to inhibit transcription (18). The mechanism responsible for the NADP-dependent stimulation of CcpA in vitro activity is not known.

CCR of gene expression is also deficient in B. subtilis glnA mutants that synthesize no or low levels of active glutamine synthetase (GS) (10). This class of glnA mutants has a pleiotropic phenotype that includes altered regulation of nitrogen and carbon metabolism and growth defects in minimal medium (10). While the defect in the regulation of carbon metabolism in these mutants is not understood, several lines of evidence indicate that GS acts as a sensor of nitrogen availability in B. subtilis. The TnrA regulatory protein controls gene expression in response to nitrogen availability (38). The TnrA protein is active during nitrogen-limited conditions, where it positively regulates the expression of some genes and negatively regulates the expression of other genes (12, 38). Since TnrA-activated genes are expressed constitutively in this class of glnA mutants, it has been proposed that GS produces or transmits an inhibitory regulatory signal to TnrA during growth with excess nitrogen (38).

In this report, examination of hut expression in wild-type and mutant strains grown in minimal medium revealed that CCR of hut expression requires either Crh or HPr(ser-P) but not glucose kinase or CcpB. In contrast, HPr is required for hut CCR in cells grown in Luria-Bertani (LB) medium. Since mutational inactivation of tnrA suppresses the defects in hut CCR and growth seen in a glnA mutant, GS is not directly involved in the mechanism of hut CCR in B. subtilis.

	MATERIALS AND METHODS

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Bacterial strains. Table 1 lists B. subtilis strains used in this study. All lacZ transcriptional fusions were transformed into strain 168 (trpC2) with plasmid DNA as previously described (40). The tnrA62::Tn917 insertion (38) was transferred by transformation with selection for transposon-encoded erythromycin resistance. Transformation with selection for spectinomycin (spc) resistance was used to transfer the glnA::spc (38), glcK::spc (43), crh::spc, ccpA::Tnspc, and ccpB::spc (43) mutations. The ptsH1 mutation (6) was transferred by transformation with selection for the genetically linked chloramphenicol resistance gene. Transformants containing the ptsH1 mutation were identified by their lack of growth on mannitol minimal medium plates containing ammonium as the nitrogen source.

Cell growth, media, and enzyme assays. The methods used for bacterial cultivation have been previously described (1). Liquid minimal cultures were grown in the morpholinepropanesulfonic acid (MOPS) minimal medium of Neidhardt et al. (26). Glucose was added at 0.5% to MOPS minimal medium and at 1% to LB medium (27). Lactate and citrate were added to a final concentration of 0.2% to MOPS minimal medium. All nitrogen sources were added at 0.2% to MOPS minimal medium. Histidase was induced by addition of L-histidine to 0.1% in the growth medium.

Uptake of -methyl glucoside. -Methyl glucoside uptake was assayed in cells grown to mid-log phase in MOPS minimal medium containing glucose and glutamine as the carbon and nitrogen sources, respectively. For one generation prior to harvesting, the cultures were grown in the presence of 1% glycerol. Cells were harvested by filtration on a 0.45-µm-pore-size filter (Millipore) and washed with buffer A (MOPS minimal salts containing 0.2% glutamine and 1% glycerol). The washed cells were resuspended in buffer A by shaking at 26°C. Uptake was initiated by the addition of ¹⁴C-labeled -methyl glucoside (New England Nuclear) to a final concentration of 100 µM, which yielded a specific activity of 2 Ci of ¹⁴C per mol. Samples of 0.5 ml were removed and filtered through 0.45-µm-pore-size filters which had been presoaked in buffer A. The filters were washed with 10 ml of buffer A containing 100 µM -methyl glucoside and counted in Ecolite(+) (ICN) scintillation fluid. The uptake rate was determined from the initial linear portion of the uptake curve.

lacZ fusions. pSFL6 and pSFL7 are neomycin-resistant lacZ transcriptional fusion vectors that integrate into the amyE locus and contain promoterless trpA-lacZ and spoVG-lacZ genes, respectively (42). The TMS922 lacZ fusion contains the tms promoter (24) cloned into pSFL7 (42). The HUT924 lacZ fusion is a derivative of pSFL7 that contains a DNA fragment with hut cre located downstream of the tms promoter (42). The HUT646 and BGL2 lacZ fusions contain the B. subtilis hut and bglPH promoters, respectively, cloned into pSFL6 (42). The NRG407 fusion contains the nrgAB promoter cloned into pSFL7 (41).

	RESULTS AND DISCUSSION

Role of HPr and Crh in CCR of hut expression in cells grown in minimal medium. To determine whether HPr and Crh are involved in CCR of hut expression, we examined the expression of histidase, the first enzyme in the histidine-degradative pathway, in B. subtilis strains containing the ptsH1 and crh mutations. Because no alteration in CCR of histidase expression was seen in strains containing either the ptsH1 or the crh mutation during growth in glucose minimal medium (data not shown), either Crh or HPr is sufficient for hut CCR under these growth conditions. CCR of histidase expression could not be evaluated in a ptsH1 crh double mutant because addition of histidine to 0.1% in glucose minimal medium (which is required for the induction of the hut operon) completely inhibited growth of the ptsH1 crh double mutant. To circumvent the histidine-sensitive phenotype of the ptsH1 crh double mutant, the crh and ptsH1 mutations were transferred into B. subtilis strains containing the HUT646 (hut-lacZ) fusion (42). The HUT646 lacZ fusion contains the hut promoter and downstream cre but lacks the downstream transcriptional terminator involved in histidine-dependent hut induction (39). As a result, high-level -galactosidase expression occurs from this fusion in cells grown in the absence of histidine.

View this table: [in this window] [in a new window]   TABLE 2.   -Galactosidase levels of lacZ fusions in wild-type and mutant strains grown in minimal medium

Role of HPr in CCR of hut expression in LB medium-grown cells. Although the ptsH1 mutation only partially relieves CCR of gnt expression in cells grown in minimal medium, no CCR of gnt expression occurs when the ptsH1 mutant is grown in LB medium (6). To determine whether the wild-type ptsH gene is required for CCR of hut expression in cells grown in LB medium, we examined CCR of hut expression in LB medium-grown cells. The HUT924 (P_tms-hut cre-lacZ) fusion (42) was used in these experiments instead of the HUT646 lacZ fusion because expression of the hut promoter is severely repressed by the CodY regulatory protein during rapid growth in the presence of amino acids (9). In the HUT924 fusion, hut cre is cloned between the unregulated tms promoter and lacZ. CCR of HUT924 expression is dependent upon the hut cre site because expression of the TMS922 (P_tms-lacZ) fusion, which contains the tms promoter transcriptionally fused to lacZ, is not regulated by CCR in cells grown in either minimal or LB medium (Table 3 and data not shown). -Galactosidase expression from the HUT646 and HUT924 fusions is subject to similar levels of CCR in wild-type cells and ptsH1, crh, and ptsH1 crh mutant cells grown in minimal medium (Table 2). Thus, the HUT924 fusion can be used to monitor cre-dependent hut CCR in LB medium-grown cells.

View this table: [in this window] [in a new window]   TABLE 3.   -Galactosidase levels from lacZ fusions in wild-type and mutant strains grown in LB medium

Growth defects of the ptsH1 and crh mutant strains. Compared to the wild-type, ptsH1 mutant, or crh mutant strain, the ptsH1 crh double mutant exhibited several growth defects in minimal medium. The ptsH1 crh mutant grew more slowly in glucose minimal medium than wild-type cells. For example, when the glucose minimal medium contained glutamine as the sole nitrogen source, the doubling time of ptsH1 crh cultures (80 min) was higher than the doubling time of wild-type, ptsH1, and crh cultures (60 min). In contrast, we observed no significant difference in the growth rates of wild-type and ptsH1 crh cells in glutamine minimal medium containing either malate or citrate as the sole carbon source (data not shown). Furthermore, growth of the ptsH1 crh double mutant in minimal and LB media was severely inhibited in the presence of 0.4% glycerol.

Role of GlnA and TnrA in CCR of hut expression. CCR is partially relieved in Gln B. subtilis mutants which synthesize no or low levels of active GS (10). Several lines of evidence indicate that the altered CCR seen in these Gln mutants may result from the defective utilization of glucose. First, measurement of intracellular metabolite pools in wild-type and glnA22 mutant cells grown with excess glucose showed that the pool sizes of Glc-6-P, pyruvate, and 2-ketoglutarate are smaller in the glnA22 mutant than in wild-type cells (10). The pool sizes of these glycolytic intermediates are decreased in wild-type cells grown with limiting amounts of glucose (8). Second, the doubling time of this class of Gln mutants in glutamine minimal medium containing excess glucose as the carbon source (101 min) is higher than that of wild-type cells (70 min) (Table 4) (10). The observation that the level of Glc-6-P is reduced in this class of Gln mutants suggested that glucose transport might be impaired. This possibility was tested by examining the uptake of the glucose analog -methyl glucoside in wild-type and Gln cells grown in minimal medium containing glucose and glutamine as the carbon and nitrogen sources. Since similar rates of -methyl glucoside uptake were seen in wild-type SF10 (1.25 nmol per min per mg) and SF22 (glnA22) (1.20 nmol per min per mg) cells, no defect in glucose transport appeared to be present in this Gln mutant.

View this table: [in this window] [in a new window]   TABLE 4.   Carbon catabolite repression of histidase expression in wild-type, tnrA, and glnA strains

TnrA-dependent regulation of gene expression in a ccpA mutant. The growth defects seen in the ccpA and ptsH1 crh mutants in glucose minimal medium may result from altered expression of CCR-regulated genes. However, because constitutive expression of TnrA-regulated genes causes a growth phenotype in glnA mutants, it is possible that inappropriate expression of TnrA-dependent genes impairs the growth of ccpA and ptsH1 crh mutants. The latter possibility was tested by examining the expression of the (nrgAB-lacZ)407 fusion in wild-type and ccpA mutant cells grown in glucose minimal medium with an excess (glutamine) or a limited (glutamate) nitrogen source. Transcription from the nrgAB promoter occurs only during nitrogen-limited conditions and is completely dependent upon TnrA (38). The ccpA mutant strain grows more slowly than the wild-type strain in glucose minimal medium containing glutamine (Table 5). Since nrgAB is expressed at low levels in both wild-type and ccpA mutant cells grown in glucose glutamine minimal medium (Table 5), the growth defect seen in the ccpA mutant in this medium does not result from constitutive expression of TnrA-regulated genes. Surprisingly, although wild-type and ccpA cultures exhibited similar growth rates under nitrogen-limited conditions (i.e., glucose glutamate minimal medium), nrgAB expression increased almost 10,000-fold in the wild-type strain but only 12-fold in the ccpA mutant (Table 5).

View this table: [in this window] [in a new window]   TABLE 5.   -Galactosidase expression of an nrgAB-lacZ fusion in wild-type and ccpA mutant strains

Role of glucose kinase in CCR of hut expression. Glc-6-P has been proposed to be involved in CCR of the B. subtilis gnt and B. megaterium xyl operons (16, 22). To determine whether Glc-6-P is required for wild-type levels of CCR of hut expression, expression of the HUT646 (hut-lacZ) fusion was examined in wild-type and glucose kinase (glcK) mutant cultures. The observation that -galactosidase is expressed from the HUT646 fusion at similar levels in wild-type and glcK strains (Table 2) indicates that glucose kinase most likely does not participate in CCR of hut expression in B. subtilis.

Role of CcpB in CCR in hut expression. CcpB, a homolog of the CcpA protein, has been reported to be involved in CCR of gnt and xyl expression in cells grown in liquid cultures with low levels of aeration or on solid medium (3). No differences in histidase expression from the hut operon were seen in the wild-type and ccpB mutant strains grown in minimal medium (data not shown). Furthermore, as judged by colony color, no difference in -galactosidase expression from the HUT924 lacZ fusion was observed in wild-type and ccpB cells grown on X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) plates containing either LB glucose medium or glucose glutamine minimal medium (data not shown). Thus, there is no evidence that CcpB participates in CCR of hut expression.