trans-Acting Factors Affecting Carbon Catabolite Repression of the hut Operon in Bacillus subtilis

Abstract

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

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 β-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 ΔglnA mutant, GS is not directly involved in the mechanism of hut CCR in B. subtilis.

MATERIALS AND METHODS

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 ΔglnA::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.

TABLE 1.

B. Subtilis strains used in this study

Strain	Genotypea	Source, reference, and/or derivationb
168	trpC2	This laboratory
QB7097	trpC2 crh::spc	I. Martin-Verstraete
GM1222	trpC2 pheA1 Δ(bgaX) ptsH1(cat) ΔamyE:: (gntRK′-lacZ)	6
JZ4	trpC2 crh::spc	168 × QB7097 DNAs
JZ5	trpC2 ptsH1(cat)	168 × GM1222 DNAs
JZ6	trpC2 glcK::spc	43
JZ7	trpC2 ccpB::spc	43
TH256	ccpA::Tn917lac lys-3 trpC2 metB10	T. Henkin
SF168CP	trpC2 ccpA::Tn917lac	168 × TH256 DNAs
SF168CPS	trpC2 ccpA::Tn917lacΔ(lacZ erm)::spc	SF168CP × p917::Sp DNAs, 35
SF62	trpC2 tnrA62::Tn917	38
SF168A	trpC2 ΔglnA::spc	168 × pGLN14 DNAs, 38
SF62A	trpC2 tnrA62::Tn917 ΔglnA::spc	SF62 × pGLN14 DNAs 38
SF10	Wild type	10
SF22	glnA22	10

^aThe genotype symbols are those of Biaudet et al. (2) with the addition of crh (14), tnrA (38), and glcK (32). 

^bStrains were derived by transforming the first strain listed with DNA from the second strain or the plasmid listed. 

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.

Extracts for enzyme assays were prepared as previously described (1) from cells harvested during exponential growth (70 to 90 Klett units). Histidase and β-galactosidase were assayed as previously described (1). β-Galactosidase activity was always corrected for endogenous β-galactosidase activity present in B. subtilis 168 cells containing the promoterless lacZ gene from pSFL6 or pSFL7 integrated at the amyE site. The assay for gluconate kinase has been described previously (11).

Uptake of α-methyl glucoside.

α-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 α-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 α-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 β-galactosidase expression occurs from this fusion in cells grown in the absence of histidine.

In cells grown in glucose minimal medium, β-galactosidase expression from the HUT646 lacZ fusion is repressed to similar levels in wild-type, ptsH1, and crh strains (Table 2). In contrast, CCR of the HUT646 lacZ fusion is completely relieved when the ptsH1 crh double mutant is grown in minimal medium with glucose as the carbon source (Table 2). The latter result agrees with published results showing that both the ptsH1 and crh mutations are required for complete relief of CCR of acsA, iol, lev, and β-xylosidase expression in cells grown in glucose minimal medium (14, 43). However, the observation that wild-type levels of hut CCR are seen in the ptsH1 mutant was surprising because the ptsH1 mutation was previously shown to partially relieve CCR of acsA, gnt, and bglPH expression in cells grown in minimal medium (19, 22, 43). This difference is not due to our growth medium or conditions because we also found that the ptsH1 mutation partially relieves CCR of the expression of β-galactosidase from the bglPH-lacZ BGL2 fusion (Table 2) and of gluconate kinase, the first enzyme in the gluconate-degradative pathway (data not shown). The observation that during growth in minimal medium the ptsH1 mutation does not affect hut CCR but that it partially relieves bglPH and gnt CCR indicates that some cre sites are more sensitive to CCR mediated by HPr than by Crh. One possible explanation is that complexes of CcpA with HPr(ser-P) and Crh(ser-P) assume different conformations and that the CcpA–HPr(ser-P) complex has a higher affinity for the bglPH and gnt cre sequences than the CcpA–Crh(ser-P) complex but that both complexes bind with similar affinities to the hut cre sequence.

TABLE 2.

β-Galactosidase levels of lacZ fusions in wild-type and mutant strains grown in minimal medium

lacZ fusiona	Relevant genotype	β-Galactosidase sp act (U/mg of protein) in cells grown onb:		Glucose repression ratioc
		Glucose	Citrate
HUT646	Wild type	0.9	11.6	13
	ptsH1	0.7	11.4	16
	crh	1.0	19.4	19
	crh ptsH1	15.4	19.0	1.2
	glcK	1.2	12.0	10
HUT924	Wild type	2.2	28.8	13
	ptsH1	3.1	29.1	9.4
	crh	2.5	39.9	16
	crh ptsH1	27.1	41.9	1.5
BGL2	Wild type	1.3	22.7	17
	ptsH1	4.2	20.7	4.9

^aAll strains were derivatives of strain 168 and had the indicated lacZ fusion integrated as a single copy at the amyE locus. 

^bData are the averages of three or more determinations, which did not vary by more than 15%. Cultures were grown in MOPS minimal medium containing glutamine as the nitrogen source and the indicated carbon source. 

^cThe glucose repression ratio was calculated by dividing the enzyme activity found in the culture grown with citrate by the enzyme activity found in the culture grown with glucose. 

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). β-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.

TABLE 3.

β-Galactosidase levels from lacZ fusions in wild-type and mutant strains grown in LB medium

lacZ fusiona	Relevant genotype	β-Galactosidase sp act (U/mg of protein) in cells grown in LB mediumb:		Glucose repression ratioc
		Without glucose	With glucose
TMS922	Wild type	44.0	36.6	1.2
	ptsH1	33.6	33.5	1.0
HUT924	Wild type	21.3	4.5	4.7
	ptsH1	20.6	20.0	1.0
BGL2	Wild type	14.2	2.2	6.5
	ptsH1	15.2	10.5	1.4

^aAll strains were derivatives of strain 168 and had the indicated lacZ fusion integrated as a single copy at the amyE locus. 

^bData are the averages of three or more determinations, which did not vary by more than 25%. Cultures were grown in LB medium with or without glucose. 

^cThe glucose repression ratio was calculated by dividing the enzyme activity found in the culture grown without glucose by the enzyme activity found in the culture grown with glucose. 

During growth in LB medium, β-galactosidase expression from the HUT924 (P_tms-hut cre-lacZ) fusion was repressed 4.7-fold by glucose in wild-type cells while no CCR occurred in the ptsH1 mutant strain (Table 3). The ptsH1 mutation also completely relieved CCR of BGL2 (bglPH-lacZ) (Table 3) and ASC7 (acsA-lacZ) (data not shown) during growth of these strains in LB medium. Interestingly, the HUT924, BGL2, and ACS7 fusions exhibited lower levels of CCR in wild-type cells grown in LB medium than in those grown in minimal medium (Tables 2 and 3 and data not shown).

It is difficult to explain why CCR of hut, gnt, and bglPH expression is completely dependent upon HPr in cells grown in LB medium but not in minimal medium. These results suggest that HPr, but not Crh, is phosphorylated in LB medium-grown cells. However, phosphorylation of HPr and Crh both in vivo and in vitro has been shown to be dependent on the same protein kinase, HprK (15, 30). To explain the difference in the HPr and Crh dependence of CCR during growth in LB and minimal media, it is necessary to postulate that factors in addition to HprK are involved in phosphorylation of these two proteins. One possibility is that the relative phosphorylation states of HPr and Crh are controlled by phosphatases specific for either HPr(ser-P) or Crh(ser-P). If the activities of the HPr(ser-P) and Crh(ser-P) phosphatases are regulated by different metabolites, differences in the levels of these metabolites in cells grown in LB and minimal media might result in the presence of only HPr(ser-P) in LB medium-grown cells. Since the doubling time of wild-type cells in LB medium plus glucose (20 to 25 min) is lower than in glucose-glutamine minimal medium (45 to 70 min), different metabolite levels may be present in wild-type cells grown in these two media. Decreased levels of HPr(ser-P) together with the absence of Crh(ser-P) in LB medium-grown cells may also explain the lower levels of hut and bglPH CCR seen in cells grown in LB medium than in cells grown in glucose minimal 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.

Since it has been proposed that CCR mediated by CcpA requires HPr(ser-P) and Crh(ser-P), any growth defects caused by the relief of CCR in the ptsH1 crh mutant should also be observed with a ccpA mutant. Although the growth phenotypes of these two strain are similar, there are differences between the ccpA and ptsH1 crh mutants. High levels of histidine completely inhibit growth of the ptsH1 crh mutant but only partially inhibit growth of the ccpA mutant (40). Similarly, high levels of glycerol severely inhibit growth of the ptsH1 crh mutant but not the ccpA mutant. The ccpA mutant is unable to grow on glucose minimal medium containing ammonium as the nitrogen source (40), but the ptsH1 crh mutant is able to grow on this medium, with a higher doubling time than that seen with wild-type cells. These differences suggest that CcpA and/or the HPr and Crh proteins have additional independent roles in regulating B. subtilis cellular metabolism. The growth phenotype of the ccpA and ptsH1 crh mutants explains why CCR of hut expression, which is subject to carbon regulation by both CcpA and CodY, is completely relieved in the ccpA mutant and the ptsH1 crh double mutant. Since CodY regulation is responsive to growth rate, no CodY-dependent regulation is expected to occur in the ccpA and ptsH1 crh mutants, which exhibit growth defects.

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 α-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 α-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.

TABLE 4.

Carbon catabolite repression of histidase expression in wild-type, tnrA, and ΔglnA strains

Relevant genotypea	Value for cells grown onb:
	Glucose		Citrate
	Doubling time (min)	Histidase sp act (U/mg of protein)	Doubling time (min)	Histidase sp act (U/mg of protein)
Wild type	70	3.3	102	180
tnrA	75	3.4	100	256
ΔglnA	101	54.5	NGc
tnrA ΔglnA	74	3.1	107	184

^aAll strains were derivatives of strain 168. 

^bHistidase levels are the averages of two or more determinations, which did not vary by more than 25%. Cultures were grown in MOPS minimal medium containing glutamine as the nitrogen source and the indicated carbon source. Histidine was added to induce hut expression. 

^cNG, no growth. The ΔglnA strain grows only for several generations before entering stationary phase in this medium. 

The global nitrogen regulatory protein TnrA activates the expression of many genes during nitrogen-limited growth of B. subtilis (38). Unexpectedly, mutational inactivation of tnrA was found to suppress the growth defect of a ΔglnA mutant in glucose minimal medium (Table 4). To determine whether the tnrA mutation also restores CCR to wild-type levels in the ΔglnA mutant, histidase expression was examined in the wild type and in ΔglnA and tnrA mutant cells. In glucose-grown cultures, histidase levels were 16-fold higher in extracts of ΔglnA cells than in extracts of wild-type or tnrA mutant cells (Table 4). In contrast, histidase levels in the ΔglnA tnrA double mutant were similar to the histidase levels in wild-type or tnrA mutant cells. Although the ΔglnA strain grew for only two generations in citrate minimal medium before growth ceased, the ΔglnA tnrA strain grew like the wild-type and tnrA strains in minimal medium with citrate as the carbon source (Table 4).

Since mutational inactivation of tnrA suppresses the CCR and growth phenotype of this class of glnA mutants, the defects in growth and CCR seen in the ΔglnA mutant appear to result from the inappropriate expression of TnrA-regulated genes. It is possible that constitutive expression of TnrA-regulated genes in the ΔglnA mutant results in a low rate of cellular growth and relief of CCR due to the depletion of intracellular pools of carbon metabolites. For example, the expression of xanthine dehydrogenase, a key enzyme in purine catabolism, is nitrogen regulated by the GS-dependent nitrogen signal pathway in B. subtilis (4). Since this signal pathway is defective in ΔglnA mutants, purine degradation may occur constitutively in these mutants and result in a futile cycle of purine synthesis and degradation that depletes intracellular carbon pools. If TnrA activates the expression of xanthine dehydrogenase, this futile cycle would not occur in the ΔglnA tnrA mutant. Thus, because the defects in growth and CCR seen in the ΔglnA mutant are the indirect result of the ΔglnA mutation, GS does not directly participate in the mechanism of CCR of hut expression in B. subtilis.

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).

TABLE 5.

β-Galactosidase expression of an nrgAB-lacZ fusion in wild-type and ccpA mutant strains

Relevant genotypea	Carbon source(s)	Value for cells grown with nitrogen sourceb:
		Glutamine		Glutamate
		Doubling time (min)	β-Galactosidase sp act (U/mg of protein)	Doubling time (min)	β-Galactosidase sp act (U/mg of protein)
Wild type	Glucose	53	0.04	165	288
	Glucose + lactate	55	0.06	180	310
ccpA	Glucose	86	0.04	155	0.5
	Glucose + lactate	85	0.05	195	399

^aAll strains were derivatives of 168 and contained the (nrgA-lacZ)407 fusion. 

^bβ-Galactosidase levels are the averages of three or more determinations, which did not vary by more than 25%. Cultures were grown in MOPS minimal medium containing the indicated carbon and nitrogen sources. 

The pattern of nrgAB expression in the ccpA mutant is similar to that previously seen in a B. subtilis ptsI mutant (41). The ptsI gene encodes the enzyme I component of the PTS and is required for transport of all PTS-dependent carbohydrates (29). Since glucose transport is deficient in ptsI mutants, the ptsI mutant grows at a reduced rate in glucose minimal medium. The expression of the TnrA-dependent nrgAB promoter is not activated in a ptsI mutant grown in glucose minimal medium containing the limiting nitrogen source glutamate (41). One explanation for this result is that because TnrA regulation occurs only during nitrogen limitation, cells whose growth is carbon limited are unable to sufficiently deplete intracellular nitrogen pools to allow high-level expression of TnrA-regulated genes. If reduced intracellular carbon pools are responsible for the inability to completely derepress nrgAB expression in the ccpA mutant, high-level nrgAB expression might be observed in ccpA cultures grown with glucose and additional carbon sources. Indeed, nrgAB was expressed at similar levels in wild-type and ccpA mutant strains grown in minimal medium with a limiting nitrogen source (glutamate) and either glucose plus lactate or glucose plus citrate as carbon sources (Table 5 and data not shown). This suggests that the ccpA mutant may contain lower levels of intracellular tricarboxylic acid cycle intermediates than wild-type cells. However, because neither lactate nor citrate was able to suppress the growth phenotype of the ccpA mutant in glucose-plus-glutamine minimal medium (Table 5 and data not shown) or restore CCR of hut expression (data not shown), additional metabolic perturbations must be present in the ccpA mutant.

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 β-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.

The experimental evidence that Glc-6-P functions as an in vivo signal for carbon availability is equivocal for several reasons. First, the Glc-6-P-dependent in vitro binding of B. megaterium CcpA to xyl cre (16) was observed only with acidic conditions (pH 4.1 to 5.4) that differ significantly from the experimentally observed cytoplasmic pH of B. megaterium (20). Second, the concentration of Glc-6-P used to demonstrate in vitro binding of B. subtilis CcpA to gnt cre (30 mM) (22) is significantly higher than the in vivo concentration measured in glucose-grown B. subtilis cells (1.35 to 4.4 mM) (7, 10). Finally, since the intracellular concentrations of Glc-6-P are similar in glucose-grown B. subtilis and E. coli cells (25), the observation that CcpA is unable to mediate CCR regulation of gnt expression in E. coli cells (23) argues that Glc-6-P does not function as an in vivo corepressor for CcpA.

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 β-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-β-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.