ScoC mediates catabolite repression of sporulation in Bacillus subtilis

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and sigmaH in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucose.     

ScoC Mediates Catabolite Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and Ï^H in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucoseReceived: 12 November 2002 / Accepted: 13 December 2002     

Beziehungen zwischen katabolischer Repression und Sporulation bei Bacillus subtilis

Acetoin dehydrogenase can be catabolite repressed by numerous sources of carbon. The following results point out that the catabolite repression of this enzyme and the inhibition of sporulation are mediated by the same mechanism:

1. Mutants, able to synthesize acetoin dehydrogenase in the presence of glucose, sporulate in glucose medium at a higher rate than the standard strain.
2. The catabolite repressing effect of a compound and its ability to inhibit sporulation are in a direct relation to each other.
3. The limitation of inorganic phosphate in the growth medium, which is known to favour sporulation, counteracts the catabolite repressing effect of glucose.

     

Analysis of tnrA alleles which result in a glucose-resistant sporulation phenotype in Bacillus subtilis

Bacillus subtilis cells cannot sporulate in the presence of catabolites such as glucose. During the analysis of Tn10-generated mutants, we found that deletion of the C-terminal region of the tnrA gene, which encodes a global regulator that positively regulates a number of genes in response to nitrogen limitation, results in a catabolite-resistant sporulation phenotype. Analyses of nrg-lacZ and nasB-lacZ, which are activated by TnrA under nitrogen limitation, showed that C-terminally truncated TnrA activates nitrogen-regulated genes constitutively. The relief of catabolite repression of sporulation may result from the uncontrolled expression of the TnrA-regulated genes.     

Catabolite repression in the D-serine deaminase system of Escherichia coli K-12

The induced synthesis of d-serine deaminase in Escherichia coli is subject to three catabolic effects: inhibition on inducer uptake, transient repression, and catabolite repression. Inhibition on d-serine uptake is not significant at the d-serine concentration normally used for induction. Transient repression and catabolite repression of d-serine deaminase synthesis are abolished by mutations in dsdCy, which appears to be an operator locus. The decline in the rate of constitutive synthesis observed in dsdCx mutants growing with glycerol as carbon source at temperatures above 37 C is due to catabolite repression. The low level of constitutivity at 37 C and the partial cis dominance of dsdCx mutants are not artifacts of catabolite repression. It is suggested that a product of one of the genes of the dsd operon may regulate the expression of the operon.     

Effect of nalidixic acid on sporulation of catabolite-resistant mutants ofBacillus subtilis

A number ofBacillus subtilis mutants that are able to sporulate in the presence of relatively high concentrations of carbon sources have been isolated in our laboratory. The present study shows that some of the mutants are also able to sporulate in the presence of nalidixic acid (Nal) under the condition where sporulation of wild-type strains is inhibited. Furthermore, it has been found that a Nal-resistant mutant is unable to sporulate normally when Nal (40 μg/ml) or glucose (55 mM) is present. Since the adverse effect of Nal on inducible enzymes is eliminated when bacterial strains carry a Nal-resistance marker, the above result suggests that the effect of this drug on sporulation might be mediated by a unique mechanism.     

Repression of sporulation in Bacillus subtilis by L-malate.

L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis. When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h. The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed. Sporulation was not repressed when malate was added to the culture at t4 or later. A mutant was selected for ability to sporulate in the presence of malate. This strain could also sporulate in the presence of glucose. The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme. This indicates a defect in the metabolism of malate in the mutant. A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate. A model for the regulation of sporulation by malate is presented and discussed. Citric acid cycle intermediates other than malate did not affect sporulation. In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block. The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate.     

Corepressor system for catabolite repression of the lac operon in Escherichia coli

Acetylated amino sugars, normally used in the biosynthesis of cell walls and cell membranes, were found to play a role as corepressors for catabolite repression of the lac operon in Escherichia coli. This conclusion was derived from studies conducted on mutants of E. coli that were able to assimilate an exogenous source of N-acetylglucosamine (AcGN) but were unable to dissimilate or grow on this compound. At concentrations less than 10(-4)m, AcGN caused severe catabolite repression of beta-galactosidase synthesis in cultures grown under either nonrepressed or partially repressed conditions. This repression occurred in the absence of any effect of AcGN on either the carbon and energy metabolism or the growth of the organism. In addition, this repression by AcGN occurred in a mutant strain that is constitutive for beta-galactosidase production, demonstrating that the AcGN effect does not involve the uptake of inducer. This model for the corepressor system of catabolite repression is discussed in relation to the existing theories on repression of the lac operon.     

Bacteriophage-enhanced sporulation: comparison of spore-converting bacteriophages PMB12 and SP10.

The previously characterized bacteriophage SP10 enhanced the frequency of wild-type sporulation by Bacillus subtilis W23 and 3-13. Comparison of SP10 with the spore-converting bacteriophage PMB12 indicated that both bacteriophages significantly increased the sporulation frequency of an oligosporogenic mutant that contained spo0J::Tn917 omega HU261. SP10 and PMB12 caused wild-type bacteria to sporulate in a liquid medium that initially contained enough glucose to inhibit the sporulation and expression of alpha-amylase by uninfected bacteria. SP10 also induced the expression of alpha-amylase in the presence of glucose, whereas PMB12 had no detectable effect. These observations were consistent with the conclusion that SP10 is a spore-converting bacteriophage and that SP10 and PMB12 relieve glucose-mediated catabolite repression of sporulation by different mechanisms.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

Phenotypes of pleiotropic-negative sporulation mutants of Bacillus subtilis

The phenotypic properties of representatives of the five genetic classes of pleiotropic-negative sporulation mutants have been investigated. Protease production, alkaline and neutral proteases, was curtailed in spoA mutants, but the remainder of mutant classes produced both proteases, albeit at reduced levels. The spoA and spoB mutants plaqued phi2 and phi15 at high efficiency, but the efficiency of plating of these phages on spoE, spoF, and spoH mutants was drastically reduced. Antibiotic was produced by the spoH mutants and to a degree by some spoF mutants, but the other classes did not produce detectable activity. The spoA mutants were less responsive to catabolite repression of histidase synthesis by glucose than was the wild type. Severe catabolite repression could be induced in spoA mutants by amino acid limitation, suggesting that the relaxation of catabolite repression observed is not due to a defect in the mechanism of catabolite repression. Although others have shown a perturbation in cytochrome regulation in spoA and spoB mutants, the primary dehydrogenases, succinate dehydrogenase and reduced nicotinamide adenine dinucleotide dehydrogenase, leading to these cytochromes are unimpaired in all mutant classes. A comparison of the structural components of cell walls and membranes of spoA and the wild type is made. The pleiotropic phenotypes of these mutants are discussed.     

The cyo operon of Pseudomonas putida is involved in carbon catabolite repression of phenol degradation

A bicistronic reporter consisting of the promoterless genes aacC1 (conferring gentamycin resistance) and lacZ fused to the catabolic promoter of the phenol degradation genes was used to identify and analyse mutants of Pseudomonas putida with altered carbon catabolite repression (CR) of phenol degradation. Out of approximately 2500 mini-Tn5 mutants analysed so far, 12 mutants that were resistant to gentamycin during growth on succinate were identified. In eight of these mutants mini-Tn5 was inserted into one of the genes of the cyo operon. The cyo operon encodes the cytochrome o ubiquinol oxidase, the terminal oxidase of the cyanide-sensitive branch of the respiratory chain. In these mutants the activity of the PphlA promoter was significantly increased during growth on succinate and reached 15-20% of that found during growth with the non-repressing carbon source pyruvate. During growth on glucose the reduction of CR was less obvious, during growth on lactate CR was unchanged. The possible significance of the cyo operon for the generation of signal(s) for carbon catabolite repression is discussed.     

Catabolite-induced repression of sporulation in Bacillus subtilis

In response to nutrient limitations, Bacillus subtilis cells undergo a series of morphological and genetic changes that culminate in the formation of endospores. Conversely, excess catabolites inhibit sporulation. It has been demonstrated previously that excess catabolites caused a decrease in culture medium pH in a process that required functional AbrB. Culture medium acidification was also shown to inhibit sigmaH-dependent sporulation gene expression. The studies reported here investigate the effects of AbrB-mediated pH sensing on B. subtilis developmental competence. We have found that neither addition of a pH stabilizer, MOPS (pH 7.5), nor null mutations in abrB blocked catabolite repression of sporulation. Moreover, catabolite-induced culture medium acidification was observed in cultures of catabolite-resistant sporulation mutants, crsA47, rvtA11, and hpr-16, despite their efficient sporulation. These results suggest that AbrB-mediated pH sensing is not the only mechanism regulating catabolite repression of sporulation. The AbrB pathway may function to channel cells toward genetic competence, as opposed to other postexponential differentiation pathways.     

Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae

Summary Glycolytic parameters were determined in recessive yeast mutants with partial defects in carbon catabolite repression. Specific activities of pyruvate kinase and pyruvate decarboxylase in glucose grown cells of all mutant and wild type stains were 4–5 times higher than in ethanol grown cells. Mutants of gene HEX1 had a reduced hexose phosphorylating activity on allmedia wheras those of gene HEX2 had elevated levels but only in glucose grown cells. Mutants of gene CAT80 were normal in this respect. All other glycolytic enzymes were normal in all mutants. This was also true for glycolytic intermediates. Only hexlmutants showed a reduced fermentation of repressing sugars. The three genes appear to be involved in catabolite repression of several but not of all repressible enzymes. Even though all three types of mutants show a limited overlap in their effects on certain enzymes, they still are distinctly different in their action spectra. Carbon catabolite repression apparently does not depend on the sole accumulation of glycolytic intermediales. The activity of the products of the three genes HEX1, HEX2 and CAT80 are required directly or indirectly for triggering carbon catabolite repression. Even a small segment of carbon catabolite repression is controlled by several genes with regulatory functions indicating that the entire regulatory circuit is highly complex.     

The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.     

Interaction between mutant alleles of araC of the Escherichia coli B/r L-arabinose operon.

Strains were constructed that contain mutational alterations affecting two distinct functional domains within the araC gene protein. The araCi (catabolite repression insensitivity) and araCh (catabolite repression hypersensitivity) mutations were used to alter the catabolite repression sensitivity domain, and mutation to D-fucose resistance was used to alter the inducer binding domain. araCh, D-fucose-resistant double mutants never exhibited constitutive ara operon expression, whereas all of the araCi, D-fucose-resistant double mutants did exhibit constitutivity. When L-arabinose was used as an inducer, most of the double mutants exhibited the sensitivity to catabolite repression associated with the araCi or araCh mutation. However, when D-fucose was used as an inducer, changes in sensitivity to catabolite repression were observed that were attributed to interactions between the two protein domains. The roles of catabolite activator protein and araC gene protein in the induction of the araBAD operon were discussed.     

Histidine utilization by Alcaligenes eutrophus: Regulation of histidase formation under heterotrophic conditions of growth

Histidine supported good growth of Alcaligenes eutrophus strain H 16 as a nitrogen source, but only poor growth as a carbon and energy source. The facultative chemolithoautotrophic bacterium was also able to utilize urocanic acid, the first intermediate of histidine catabolism. The products of histidine degradation were ammonium, formate and glutamate. Three enzymes of the pathway, histidase, urocanase and formiminoglutamate hydrolase, were present in histidine-grown cells. Two types of spontaneous mutants, derived from the wild type, were characterized by an increased growth rate on histidine. One of these types was found to produce histidase constitutively and at a higher activity compared with the parental strain. The second type of mutant had apparently gained an improved histidine uptake system, which is supposed to be growth rate-limiting in the wild type. From the physiological studies the conclusion was drawn that the control of histidine-degrading enzymes is based on induction by urocanate and catabolite repression by carbon sources supporting fast growth, such as succinate or pyruvate. Ammonium was found not to affect catabolite repression, however, we obtained evidence that histidine uptake is subject to a nitrogen control.Abbreviation CTAB hexadecyltrimethylammonium bromide