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PubMed1969

Catabolite repression of the lac operon. Separt epressionof two enzymes.

1. Catabolite repression of beta-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol-minimal medium and in glucose-minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for beta-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for beta-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of beta-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of beta-galactosidase and thiogalactoside transacetylase is separate but equal.

Acyltransferases↗
PubMed1998

Expression of the Bacillus subtilis acsA gene: position and sequence context affect cre-mediated carbon catabolite repression.

In Bacillus subtilis, carbon catabolite repression (CCR) of many genes is mediated at cis-acting carbon repression elements (cre) by the catabolite repressor protein CcpA. Mutations in transcription-repair coupling factor (mfd) partially relieve CCR at cre sites located downstream of transcriptional start sites by abolishing the Mfd-mediated displacement of RNA polymerase stalled at cre sites which act as transcriptional roadblocks. Although the acsA cre is centered 44.5 bp downstream of the acsA transcriptional start site, CCR of acsA expression is not affected by an mfd mutation. When the acsA cre is centered 161.5 bp downstream of the transcriptional start site for the unregulated tms promoter, CCR is partially relieved by the mfd mutation. Since CCR mediated at an acsA cre centered 44.5 bp downstream of the tms start site is not affected by the mfd mutation, the inability of Mfd to modulate CCR of acsA expression most likely results from the location of the acsA cre. Higher levels of CCR were found to occur at cre sites flanked by A+T-rich sequences than at cre sites bordered by G and C nucleotides. This suggests that nucleotides adjacent to the proposed 14-bp cre consensus sequence participate in the formation of the CcpA catabolite repression complex at cre sites. Examination of CCR of acsA expression revealed that this regulation required the Crh and seryl-phosphorylated form of the HPr proteins but not glucose kinase.

Bacillus subtilis↗
PubMed1993

Promoter-independent catabolite repression of the Bacillus subtilis gnt operon.

The mechanism underlying catabolite repression in Bacillus species remains unknown. A recent study of the promoter-independent catabolite repression of the gnt operon implicated a consensus sequence (ATTGAAAG) in catabolite repression in the genus Bacillus. The introduction of base-substitutions into the ATTGAAAG sequence in the chromosomal gnt operon affected catabolite repression of the gnt operon. Deletion analysis indicated that the ATTGAAAG sequence is probably part of a cis sequence necessary for the promoter-independent catabolite repression of the gnt operon. Furthermore, we subjected gnt transcripts synthesized with and without glucose to S1 nuclease and slot blotting analyses. The results indicated that the gnt transcripts decreased in the region (+93 to +203; +1, the transcription initiation nucleotide) only in the presence of glucose. Mechanisms underlying this promoter-independent catabolite repression are discussed.

Bacillus subtilis↗
PubMed1997

Catabolite repression by glucose 6-phosphate, gluconate and lactose in Escherichia coli.

While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIA(Glc) of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of beta-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP* protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these non-PTS carbon sources.

Adenylyl Cyclases↗
PubMed2004

Relief of catabolite repression in a cAMP-independent catabolite gene activator mutant of Escherichia coli.

We isolated and characterized a new catabolite gene activator mutant (crp*) of Escherichia coli that confers cAMP-independent expression and total relief of catabolite repression of beta-galactosidase and tryptophanase synthesis. The two mutations responsible for this phenotype change the amino acids at codon 72 from Glu to Ala and at codon 144 from Ala to Thr in the corresponding CAP* protein.

Cyclic AMP↗
PubMed1998

CcpB, a novel transcription factor implicated in catabolite repression in Bacillus subtilis.

Recent work has shown that in Bacillus subtilis catabolite repression of several operons is mediated by a mechanism dependent on DNA-binding protein CcpA complexed to a seryl-phosphorylated derivative of HPr [HPr(Ser-P)], the small phosphocarrier protein of the phosphoenolpyruvate-sugar phosphotransferase system. In this study, it was found that a transposon insertional mutation resulted in the partial loss of gluconate (gnt) and xylose (xyl) operon catabolite repression by glucose, mannitol, and sucrose. The transposon insertion was localized to a gene, designated ccpB, encoding a protein 30% identical to CcpA, and relief from catabolite repression was shown to be due to the absence of CcpB rather than to the absence of a protein encoded by a downstream gene within the same operon. The relative intensities of CcpA- and CcpB-mediated catabolite repression depended on growth conditions. On solid media, and when cells were grown in liquid media with little agitation, CcpB and CcpA both proved to function in catabolite repression. However, when cells were grown in liquid media with much agitation, CcpA alone mediated catabolite repression. Like CcpA, CcpB appears to exert its catabolite-repressing effect by a mechanism dependent on the presence of HPr(Ser-P).

Amino Acid Sequence↗
PubMed1976

The role of mitochondria in carbon catabolite repression in yeast.

The role of mitochondria in carbon catabolite repression in Saccharomyces cerevisiae was investigated by comparing normal, respiratory competent (RHO) strains with their mitochondrially inherited, respiratory deficient mutant derivatives (rho). Formation of maltase and invertase was used as an indicator system for the effect of carbon catabolite repression on carbon catabolic reactions. Fermentation rates for glucose, maltose and sucrose were the same in RHO and rho strains. Specific activities of maltase and invertase were usually higher in the rho-mutants. A very pronounced difference in invertase levels was observed when cells were grown on maltose; rho-mutants had around 30 times more invertase than their RHO parent strains. The fact that rho-mutants were much less sensitive to carbon catabolite repression of invertase synthesis than their RHO parents was used to search for the mitochondrial factor(s) or function(s) involved in carbon catabolite repression. A possible metabolic influence of mitochondria on this system of regulation was tested after growth of RHO strains under anaerobic conditions (no respiration nor oxidative phosphorylation), in the presence of KCN (respiration inhibited), dinitrophenol (uncoupling of oxidative phosphorylation) and of both inhibitors anaerobic conditions and dinitrophenol had no effect on the extent of invertase repression. KCN reduced the degree of repression but not to the level found in rho-mutants. A combination of both inhibitors gave the same results as with KCN alone. Erythromycin and chloramphenicol were used as specific inhibitors of mitochondrial protein synthesis. Erythromycin prevented the formation of mitochondrial respiratory systems but did not induce rho-mutants under the conditions used. However, repression of invertase was as strong as in the absence of the inhibitor. Chloramphenicol led only to a slight reduction of the respiratory systems and did not affect invertase levels. A combination of both antibiotics had about the same effect as growth in the presence of KCN. The results showed that mitochondria are involved in carbon catabolite repression and they cause an increase in the degree of repression. These effects cannot be due to mere metabolic activities nor to factors made on the mitochondrial protein synthesizing machinery. This regulatory role of mitochondria is observed as long as an intact mitochondrial genome is maintained.

Carbon↗
PubMed1978

Regulation of lac operon expression: reappraisal of the theory of catabolite repression.

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of beta-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3',5'-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of beta-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.

Culture Media↗
PubMed1978

Catabolite repression in Escherichia coli mutants lacking cyclic AMP.

The regulation of catabolite repression of beta-galactosidase has been studied in Escherichia coli mutants deleted for the adenyl cyclase gene (cya delta), and thus unable to synthesize cyclic AMP. It has been found that, provided a second mutation occurs either in the crp gene coding for the catabolite gene activator protein (CAP) or in the Lactose region, these mutants exhibit catabolite repression. If the catabolite repression seen in the mutant strains corresponds to the mechanism operating in wild-type cells the results would suggest that the intracellular concentration of cyclic AMP cannot be the unique regulator of catabolite repression.

Cyclic AMP↗
PubMed1995

Glucose kinase-dependent catabolite repression in Staphylococcus xylosus.

By transposon Tn917 mutagenesis, 16 mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild-type strain. The transposons were found to reside in three adjacent locations in the genome of S. xylosus. The nucleotide sequence of the chromosomal fragment affected by the Tn917 insertions yielded an open reading frame encoding a protein with a size of 328 amino acids with a high level of similarity to glucose kinase from Streptomyces coelicolor. Weaker similarity was also found to bacterial fructokinases and xylose repressors of gram-positive bacteria. The gene was designated glkA. Immediately downstream of glkA, two open reading frames were present whose deduced gene products showed no obvious similarity to known proteins. Measurements of catabolic enzyme activities in the mutant strains grown in the presence or absence of sugars established the pleiotropic nature of the mutations. Besides beta-galactosidase activity, which had been used to detect the mutants, six other tested enzymes were partially relieved from repression by glucose. Reduction of fructose-mediated catabolite repression was observed for some of the enzyme activities. Glucose transport and ATP-dependent phosphorylation of HPr, the phosphocarrier of the phosphoenolpyruvate:carbohydrate phosphotransferase system involved in catabolite repression in gram-positive bacteria, were not affected. The cloned glkA gene fully restored catabolite repression in the mutant strains in trans. Loss of GlkA function is thus responsible for the partial relief from catabolite repression. Glucose kinase activity in the mutants reached about 75% of the wild-type level, indicating the presence of another enzyme in S. xylosus. However, the cloned gene complemented an Escherichia coli strain in glucose kinase. Therefore, the glkA gene encodes a glucose kinase that participates in catabolite repression in S. xylosus.

Amino Acid Sequence↗
PubMed1996

Cyclic AMP-independent catabolite repression in bacteria.

Until recently, only one mechanism of catabolite repression in bacteria, a mechanism dependent on the cyclic AMP receptor protein of Escherichia coli, was understood in molecular detail. Two cyclic AMP-independent catabolite repression mechanisms are currently under study. One such mechanism, found in E. coli, involves the catabolite repressor/activator (Cra) protein (formerly designated the fructose repressor FruR) which represses sugar catabolic systems and activates sugar anabolic systems. When catabolites bind to Cra, Cra dissociates from the DNA causing catabolite activation and catabolite repression, respectively. The second such mechanism, found in Bacillus subtilis, involves a catabolite-activated, ATP-dependent protein kinase that phosphorylates a specific seryl residue in the small phosphocarrier protein, HPr, of the phosphotransferase system. HPr(ser-P) binds to a transcription factor, CcpA, to promote DNA binding. DNA binding of the complex in turn promotes catabolite repression or catabolite activation, depending on the target operon. The characterization of these novel mechanisms establishes that cyclic AMP-independent catabolite control is operative in bacteria, and that multiple mechanisms of catabolite control evolved independently of each other.

Bacillus subtilis↗
PubMed1969

Catabolite repression gene of Escherichia coli.

A catabolite repression gene (cat) which alters the sensitivity of Escherichia coli to catabolite repression has been mapped by transduction and shown to be located between the pyrC and purB genes. When the cat-1 mutation was studied in a number of genetic backgrounds, the results showed that this mutation affects the synthesis of more than one catabolic enzyme but does not completely eliminate catabolic repression under all conditions. It is suggested that this mutation may cause a block in the accumulation of the catabolite effector. Our experiments show that this effector is not glucose-6-phosphate.

Bacterial Proteins↗
PubMed2000

Crc is involved in catabolite repression control of the bkd operons of Pseudomonas putida and Pseudomonas aeruginosa.

Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1972

Polycistronic effects of catabolite repression on the lac operon.

The catabolite repression caused by glucose and glucose-6-phosphate has been studied for both beta-galactosidase and thiogalactoside transacetylase, the products of the operator proximal and distal cistrons of the lac operon, respectively. We find that both cistrons are affected coordinately by this form of repression. We also find that a single alteration at the lac promoter region is sufficient to abolish sensitivity to repression of both cistrons. From this, we conclude that there is only one target site for catabolite repression in the lac operon.

Acetyltransferases↗
PubMed1996

Catabolite repression resistance of gnt operon expression in Bacillus subtilis conferred by mutation of His-15, the site of phosphoenolpyruvate-dependent phosphorylation of the phosphocarrier protein HPr.

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.

Bacillus subtilis↗
PubMed1980

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

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 strains were 4--5 times higher than in ethanol grown cells. Mutants of gene HEX1 had a reduced hexose phosphorylating activity on all media whereas 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 hex1-mutants 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 intermediates. 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.

Carbon↗
PubMed2003

CcpA-dependent carbon catabolite repression in bacteria.

Carbon catabolite repression (CCR) by transcriptional regulators follows different mechanisms in gram-positive and gram-negative bacteria. In gram-positive bacteria, CcpA-dependent CCR is mediated by phosphorylation of the phosphoenolpyruvate:sugar phosphotransferase system intermediate HPr at a serine residue at the expense of ATP. The reaction is catalyzed by HPr kinase, which is activated by glycolytic intermediates. In this review, the distribution of CcpA-dependent CCR among bacteria is investigated by searching the public databases for homologues of HPr kinase and HPr-like proteins throughout the bacterial kingdom and by analyzing their properties. Homologues of HPr kinase are commonly observed in the phylum Firmicutes but are also found in the phyla Proteobacteria, Fusobacteria, Spirochaetes, and Chlorobi, suggesting that CcpA-dependent CCR is not restricted to gram-positive bacteria. In the alpha and beta subdivisions of the Proteobacteria, the presence of HPr kinase appears to be common, while in the gamma subdivision it is more of an exception. The genes coding for the HPr kinase homologues of the Proteobacteria are in a gene cluster together with an HPr-like protein, termed XPr, suggesting a functional relationship. Moreover, the XPr proteins contain the serine phosphorylation sequence motif. Remarkably, the analysis suggests a possible relation between CcpA-dependent gene regulation and the nitrogen regulation system (Ntr) found in the gamma subdivision of the Proteobacteria. The relation is suggested by the clustering of CCR and Ntr components on the genome of members of the Proteobacteria and by the close phylogenetic relationship between XPr and NPr, the HPr-like protein in the Ntr system. In bacteria in the phylum Proteobacteria that contain HPr kinase and XPr, the latter may be at the center of a complex regulatory network involving both CCR and the Ntr system.

Amino Acid Motifs↗
PubMed2007

Carbon catabolite repression of sucrose utilization in Staphylococcus xylosus: catabolite control protein CcpA ensures glucose preference and autoregulatory limitation of sucrose utilization.

Sucrose utilization in Staphylococcus xylosus is dependent on two genes, scrA and scrB; encoding a PTS permease and a sucrose phosphate hydrolase, respectively. The genes are encoded on separate loci and are transcribed from two promoters, P(scrA) and P(scrB), both of which are controlled by the repressor ScrR by binding to the operator sequences O(A) and O(B). In the scrA promoter region, a catabolite-responsive element (cre), operator for the global catabolite control protein CcpA, is also present, but its contribution to scrA regulation has not been determined. Using an integrative promoter probe plasmid, the activities of the promoters P(scrA) and P(scrB) were determined under different growth conditions. Both promoters are induced by sucrose and induction is prevented when glucose is also present. Without a functional CcpA, glucose-mediated prevention of induction is lost, clearly demonstrating that CcpA ensures hierarchical sugar utilization with glucose as preferred substrate. Measurements of promoter activities in the absence of a functional ScrR repressor indicated that CcpA also acts upon the operators O(A) and O(B), albeit not as efficiently as on the genuine cre in P(srcA). Besides determining the choice of the carbon source, CcpA has a second effect on sucrose gene expression. When sucrose is the sole carbon source, sucrose catabolism activates carbon catabolite repression and CcpA prevents full induction of the sucrose utilization genes by partially repressing the scrA promoter. Thus, CcpA-dependent regulation serves as a built-in autoregulatory device to restrict sucrose uptake.

Bacterial Proteins↗