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Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium.

Different pleiotropic transcriptional regulators are known to function in the coordination of regulons concerned with carbon, nitrogen, sulfur, phosphorus and iron metabolism, but how expression profiles of these different regulons are coordinated with each other is not known. The basis for the effects of cysB mutations on carbon utilization in Escherichia coli and Salmonella typhimurium was examined. cysB mutations affected the utilization of some carbon sources more than others and these effects could be partially, but not completely, reversed by the inclusion of cysteine or djenkolate in the growth medium. Assays of transport systems and enzymes concerned with glucitol and alanine utilization showed that these activities were depressed in cysB mutants relative to isogenic wild-type strains, and cysteine or djenkolate present in the growth media partially restored these activities. Using transcriptional fusions to the fdo (formate dehydrogenase) and gut (glucitol) operons, it was shown that decreased expression resulted from defects at the transcriptional level. Furthermore, the effects of loss of CysB were much less pronounced under conditions of catabolite repression than in the absence of a catabolite-repressing carbon source, and cAMP largely reversed the effect of the loss of CysB. Comparable effects were seen for E. coli lacZ gene expression under the control of its own native promoter, and sulfur limitation in a cysB mutant depressed net cAMP production in a cAMP phosphodiesterase mutant. Adenylate cyclase thus appears to be responsive to sulfur deprivation. These observations may have physiological significance allowing carbon and sulfur regulon coordination during the growth of enteric bacteria in response to nutrient availability.

Adenylyl Cyclases↗

Regulation of pectate lyase synthesis in Pseudomonas fluorescens and Erwinia carotovora.

Inducible synthesis of extracellular pectate lyase occurs in Erwinia carotovora, a bacterial soft-rot pathogen of plants, and, to a lesser extent, in a nonpathogenic isolate of Pseudomonas fluorescens. A combination of pectin and a heat-labile factor in fresh potato tissue or acetone powders of the tissue provided the best carbon source for induction. Yields of inducible pectate lyase were much greater than those usually reported. The pathogen, but not the saprophyte, produced a small amount of constitutive enzyme when grown on glucose. The relatively low level or absence of constitutive synthesis in these bacteria did not result from catabolite repression. Attempts were made to relieve any existing catabolite repression by restricting growth through slow feeding of glucose or by growing the organisms on glycerol. These conditions did not significantly alter the differential rate of lyase synthesis compared with changes observed in the presence of inducers. Previous growth history did not affect induction in the pathogen. However, P. fluorescens previously cultured on glucose required 10 to 20 generations of growth on inducing medium before appreciable lyase synthesis occurred. Differences between the pathogen and nonpathogen suggest that regulation of pectate lyase synthesis is related to pathogenicity of soft-rot bacteria.

Culture Media↗

Identification of a co-repressor binding site in catabolite control protein CcpA.

The catabolite control protein CcpA is the central regulator of carbon catabolite repression in Bacilli and other Gram-positive bacteria. A comparison of 12 CcpA-like sequences with regulators from the LacI/GalR family defines a CcpA subfamily based on extensive similarities found among CcpAs and not in 32 other members of the family. These amino acids are clustered in three blocks in the CcpA sequence. Their interpretation, assuming a PurR-like fold, reveals that almost all of them are surface exposed and form a continuous patch on the N-terminal subdomain of the protein core extending into the DNA reading head. We introduced nine single amino acid exchanges in the subfamily specific residues of CcpA from Bacillus megaterium. Six mutants, namely CcpA47RS, 79AE, 89YE, 295YR, 299YE and 303RD, are inactive or severely impaired in catabolite repression, underlining their relevance for CcpA function. They are negatively transdominant over wild-type CcpA demonstrating their ability to correctly fold for dimerization. Five of them are unable or impaired in binding HPr-Ser-46-P in vitro, establishing a correlation between catabolite repression efficiency and HPr-Ser-46-P binding. These results support the hypothesis that the conserved region in CcpA is the HPr-Ser-46-P binding site.

Amino Acid Sequence↗

Fructose-1,6-bisphosphatase of the yeast Kluyveromyces lactis.

The fructose-1,6-bisphosphatase [Fru(1,6)P2ase] gene of the budding yeast, Kluyveromyces lactis, was cloned and sequenced. The gene encodes one open reading frame predicting a 354-amino-acid polypeptide. The polypeptide is different from other Fru(1,6)P2ases in that it contains a short amino-acid-insert region close to a basic residue located at the binding site for the allosteric inhibitor AMP. Comparison of the biochemical properties of the K. lactis enzyme with its closest homolog, the Saccharomyces cerevisiae Fru(1,6)P2ase (74% amino acid identity), reveals that the K. lactis enzyme is significantly less sensitive to AMP (Ki = 540 microM) than the S. cerevisiae enzyme (Ki = 190 microM). However, studies with a K. lactis Fru(1,6)P2ase mutant, in which the insert region (amino acids 152-160) was deleted by site-directed mutagenesis [(des-152-160)Fru(1,6)P2ase], showed that the mutant enzyme had higher sensitivity to AMP inhibition (Ki = 280 microM) than the control K. lactis enzyme. Thus, the nine-amino-acid insert region appears to be responsible for the decreased AMP inhibition shown by the K. lactis wild-type enzyme. Catabolite-repression and catabolite-inactivation studies show that, unlike the complete repression of FBP1 mRNA and inactivation of enzyme activity by glucose seen in S. cerevisiae, mRNA levels and enzyme activity of K. lactis Fru(1,6)P2ase decreased only about 2-4-fold due to the presence of glucose in the cell-culture medium.

Amino Acid Sequence↗

Glucose effect in tgl mutant of Escherichia col K12 defective in methyl-alpha-D-glucoside transport.

1. The dependence of the rate of accumulation of methyl-alpha-D-glucoside on its extracellular concentration was studied in the tgl mutant of Escherichia coli K12, isolated earlier. It has been shown that the kinetics of methyl-alpha-D-glucoside transport differ sharply from those in wild-type bacteria. 2. The beta-galactosidase synthesis in tgl strain is much less sensitive both to permanent and transient glucose catabolite repression. The level of cyclic AMP in mutant cells under the conditions of glucose catabolite repression is several times higher than in the parent strain. 3. The tgl mutation does not affect the manifestation of catabolite inhibition and inducer exclusion with glucose. 4. The data obtained are discussed in the light of a hypothesis concerning the existence of two sites, binding and pecific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system. The tgl mutation alters the first site, and the second one is damaged by the pgt mutation. 5. It is suggested that the products of the tgl and gpt genes are necessary for the manifestation of the phenomena of glucose permanent and transient repression. The effects of catabolite inhibition and inducer exclusion are realized irrespective of the existence or absence of the tgl product.

Amino Acids↗

Regulation of histidase synthesis in intergeneric hybrids of enteric bacteria.

Regulation of the expression of the histidase coded by hutk of Klebsiella aerogenes in Salmonella typhimurium and in Escherichia coli and of the expression of the histidase coded by huts of S. typhimurium in E. coli was investigated. The hutk histidase was found to be sensitive to catabolite repression in K. aerogenes and in E. coli, but insensitive to catabolite repression in S. typhimurium; huts histidase has previously been shown to be catabolite sensitive in all three organisms. The expression of both hutk and huts histidase in E. coli was activated by nitrogen starvation. Apparently, the glutamine synthetase of E. coli may activate the formation of some glutamate- and ammonia-producing enzymes.

Ammonia-Lyases↗

Regulation of alcR, the positive regulatory gene of the ethanol utilization regulon of Aspergillus nidulans.

The alcR positive control gene is necessary for the expression of both alcA (coding for alcohol dehydrogenase ADH I), and aldA (coding for aldehyde dehydrogenase, AldDH) in Aspergillus nidulans. Using a cloned alcR probe and Northern blots analysis we show that: (1) alcR itself is inducible; (2) alcR inducibility depends on the expression of the alcR gene itself; and (3) alcR is subject to carbon catabolite repression and its expression is controlled by the negatively acting creA wide specificity gene. The repression of alcR is sufficient to explain the carbon catabolite repression of ADH I and AldDH.

Alcohol Dehydrogenase↗

Genetics of L-sorbose transport and metabolism in Lactobacillus casei.

Genes encoding L-sorbose metabolism of Lactobacillus casei ATCC 393 have been identified on a 6.8-kb chromosomal DNA fragment. Sequence analysis revealed seven complete genes and a partial open reading frame transcribed as two units. The deduced amino acid sequences of the first transcriptional unit (sorRE) showed high similarity to the transcriptional regulator and the L-sorbose-1-phosphate reductase of the sorbose (sor) operon from Klebsiella pneumoniae. The other genes are transcribed as one unit (sorFABCDG) in opposite direction to sorRE. The deduced peptide sequence of sorF showed homology with the D-sorbitol-6-phosphate dehydrogenase encoded in the sor operon from K. pneumoniae and sorABCD to components of the mannose phosphotransferase system (PTS) family but especially to domains EIIA, EIIB, EIIC and EIID of the phosphoenolpyruvate-dependent L-sorbose PTS from K. pneumoniae. Finally, the deduced amino acid sequence of a truncated gene (sorG) located downstream of sorD presented high similarity with ketose-1,6-bisphosphate aldolases. Results of studies on enzyme activities and transcriptional analysis revealed that the two gene clusters, sorRE and sorFABCDG, are induced by L-sorbose and subject to catabolite repression by D-glucose. Data indicating that the catabolite repression is mediated by components of the PTS elements and by CcpA, are presented. Results of sugar uptake assays in L. casei wild-type and sorBC mutant strains indicated that L-sorbose is taken up by L-sorbose-specific enzyme II and that L. casei contains an inducible D-fructose-specific PTS. Results of growth analysis of those strains and a man sorBC double mutant suggested that L-sorbose is probably also transported by the D-mannose PTS. We also present evidence, from studies on a sorR mutant, suggesting that the sorR gene encodes a positive regulator of the two sor operons. Sequence alignment of SorR, SorC (K. pneumoniae), and DeoR (Bacillus subtilis) revealed that they might constitute a new group of transcriptional regulators.

Amino Acid Sequence↗

The regulation of ubiquinone-6 biosynthesis by Saccharomyces cerevisiae.

Increasing concentrations of glucose (1-5%) in the growth medium depressed ubiquinone-6 biosynthesis in continuously cultured wild type Saccharomyces cerevisiae. In addition, an early intermediate in the pathway of ubiquinone-6 biosynthesis, i.e. 3,4-dihydroxy-5-hexaprenylbenzoate (3,4-DHHB), was found to accumulate. The increase in 3,4-DHHB levels varied inversely with the diminished levels of ubiquinone-6, suggesting that O-methylation of 3,4-DHHB is a regulated step in catabolite repression. Experiments using protoplasts demonstrated that the effect of catabolite repression on this pathway was reversible by 1.2 mM cAMP but not by other nucleotides and cyclic nucleotides. This response to cAMP was unaltered by the protein synthesis inhibitor cycloheximide, indicating that the regulatory control for this reaction must occur at the enzymatic level. Additional experiments demonstrated the presence of a heat-labile component of the cytoplasm, which was essential for this effect of cAMP. This observation suggests that this cytosolic effector may be translocated to the inner membrane of the mitochondria, the intracellular site for ubiquinone-6 biosynthesis.

Cyclic AMP↗

A yeast gene that is essential for release from glucose repression encodes a protein kinase.

The SNF1 gene plays a central role in carbon catabolite repression in the yeast Saccharomyces cerevisiae, namely that SNF1 function is required for expression of glucose-repressible genes. The nucleotide sequence of the cloned SNF1 gene was determined, and the predicted amino acid sequence shows that SNF1 encodes a 72,040-dalton polypeptide that has significant homology to the conserved catalytic domain of mammalian protein kinases. Specific antisera were prepared and used to identify the SNF1 protein. The protein was shown to transfer phosphate from adenosine triphosphate to serine and threonine residues in an in vitro autophosphorylation reaction. These findings indicate that SNF1 encodes a protein kinase and suggest that protein phosphorylation plays a critical role in regulation by carbon catabolite repression in eukaryotic cells.

Amino Acid Sequence↗

Priority of pentose utilization at the level of transcription: arabinose, xylose, and ribose operons.

When E. coli cells were grown in minimal medium supplemented with D-ribose and D-xylose, a diauxic growth preferring D-xylose was observed. Transcription of the ribose (rbs) operon was repressed in the presence of D-xylose, phenotypically similar to catabolite repression by D-glucose, although D-ribose did not affect transcription of the xylose (xyl) operon. Complementation analysis with xylR revealed that the repression of the rbs operon by D-xylose is exerted at the transcriptional level through XylR, suggesting a novel mechanism for catabolite repression. Furthermore, it was shown that L-arabinose reduced transcriptions of both xyl and rbs operons, whereas the arabinose operon was not affected by D-xylose or D-ribose, suggesting a priority mechanism for pentose utilization.

Arabinose↗

Elements from the cAMP signaling pathway are involved in the control of expression of the yeast gluconeogenic gene FBP1.

cAMP represses the transcription of some Saccharomyces cerevisiae genes sensitive to catabolite repression. The effect of cAMP on the expression of FBP1, encoding fructose-1,6-bisphosphatase (FbPase), has been further investigated. In yeast cells shifted to a derepressing medium, synthesis of FbPase was delayed if the strong decrease in intracellular cAMP, which occurs during the shift, was prevented. A similar delay occurred in a RAS2val19 strain, while in a tpk1w strain, with weak protein kinase A activity, induction of FbPase occurred earlier than in a TPK1 strain. In the tpk1w strain, proteins which bind the UAS1 element of FBP1 were present during growth on glucose but they were only weakly operative. Expression of CAT8 and SIP4, encoding proteins which bind the UAS2 element, was blocked by a high concentration of cAMP, but catabolite repression of these genes was not much relieved in a tpk1w strain. We conclude that in S. cerevisiae, as reported for Schizosaccharomyces pombe, control of FBP1 requires both cAMP-dependent and independent pathways; however, the mechanisms operating in the two yeasts are different.

Cyclic AMP↗

Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans.

Acetate inducible genes of Aspergillus nidulans were cloned via differential hybridization to cDNA probes. Using transformation of mutant strains the genes were identified as facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase). The levels of RNA encoded by these genes were shown to be acetate inducible and subject to carbon catabolite repression. Induction is abolished in a facB mutant and carbon catabolite repression is relieved in a creA mutant.

Acetate-CoA Ligase↗

[Knockout of the ccpA gene in Bacillus subtilis and influence on riboflavin production].

Bacillus subtilis uses glucose as preferred source of carbon and energy, but expression of many genes involved in central pathways is often repressed or activated in the presence of glucose. This phenomenon is called carbon catabolite repression (CCR) in B. subtilis, which is mainly mediated by the global regulator CcpA encoded by ccpA gene. In the present of glucose, the genes encoding glycolytic enzymes are induced; the genes responsible for the production of acetate from pyruvate are induced also, however, the genes required for Krebs cycle are repressed. Sometimes, this leads to lower production in fed-batch fermentation. In this study, ccpA gene of the riboflavin producing train B. subtilis24/ pMX45 was disrupted using homologous recombination technology and a ccpA mutant B. subtilis24A1/pMX45 was constructed. The comparison of shake-flask fermentation indicates B. subtilis24A1/pMX45 could grow normally in culture medium containing excess glucose; and the ratio of glucose consumption increased remarkably, for example, 10% glucose can be consumed within 70 hours, biomass increased to 1.5 x 10(9)cell/mL; at the same time the accumulation amount of overflow metabolite decreases. Especially, when fermenting with culture medium containing 8% and 10% glucose respectively, the yields of riboflavin increase 62% and 95% than B. subtilis24/pMX45. These results demonstrate knockout of ccpA gene can relieve carbon catabolite repression and lead to the alteration of central catabolite flux in favor of synthesis of riboflavin, so the yield of riboflavin can be enhanced significantly.

Bacillus subtilis↗

Role of the DNA sequence downstream of the Bacillus subtilis hut promoter in regulation of the hut operon.

To identify the role of the downstream region of a hut promoter in regulation of the Bacillus subtilis hut operon, three single-base substitutions (+9G-->A, +14C-->T, and +23T-->G) were introduced into the hut operon. Analysis of expression of the hut operon containing each of these three single-base substitutions and the hut-lacZ fusions with the single-base substitutions at position +14 showed that the position at +14 and probably the position at +23 were required for amino acid repression at the hut promoter, while the position at +14 was not required for catabolite repression at the hut promoter. The position at +9 was required for a histidine-dependent increase of activity of the hut promoter. Analysis of expression of the hut-lacZ fusions and the hut operon in the codY mutant indicated that the position at +14 and probably the position at +23 were involved in CodY-mediated amino acid repression at the hut promoter and that CodY was not required for catabolite repression at the hut promoter.

Bacillus subtilis↗

Genomic variation in Streptococcus mutans: deletions affecting the multiple pathways of beta-glucoside metabolism.

The genome of Streptococcus mutans UA159 contains two phospho-beta-glucosidase genes, bglA and celA, which occur in operon-like arrangements along with genes for components of phosphotransferase transport systems and a third phospho-beta-glucosidase encoded by the arb gene, which does not have its own associated transport system but relies on uptake by the bgl or cel systems. Targeted inactivation of each of the phospho-beta-glucosidase genes revealed that bglA is involved in aesculin hydrolysis, celA is essential for utilisation of cellobiose, amygdalin, gentobiose and salicin, and arb is required for utilisation of arbutin. Inactivation of genes for the phosphotransferase systems revealed an overlap of specificity for transport of beta-glucosides and also indicated that further, unidentified transport systems exist. The cel and arb genes are subject to catabolite repression by glucose, but the regM gene is not essential for catabolite repression. Screening a collection of isolates of S. mutans revealed strains with deletions affecting the msm, bgl and/or cel operons. The phenotypes of these strains could largely be explained on the basis of the results obtained from the knockout mutants of S. mutans UA159 but also indicated the existence of other pathways apparently absent from UA159. The extensive genetic and phenotypic variation found in beta-glucoside metabolism indicates that there may be extensive heterogeneity in the species.

Amygdalin↗

Mutational analysis of the bglH catabolite-responsive element (cre) in Lactobacillus plantarum.

Catabolite repression of the Lactobacillus plantarum bglH gene is mediated by the catabolite control protein A (CcpA). Here we show that the binding site for the protein is a catabolite-responsive element (cre) sequence overlapping the start site of transcription. Two single and one double base substitutions in the cre sequence caused derepression of a plasmid-borne bglH-cat transcriptional fusion in L. plantarum cells grown on glucose. Analysis of the single mutations indicates that the G and C nucleotide residues in positions 2 and 13, respectively, of the 14-bp cre sequence are required for catabolite repression.

Bacterial Proteins↗

Dual role for N-2-acetylornithine 5-aminotransferase from Pseudomonas aeruginosa in arginine biosynthesis and arginine catabolism.

In Pseudomonas aeruginosa N-2-acetylornithine 5-aminotransferase (ACOAT), the fourth enzyme of arginine biosynthesis is induced about 15-fold by cultivating the organism on a medium with L-arginine as the sole carbon and nitrogen source. Synthesis of the enzyme is subject to catabolite repression and nitrogen source. Synthesis of the enzyme is subject to catabolite repression by a variety of carbon sources. ACOAT from strain PAO 1 was purified over 40-fold to electrophoretic homogeneity. A molecular weight of approximately 110,000 was obtained by thin-layer gel filtration. Electrophoresis in sodium dodecyl sulfate gels gave a single band corresponding to a molecular weight of 55,000. Purified ACOAT catalyzes the transamination of N-2-acetyl-L-ornithine as well as of L-ornithine with 2-oxoglutarate (Km values of 1.1, 10.0, and 0.7 mM, respectively). With N-2-acetyl-L-ornithine as amino donor, the pH-optimum of the enzymatic reaction is 8.5; with L-ornithine as amino donor, 9.5. The catalytic properties of ACOAT as well as the regulation of its synthesis indicate that in P. aeruginosa this enzyme functions in the biosynthesis as well as in the catabolism of L-arginine.

Arginine↗