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PubMed1998

Transcriptional regulation and organization of the dcuA and dcuB genes, encoding homologous anaerobic C4-dicarboxylate transporters in Escherichia coli.

The dcuA and dcuB genes of Escherichia coli encode homologous proteins that appear to function as independent and mutually redundant C4-dicarboxylate transporters during anaerobiosis. The dcuA gene is 117 bp downstream of, and has the same polarity as, the aspartase gene (aspA), while dcuB is 77 bp upstream of, and has the same polarity as, the anaerobic fumarase gene (fumB). To learn more about the respective roles of the dcu genes, the environmental and regulatory factors influencing their expression were investigated by generating and analyzing single-copy dcuA- and dcuB-lacZ transcriptional fusions. The results show that dcuA is constitutively expressed whereas dcuB expression is highly regulated. The dcuB gene is strongly activated anaerobically by FNR, repressed in the presence of nitrate by NarL, and subject to cyclic AMP receptor protein (CRP)-mediated catabolite repression. In addition, dcuB is strongly induced by C4-dicarboxylates, suggesting that dcuB is under the control of an uncharacterized C4-dicarboxylate-responsive gene regulator. Northern blotting confirmed that dcuA (and aspA) is expressed under both aerobic and anaerobic conditions and that dcuB (and fumB) is induced anaerobically. Major monocistronic transcripts were identified for aspA and dcuA, as well as a minor species possibly corresponding to an aspA-dcuA cotranscript. Five major transcripts were observed for dcuB and fumB: monocistronic transcripts for both fumB and dcuB; a dcuB-fumB cotranscript; and two transcripts, possibly corresponding to dcuB-fumB and fumB mRNA degradation products. Primer extension analysis revealed independent promoters for aspA, dcuA, and dcuB, but surprisingly no primer extension product could be detected for fumB. The expression of dcuB is entirely consistent with a primary role for DcuB in mediating C4-dicarboxylate transport during anaerobic fumarate respiration. The precise physiological purpose of DcuA remains unclear.

Amino Acid Sequence↗
PubMed1986

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.

Amidohydrolases↗
PubMed1994

Glucitol induction in Bacillus subtilis is mediated by a regulatory factor, GutR.

Expression of the glucitol dehydrogenase gene (gutB) is suggested to be regulated both positively and negatively in Bacillus subtilis. A mutation in the gutR locus results in the constitutive expression of gutB. The exact nature of this mutation and the function of gutR are still unknown. Cloning and characterization of gutR indicated that this gene is located immediately upstream of gutB and is transcribed in the opposite direction relative to gutB. GutR is suggested to be a 95-kDa protein with a putative helix-turn-helix motif and a nucleotide binding domain at the N-terminal region. At the C-terminal region, a short sequence of GutR shows homology with two proteins, Cyc8 (glucose repression mediator protein) and GsiA (glucose starvation-inducible protein), known to be directly or indirectly involved in catabolite repression. Part of the C-terminal conserved sequence from these proteins shows all the features observed in the tetratricopeptide motif found in many eucaryotic proteins. To study the functional role of gutR, chromosomal gutR was insertionally inactivated. A total loss of glucitol inducibility was observed. Reintroduction of a functional gutR to the GutR-deficient strain through integration at the amyE locus restores the inducibility. Therefore, GutR serves as a regulatory factor to modulate glucitol induction. The nature of the gutR1 mutation was also determined. A single amino acid change (serine-289 to arginine-289) near the putative nucleotide binding motif B in GutR is responsible for the observed phenotype. Possible models for the action of GutR are discussed.

Amino Acid Sequence↗
PubMed1998

Transcriptional activation of the Bacillus subtilis ackA gene requires sequences upstream of the promoter.

Transcriptional activation of the Bacillus subtilis ackA gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the ackA promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in B. subtilis and other gram-positive bacteria, recognizes a cis-acting consensus sequence, designated cre (catabolite response element), generally located within or downstream of the promoter of the repressed gene. Two sites resembling this sequence are centered at positions -116.5 and -56.5 of the ackA promoter and have been termed cre1 and cre2, respectively. Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose. In this study, cre2, the site closer to the promoter, and the region upstream of cre2 were shown to be indispensable for CcpA-dependent transcriptional activation of ackA, whereas cre1 was not required. In addition, insertion of 5 bp between cre2 and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of cre2 and/or upstream sequences. DNase footprinting experiments demonstrated binding of CcpA in vitro to cre2 but not cre1, consistent with the genetic data. Activation of ackA transcription was blocked in a ptsH1/crh double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.

Acetate Kinase↗
PubMed1970

Transient repression of the lac operon - the effect of a lac promoter deletion.

Experiments have been done to show whether the lac promoter delection L1, which partly alleviates catabolite repression, also affects transient repression of lac. In stain L1/F'M15 all of the beta-galactosidase is synthesized from a chromosomal gene cis to L1, whereas 98% of the thiogalactosidase transacetylase is synthesized from an episomal gene cis to an intact i-p-o region. The addition of glucose to induced cultures of strain L1/F'M15 growing in glycerol medium caused extensive transient repression of transacetylase but almost no transient repression of beta-galactosidase. In control experiments with a diploid stain of genotype p(+)z(+)a(-)/F'p(+)z(-)a(+) the two enzymes suffered equal transient repression. Thus L1 substantially relieves transient repression.

Journal Article↗
PubMed1982

Enzymes of arginine utilization and their formation in Aeromonas formicans NCIB 9232.

In Aeromonas formicans two inducible catabolic pathways of L-arginine have been characterized. The arginine decarboxylase is induced by arginine which also induces the three enzymes of the arginine deiminase pathway but only in stress conditions such as a shift from aerobic growth conditions to very low oxygen tension. Addition of glucose to medium containing arginine leads to repression of the enzymes involved in the arginine deiminase pathway while exogenous cAMP prevents that repression of enzyme synthesis by glucose. This suggests that the induction of arginine deiminase pathway is regulated by carbon catabolite repression and the energetic state of the cell.

Adenosine Triphosphate↗
PubMed1999

Characterization of a prolidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 with an unusual regulation of biosynthesis.

Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 (Lb. bulgaricus) is characterized by a high level of peptidase activities specific to proline-containing peptides. A prolidase (PepQ, EC 3.4.13.9) was purified to homogeneity and characterized as a strict dipeptidase active on X-Pro dipeptides, except Gly-Pro and Pro-Pro. The values for Km and Vmax were, respectively, 2.2 mM and 0.33 mmol min(-1) mg(-1), with Leu-Pro as the substrate. The enzyme exhibited optimal activity at 50 degrees C and pH 6.0, and required the presence of Zn2+. Size exclusion chromatographies and SDS-PAGE analysis led to the conclusion that this prolidase was a homodimer. Antibodies raised against the purified protein allowed the detection of PepQ among several Lactobacillus species but not lactococci. The pepQ gene and the upstream region were isolated and sequenced. The deduced peptide sequence showed that PepQ belongs to the M24 family of metallopeptidases. The pepR1 gene is located immediately upstream of pepQ and its product is homologous to the transcription factor CcpA, which is involved in catabolite repression of catabolic operons from Gram-positive bacteria. The pepR1-pepQ intergenic region contains a consensus catabolite-responsive element (CRE) which could be a target for PepR1 protein. Moreover, in contrast to other proline-specific enzymes from Lb. bulgaricus, PepQ biosynthesis was shown to be dependent on the composition of the culture medium, but not on the peptide concentration. A possible regulation mechanism is discussed.

Amino Acid Sequence↗
PubMed1973

Regulation of Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase in Germinated Spores of Geotrichum candidum.

Germinating spores of Geotrichum candidum produce only a nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase. Synthesis of glutamate dehydrogenase was repressed by the presence of ammonia, whereas urea, glutamate, or glutamine were ineffective. The enzyme was not subject to catabolite repression and was localized in the cell sap fraction. The glutamate dehydrogenase has been purified 93-fold and showed maximal activity at pH 8.2 in the forward and reverse directions. When measuring the initial reaction rate at pH 7.2, a variety of tricarboxylic acid cycle intermediates displayed additive and unidirectional activation of the reductive amination reaction and inhibition of the oxidative deamination reaction. The modulating effects were pH-dependent and diminished at alkaline pH values. Substrate inhibition exerted by alpha-ketoglutarate was strongest at neutral pH.Rate-concentration plot with respect to alpha-ketoglutarate at pH 7.2 was sigmoidal, and the enzyme was entirely inactive at substrate concentrations lower than 1 mm. In the presence of fumarate, the enzyme did not exhibit the cooperative interaction described above and was markedly activated at alpha-ketoglutarate concentrations lower than 1 mm. The tricarboxylic acid cycle intermediates released the enzyme from substrate inhibition by alpha-ketoglutarate. Double reciprocal rate-concentration plots displayed noncompetitive inhibition between succinate and NADP and a mixed noncompetitive and competitive inhibition between succinate and glutamate. The binding order of substrates in the reductive amination reaction was NADPH, alpha-ketoglutarate, and subsequently ammonia.

Journal Article↗
PubMed2001

Regulation of the glv operon in Bacillus subtilis: YfiA (GlvR) is a positive regulator of the operon that is repressed through CcpA and cre.

Maltose metabolism and the regulation of the glv operon of Bacillus subtilis, comprising three genes, glvA (6-phospho-alpha-glucosidase), yfiA (now designated glvR), and glvC (EIICB transport protein), were investigated. Maltose dissimilation was dependent primarily upon the glv operon, and insertional inactivation of either glvA, glvR, or glvC markedly inhibited growth on the disaccharide. A second system (MalL) contributed to a minor extent to maltose metabolism. Northern blotting revealed two transcripts corresponding to a monocistronic mRNA of glvA and a polycistronic mRNA of glvA-glvR-glvC. Primer extension analysis showed that both transcripts started at the same base (G) located 26 bp upstream of the 5' end of glvA. When glvR was placed under control of the spac promoter, expression of the glv operon was dependent upon the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). In regulatory studies, the promoter sequence of the glv operon was fused to lacZ and inserted into the amyE locus, and the resultant strain (AMGLV) was then transformed with a citrate-controlled glvR plasmid, pHYCM2VR. When cultured in Difco sporulation medium containing citrate, this transformant [AMGLV(pHYCM2VR)] expressed LacZ activity, but synthesis of LacZ was repressed by glucose. In an isogenic strain, [AMGLVCR(pHYCM2VR)], except for a mutation in the sequence of a catabolite-responsive element (cre), LacZ activity was expressed in the presence of citrate and glucose. Insertion of a citrate-controlled glvR plasmid at the amyE locus of ccpA(+) and ccpA mutant organisms yielded strains AMCMVR and AMCMVRCC, respectively. In the presence of both glucose and citrate, AMCMVR failed to express the glv operon, whereas under the same conditions high-level expression of both mRNA transcripts was found in strain AMCMVRCC. Collectively, our findings suggest that GlvR (the product of the glvR gene) is a positive regulator of the glv operon and that glucose exerts its effect via catabolite repression requiring both CcpA and cre.

Bacillus subtilis↗
PubMed1992

Improving the production of E. coli beta-lactamase in Bacillus subtilis: the effect of glucose, pH and temperature on the production level.

Bacillus subtilis has been considered a promising host for the production of foreign proteins. However, proteases released by the host organism can often cause rapid breakdown of secreted heterologous proteins. Here we report that the addition of 6% glucose and 100 mM potassium phosphate to the growth medium significantly reduces the degradation of E. coli TEM beta-lactamase secreted from B. subtilis, when applying an expression system based on B. amyloliquefaciens alpha-amylase. The yield of beta-lactamase was increased 10-20-fold when compared to the yield in Luria medium. The promoter of B. amyloliquefaciens alpha-amylase gene is repressed by glucose. However, here we show that the repression does not take place in a multicopy plasmid, thus enabling our approach to efficiently reduce the protease action by catabolite repression. We have also studied the role of pH and temperature on the beta-lactamase production in laboratory scale bioreactors. Low temperature and low pH are both favorable for a high level beta-lactamase production by the high copy plasmid construction.

Bacillus subtilis↗
PubMed1989

Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants.

The cell wall-degrading enzymes polygalacturonase and pectate lyase have been suggested to be crucial for penetration and colonization of plant tissues by some fungal pathogens. We have found that Aspergillus nidulans (= Emericella nidulans), a saprophytic Ascomycete, produces levels of these enzymes equal to those produced by soft-rotting Erwinia species. Induction of polygacturonase and pectate lyase in A. nidulans requires substrate and is completely repressed by glucose. Surprisingly, inoculation of excised plant tissues with A. nidulans conidia leads to formation of necrotic, water-soaked lesions within which the organism sporulates. Thus, A. nidulans has phytopathogenic potential. The release of glucose and other sugars from wounded tissues may repress pectolytic enzyme production and limit disease development. Therefore, we tested creA204, a mutation that relieves glucose repression of some A. nidulans carbon utilization enzymes, for its effect on production of pectolytic enzymes. creA204 failed to relieve catabolite repression of polygalacturonase or pectate lyase and had no effect on disease severity.

Aspergillus nidulans↗
PubMed2001

Reverse genetics of Escherichia coli glycerol kinase allosteric regulation and glucose control of glycerol utilization in vivo.

Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of Escherichia coli glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (formerly known as III(glc)), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the glpK gene. Genetics methods based on homologous recombination are used to place glpK alleles with known specific mutations into the chromosomal context of the glpK gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. The E. coli strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different glpR alleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the glpK gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIA(Glc) inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.

Allosteric Regulation↗
PubMed1988

Cellular location of asparaginase activity in Saccharomyces cerevisiae and regulation of this activity by nitrogen compounds.

Three asparaginase activities have been detected in Saccharomyces cerevisiae. One is found outside the permeability barrier; a second one is found inside and is soluble in the cell, and the third one is localized in a system of membrane particles. Synthesis of the membrane and external asparaginases require "de novo" synthesis of RNA and protein. The synthesis of exocellular asparaginase is inhibited by several nitrogen compounds (catabolite repression). This inhibition might take place at the transcriptional level. Moreover, this isoenzyme is reversibly inactivated by its natural substrates (catabolite inhibition by substrates). The half life of external asparaginase mRNA was calculated by two independent methods and values of 7.5 and 9.5 min were found.

Asparaginase↗
PubMed1989

Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae.

The yeast PCK1 gene coding for phosphoenolpyruvate carboxykinase (PEPCK) was isolated by functional complementation of pck1 strains from S. cerevisiae. Only one copy of the gene was found per haploid yeast genome. An RNA of about 2 kb which hybridized with a DNA probe internal to the PCK1 gene was found only in cells growing in non-fermentable carbon sources. Yeast strains carrying multiple copies of the PCK1 gene showed normal catabolite repression of PEPCK except those carrying the shortest insertion complementing the mutation (2.2 kb) that presented an altered kinetics of derepression. Catabolite inactivation was decreased in strains transformed with multicopy plasmids carrying the PCK1 gene.

Blotting, Southern↗
PubMed2007

The phosphotransferase system of Lactobacillus casei: regulation of carbon metabolism and connection to cold shock response.

Genome sequencing of two different Lactobacillus casei strains (ATCC334 and BL23) is presently going on and preliminary data revealed that this lactic acid bacterium possesses numerous carbohydrate transport systems probably reflecting its capacity to proliferate under varying environmental conditions. Many carbohydrate transporters belong to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but all different kinds of non-PTS transporters are present as well and their substrates are known in a few cases. In L. casei regulation of carbohydrate transport and carbon metabolism is mainly achieved by PTS proteins. Carbon catabolite repression (CCR) is mediated via several mechanisms, including the major P-Ser-HPr/catabolite control protein A (CcpA)-dependent mechanism. Catabolite response elements, the target sites for the P-Ser-HPr/CcpA complex, precede numerous genes and operons. PTS regulation domain-containing antiterminators and transcription activators are also present in both L. casei strains. Their activity is usually controlled by two PTS-mediated phosphorylation reactions exerting antagonistic effects on the transcription regulators: P~EIIB-dependent phosphorylation regulates induction of the corresponding genes and P~His-HPr-mediated phosphorylation plays a role in CCR. Carbohydrate transport of L. casei is also regulated via inducer exclusion and inducer expulsion. The presence of glucose, fructose, etc. leads to inhibition of the transport or metabolism of less favorable carbon sources (inducer exclusion) or to the export of accumulated non-metabolizable carbon sources (inducer expulsion). While P-Ser-HPr is essential for inducer exclusion of maltose, it is not necessary for the expulsion of accumulated thio-methyl-beta-D-galactopyranoside. Surprisingly, recent evidence suggests that the PTS of L. casei also plays a role in cold shock response.

Biological Transport↗
PubMed2004

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation.

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate--to--moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 x 106 spores/10 g of wheat bran at a water activity (aw) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.

Bioreactors↗
PubMed1983

Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region.

The regulatory region of the cya gene from Escherichia coli has been characterized by nucleotide sequence analysis and genetic approaches. Two promoters, P1 and P2, organized in that order with respect to the beginning of the cya open reading frame, were identified. Using cya-lac operon and protein fusions, it was possible to show that both promoters are active in vivo. P1 activity seemed sensitive to catabolite repression whereas activity of the stronger promoter, P2, did not respond to inhibition by glucose. No effect of cAMP or its receptor, catabolite activator protein (CAP), could be found although the DNA sequence reveals a consensus CAP site downstream of P2. The 548 nucleotides situated at the 3' end of the sequence carry an open reading frame which can tentatively be assigned to the beginning of adenylate cyclase. Among noteworthy features of the corresponding sequence are an UUG codon as the putative start site of cyclase, and a long hydrophobic stretch of amino acids resembling leader peptides in secreted or membrane proteins.

Adenylyl Cyclases↗
PubMed1977

Urease of Klebsiella aerogenes: control of its synthesis by glutamine synthetase.

Urease was purified 24-fold from extracts of Klebsiella aerogenes. The enzyme has a molecular weight of 230,000 as determined by gel filtration, is highly substrate specific, and has a Km for urea of 0.7 mM. A mutant strain lacking urease was isolated; it failed to grow with urea as the sole source of nitrogen but did grow on media containing other nitrogen sources such as ammonia, histidine, or arginine. Urease was present at a high level when the cells were starved for nitrogen; its synthesis was repressed when the external ammonia concentration was high. Formation of urease did not require induction by urea and was not subject to catabolite repression. Its synthesis was controlled by glutamine synthetase. Mutants lacking glutamine synthetase failed to produce urease, and mutants forming glutamine synthetase at a high constitutive level also formed urease constitutively. Thus, the formation of urease is regulated like that of other enzymes of K. aerogenes capable of supplying the cell with ammonia or glutamate.

Enzyme Repression↗