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PubMed1975

Derepression in Saccharomyces cerevisiae can be dissociated from cellular proliferation and deoxyribonucleic acid synthesis.

A method has been developed that permits precise control of release from catabolite repression in Saccharomyces cerevisiae. It consists of transferring cells growing exponentially on 5% glucose to derepression medium at high cell density. Derepression then proceeds with reproducible kinetics and is complete within 6 to 7.5 h for various intra- and extramitochondrial markers, in the absence of any substantial increase in cellular dry weight or protein. Nuclear (and mitochondrial) deoxyribonucleic acid synthesis can be interrupted in certain thermosensitive (cdc) mutants at the nonpermissive temperature; a shift to this temperature before the onset of derepression has no effect on its outcome.

Bacterial Proteins↗
PubMed1986

Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation.

Mutants of Erwinia chrysanthemi impaired in pectin degradation were isolated by chemical and Mu d(Ap lac) insertion mutagenesis. A mutation in the kduD gene coding for 2-keto-3-deoxygluconate oxidoreductase prevented the growth of the bacteria on polygalacturonate as the sole carbon source. Analysis of the kduD::Mu d(Ap lac) insertions indicated that kduD is either an isolated gene or the last gene of a polycistronic operon. Some of the Mu d(Ap lac) insertions were kduD-lac fusions in which beta-galactosidase synthesis reflected kduD gene expression. In all these fusions, beta-galactosidase activity was shown to be sensitive to catabolite repression by glucose and to be inducible by polygalacturonate, galacturonate, and other intermediates of polygalacturonate catabolism. Galacturonate-mediated induction was prevented by a mutation which blocked its metabolism to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate appeared to be the true inducer of kduD expression resulting from galacturonate degradation. 5-Keto-4-deoxyuronate or 2,5-diketo-3-deoxygluconate were the true inducers, originating from polygalacturonate cleavage. These three intermediates also appeared to induce pectate lyases, oligogalacturonate lyase, and 5-keto-4-deoxyuronate isomerase synthesis.

Aldose-Ketose Isomerases↗
PubMed1998

Regulation of Drosophila Adh promoter switching by an initiator-targeted repression mechanism.

The stage-specific expression of the Drosophila alcohol dehydrogenase (Adh) gene is achieved through the alternate activation of two tandem promoters. The proximal promoter is active primarily during late embryonic development and early larval stages, while the distal promoter is active in late third instar larvae and adults. Here, we provide evidence that this Adh promoter switch is regulated by a zinc finger repressor protein (AEF-1) that is expressed predominantly in adult flies and targets the initiator region of the proximal promoter. We propose that AEF-1 plays a critical role in Adh promoter switching by blocking interactions between a component of the general transcription machinery and the initiator region of the proximal promoter.

Alcohol Dehydrogenase↗
PubMed1987

Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast.

The HEX2 gene which is necessary for glucose repression and is involved in the regulation of hexokinase PII synthesis and maltose uptake, has been cloned by complementation of a hex2 mutant, and selection for restored growth on maltose. Glucose repression in the transformants was like that in the wild type. The HEX2 gene was localized within a 2.15 kb fragment. The restriction map was confirmed by Southern hybridization of genomic DNA. Based on 30 tetrads, the linkage between HEX2 and TRP1 was determined as 10 cM. Plasmid integration directed to the genomic site of the cloned gene also gave a similar linkage distance between the amino acid auxotroph plasmid marker and genomic TRP1. Gene disruption of HEX2 yielded nonrepressible transformants with elevated hexokinase PII activity showing inhibition by maltose; this provides clear evidence that the HEX2 gene has been isolated.

DNA Restriction Enzymes↗
PubMed1984

Genetic considerations in the effects of ethanol in mice. II. A trans-acting inducibility regulator(s) affecting alcohol dehydrogenase (ADH) activity.

The inducibility of alcohol dehydrogenase (ADH) has been recognized in different systems including maize, Drosophila, and mice. Our earlier results showed strain-specific ADH responses to chronic ethanol administration relative to matched littermate controls in mice. For this study we used two strains which showed "induction" (BALB/c and S.W.) and two strains which showed "repression" (C57BL/6J and 129/ReJ) to produce three sets of F1 hybrids and their reciprocals and one set (BALB/c X C57BL/6J) of recombinant inbred (RI) lines. The ADH properties of the resulting genotypes were again evaluated following 15% ethanol treatment in drinking water (2 weeks) in relation to their littermate matched controls in replicated trials. Our F1 results suggest complete dominance for induction over repression at the phenotypic level, and the two repressed strains showed complementation. No significant differences were observed in the reciprocal F1's and all pairs of a given genotype-treatment combination yielded consistent results. The 1:1 segregation of RI lines suggests a single gene difference for ADH inducibility between BALB/c and C57BL/6J. These findings suggest the presence of a trans-acting inducibility regulator(s) for ADH which may or may not represent a single locus. Variability for such regulatory elements may provide an explanation for the commonly observed individual differences in natural populations for response to alcohol including alcohol metabolism.

Alcohol Dehydrogenase↗
PubMed1996

The 3-phosphoglycerate kinase gene of the yeast Yarrowia lipolytica de-represses on gluconeogenic substrates.

We have isolated the 3-phosphoglycerate kinase (PGK) gene of the yeast Yarrowia lipolytica by probing a genomic library with a PCR fragment amplified with primers deduced from two highly conserved regions of various PGKs. It is a unique sequence encoding a polypeptide of 417 residues with extensive homology to other PGKs, especially to that of Aspergillus nidulans (76% identity). The expression of the Y. lipolytica PGK1 gene proved to be higher on gluconeogenic substrates than on glycolytic ones. Haploid strains harboring a disrupted allele were able to grow on mixtures of a gluconeogenic carbon source and of a glycolytic one, but required proline supplementation in the presence of glucose, and were inhibited by glycerol.

Amino Acid Sequence↗
PubMed1976

The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.

Amino Acids↗
PubMed1991

Catabolite repression of alpha-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors.

Expression of the alpha-amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidly metabolizable carbon sources. Glucose-mediated repression has previously been shown to involve a site near the transcriptional start-point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression. The gene affected by this mutation, which was localized near 263 degrees on the B. subtilis chromosomal map, was isolated and its DNA sequence was determined. This gene, designated ccpA, exhibited striking homology to repressor genes of the lac and gal repressor family. The ccpA gene was found to be allelic to alsA, previously identified as a regulator of acetoin biosynthesis, and may be involved in catabolite regulation of other systems as well.

Acetoin↗
PubMed1987

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression.

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.

Animals↗
PubMed1995

Nitrate repression of the Escherichia coli pfl operon is mediated by the dual sensors NarQ and NarX and the dual regulators NarL and NarP.

The pfl operon is expressed at high levels anaerobically. Growth of Escherichia coli in the presence of nitrate or nitrite led to a 45% decrease in expression when cells were cultivated in rich medium. Nitrate repression, however, was significantly enhanced (sevenfold) when the cells were cultured in minimal medium. Regulation of pfl expression by nitrate was dependent on the NarL, NarP, NarQ, and NarX proteins but independent of FNR, ArcA, and integration host factor, which are additional regulators of pfl expression. Strains unable to synthesize any one of the NarL, NarP, NarQ, or NarX proteins, but retaining the capacity to synthesize the remaining three, exhibited essentially normal nitrate regulation. In contrast, narL narP and narX narQ double null mutants were devoid of nitrate regulation when cultured in rich medium but they retained some nitrate repression (1.3-fold) when grown in minimal medium. By using lacZ fusions, it was possible to localize the DNA sequences required to mediate nitrate repression to the pfl promoter-regulatory region. DNase I footprinting studies identified five potential binding sites for the wild-type NarL protein in the pfl promoter-regulatory region. Specific footprints were obtained only when NarL was phosphorylated with acetyl phosphate before the binding reaction was performed. Each of the protected regions contained at least one heptamer sequence which has been deduced from mutagenesis studies to be essential for NarL binding (K. Tyson, A. Bell, J. Cole, and S. Busby, Mol. Microbiol. 7:151-157, 1993).

Acetyltransferases↗
PubMed2001

Iron-Mediated regulation of alkaline proteinase production in Pseudomonas aeruginosa.

We analyzed the regulation by iron of alkaline proteinase (AP) production in Pseudomonas aeruginosa. Extracellular AP production was detected from the mid-logarithmic to the stationary phase by an antibody-based assay system, and was strongly repressed by iron in the medium. This repression was shown by Northern hybridization and primer extension to occur at the level of transcription. The primer extension analysis revealed that the start point of transcription of AP gene was the nucleotide position -84 from the start point of translation. Furthermore, we investigated whether this transcriptional repression involved PvdS protein. Using the mutant strain of pvdS, the alternative sigma factor gene revealed that the PvdS protein is required for the full expression of AP, and a previous study showed that expression of pvdS is also repressed by iron. Therefore, we thought that one mechanism of repression of AP production operated through reduction of the PvdS protein level. Purified AP decomposed the transferrin, and released iron from it. Purified AP added to the medium containing transferrin as the only iron source enhanced the growth of P. aeruginosa. Moreover, mutation in the AP gene decreased the growth rate in the medium containing the transferrin as the only iron source. These results clearly indicated that AP expression should occur in a free-iron-deficient environment and emphasized the importance of AP to iron acquisition in the infection site.

Apoproteins↗
PubMed1994

Regulation of the xylanase-encoding xlnA gene of Aspergillus tubigensis.

A gene encoding an endo-1,4-beta-xylanase from Aspergillus tubigensis was cloned by oligonucleotide screening using oligonucleotides derived from amino acid sequence data obtained from the purified protein. The isolated gene was functional as it could be expressed in the very closely related fungus Aspergillus niger. The xylanase encoded by this gene is synthesized as a protein of 211 amino acids. After cleavage of the presumed prepropeptide this results in a mature protein of 184 amino acids with a molecular weight of 19 kDa and an isoelectric point of 3.6. The regulatory region of the xlnA gene was studied with respect to the response to xylan induction and carbon catabolite repression. By deletion analysis of the 5' upstream region of the gene a 158 bp region involved in the xylan specific induction was identified. To study this regulatory element a reporter system for transcriptional activating sequences was developed that is based on the A. niger glucose oxidase-encoding gene. From the results with this reporter system it is concluded that this 158 bp fragment not only contains the information required for induction of transcription but that it also plays a role in carbon catabolite repression of the xlnA gene. The region directly upstream of this fragment contains four potential CREA target sites; deletion of this region leads to an increase in the level of transcription. These results suggest that carbon catabolite repression of the xlnA gene is controlled at two levels, directly by repression of xlnA gene transcription and indirectly by repression of the expression of a transcriptional activator. This type of mechanism would be similar to the double lock mechanism for the regulation of gene expression of alcA in Aspergillus nidulans. The reporter system was also used to study the regulation of expression via the functions located on this fragment in A. niger and in A. nidulans. Essentially the same pattern of regulation was found in both of these hosts. Therefore, regulation of xylanase gene expression is basically conserved in all three aspergilli.

Amino Acid Sequence↗
PubMed1989

Induction and metabolite regulation of levanase synthesis in Bacillus subtilis.

Levanase expression in Bacillus subtilis was studied by using transcriptional and translational fusions. It was shown that the degradative products of levan or inulin and low concentrations of fructose were able to induce levanase expression. In the wild-type strain and in a constitutive overproducing sacL mutant, levanase synthesis was repressed by glucose or fructose. This catabolite repression was partially abolished in the derepressed alpha-amylase gra-26 mutant. The levanase gene (sacC) appears to be the distal gene of an operon transcribed from a fructose-inducible promoter. Deletion mapping experiments and primer extension analysis revealed a transcriptional start point located 2.7 kilobases upstream from the sacC gene. Two constitutive sacL mutations were shown to be closely linked by transformation to the sacC gene. The sacL6 and sacL8 mutations were mapped in the promoter-proximal region of the operon.

Amino Acid Sequence↗
PubMed1978

Stable isotope fractionation by Clostridium pasteurianum. 2. Regulation of sulfite reductases by sulfur amino acids and their influence on sulfur isotope fractionation during SO32- and SO42- reduction.

In addition to an assimilatory sulfite reductase, studies of cultures of Clostridium pasteurianum supplemented with methionine, cysteine, and 35SO42- provides evidence for another reductase which is induced by SO32-. This inducible reductase appears to be dissimaltory because of the copious sulfide production arising when the cells are grown on SO32-. Cysteine can repress the assimilatory sulfite reductase but does not affect the inducible reductase. During late logarithmic growth on 1 mM SO42- + 10mM cysteine, depression of the inducible reductase occurred along with increased sulfide production. The presence of 1 mM cysteine and (or) 1 mM cysteine and (or) 1 mM methionine does not affect the inverse sulfur isotope effect for evolved H2S. However, 5 and 10 mM cysteine reduce the maximum delta34S value for released H2S from +40 to 10%. A small conversion of cysteine to H2S by C. pasteurianum occurs, but only in the stationary phase.

Cell-Free System↗
PubMed1975

Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source.

Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis.

Chromosome Mapping↗
PubMed1975

Regulation of extracellular protease secretion in Pseudomonas maltophilia.

Cells grown in minimal medium and harvested in late exponential phase secreted extracellular protease linearly when suspended at high density in fresh medium. If the cells were suspended with 0.2% (wt/vol) yeast extract and no additional carbon source, the rate of exoenzyme production was increased several-fold. When pyruvate, L-malate, succinate, or alpha-ketoglutarate was added, repression of exoenzyme secretion was observed. The most effective repressor was alpha-ketoglutarate. These compounds were also preferred substrates for growth of Pseudomonas maltophilia. The data suggest that exoenzyme secretion is controlled by a mechanism similar to catabolite repression. In support of this was the observation that alpha-ketoglutarate repressed exoenzyme secretion preferentially with respect to total protein synthesis. The addition of inhibitors that affect protein synthesis indicated that exoenzyme secretion is several times more sensitive than is total protein synthesis. The addition of chloramphenicol and rifamycin-SV to actively secreting cell suspensions suggested that de novo protein synthesis is required, but that exoenzyme secretion may be supported for at least 30 min in the absence of messenger synthesis. Rifamycin-insensitive protease secretion could be reversed by either alpha-ketoglutarate or chloramphenicol, suggesting that alpha-ketoglutarate is coupled to a post-transcriptional control mechanism.

Alanine↗
PubMed1990

Transcription of a yeast phosphoglucomutase isozyme gene is galactose inducible and glucose repressible.

The Saccharomyces cerevisiae GAL5 (PGM2) gene was isolated and shown to encode the major isozyme of phosphoglucomutase. Northern (RNA) blot hybridization revealed that the GAL5 transcript level increased three- to fourfold in response to galactose and was severely repressed in response to glucose. Total cellular phosphoglucomutase activity was likewise responsive to galactose and to glucose, and this responsiveness was found to be due primarily to variation in the activity of the major isozyme of phosphoglucomutase. These results imply that the major and minor isozymes of phosphoglucomutase have distinct roles in yeast cells. The galactose inducibility of GAL5 was found to be under the control of the GAL4, GAL80, and GAL3 genes. In striking contrast to other galactose-inducible genes, the GAL5 gene exhibited an unusually high GAL4-independent basal level of expression. These results have implications for metabolic trafficking.

Cloning, Molecular↗
PubMed1994

Catabolite repression and induction time effects for a temperature-sensitive GAL-regulated yeast expression system.

The effects of residual catabolite repression and the importance of induction timing were determined for a temperature-sensitive (ts) GAL-regulated stable yeast expression system. The Saccharomyces cerevisiae strain employed carries a reg1 mutation inhibiting catabolite repression, and a ts mutation enabling induction of the regulated GAL promoters by a temperature shift to 35 degrees C. Despite the reg1 mutation and induction method, glucose depressed lacZ expression from a GAL1 promoter during batch culture. beta-Galactosidase specific activity was consistently lower at higher initial glucose concentrations in both SDC (semi-defined) and YPDa (complex) media; decreases of 18-36% were observed as glucose concentration was increased between 1, 3, 5, and 10 g l-1. However, the reductions in beta-galactosidase specific activity due to residual catabolite repression were more than balanced by substantial improvements in biomass yield at higher glucose levels. Therefore, productivity rose with increasing glucose concentration; in YPDa medium, increasing initial glucose from 1 to 10 g l-1 resulted in a 2.6-fold increase in beta-galactosidase volumetric activity. Due to the negative effects of shifting temperature to 35 degrees C, the trade-offs between optimum growth and a lengthy induction period were also evaluated. Delaying the time of induction reduced final specific activities but improved cell yield, and waiting 14 h into batch culture to induce lacZ expression provided modest 9-15% improvements in overall productivity.

Enzyme Repression↗