Induction and catabolite repression of chondroitinase in batch and chemostat cultures of Proteus vulgaris.
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We have previously proposed that metabolic status is important in the regulation of cucumber malate synthase (MS) and isocitrate lyase (ICL) gene expression during plant development. In this article, we used a cell culture system to demonstrate that intracellular metabolic status does influence expression of both of these genes. Starvation of cucumber cell cultures resulted in the coordinate induction of the expression of MS and ICL genes, and this effect was reversed when sucrose was returned to the culture media. The induction of gene expression was closely correlated with a drop in intracellular sucrose, glucose, and fructose below threshold concentrations, but it was not correlated with a decrease in respiration rate. Glucose, fructose, or raffinose in the culture media also resulted in repression of MS and ICL. Both 2-deoxyglucose and mannose, which are phosphorylated by hexokinase but not further metabolized, specifically repressed MS and ICL gene expression relative to a third glyoxylate cycle gene, malate dehydrogenase. However, the addition of 3-methylglucose, an analog of glucose that is not phosphorylated, did not result in repression of either MS or ICL. It is proposed that the signal giving rise to a change in gene expression originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis.
Glutamine, like glucose, repressed sporulation and the synthesis of mycobacillin and dipicolinic acid by Bacillus subtilis, and these syntheses were depressed by dibutyryl cyclic GMP but not by dibutyryl cyclic AMP. Neither of these dibutyryl cyclic nucleotides affected sporulation or a number of spore-associated parameters in the strain under normal physiological conditions. Mutants insensitive to glutamine repression were indifferent to the addition of either of the dibutyryl cyclic nucleotides both in the presence and in the absence of glutamine. Sporulation resulted from the remission of repression obtained under the catabolically active state.
In Aspergillus nidulans, it is known that creB encodes a deubiquitinating enzyme that forms a complex with the WD40 motif containing protein encoded by creC, that mutations in these genes lead to altered carbon source utilization and that the creD34 mutation suppresses the phenotypic effects of mutations in creC and creB. Therefore, creD was characterized in order to dissect the regulatory network that involves the CreB-CreC deubiquitination complex. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p proteins from Saccharomyces cerevisiae. An additional gene was identified in the A. nidulans genome that also encodes an arrestin and PY motif-containing protein, which we have designated apyA, and thus two similar proteins also exist in A. nidulans. In S. cerevisiae, Rod1p and Rog3p interact with the ubiquitin ligase Rsp5p, and so the A. nidulans homologue of Rsp5p was identified, and the gene encoding this HECT ubiquitin ligase was designated hulA. CreD and ApyA were tested for protein-protein interactions with HulA via the bacterial two-hybrid system, and ApyA showed strong interaction, and CreD showed weak interaction, with HulA in this system.
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The glucose analog, 3-O-methyl-D-glucose, inhibited growth of yeast on non-fermentable carbon sources. The sugar was phosphorylated by the yeast and also in vitro by a commercial preparation of yeast hexokinase. The chromatographic behaviour of the phosphorylated product was identical in both cases. This suggests that 3-O-methyl-D-glucose is phosphorylated to form 3-O-methyl-D-glucose 6-phosphate. The inhibition of the growth appears to be due to interference with the derepression of several enzymes necessary to grow on non-fermentable carbon sources. Spontaneous mutants whose growth was unaffected by 3-O-methyl-D-glucose were isolated. In these mutants there was no significant accumulation of the phosphorylated ester and the derepression of the enzymes tested was not affected by the glucose analog.
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The luxA,B genes from the Gram-negative marine bacterium Vibrio harveyi MAV were used in Staphylococcus carnosus TM300 as a reporter system for regulated expression of xylose utilization. The luciferase genes were fused to the xyl operon from Staphylococcus xylosus C2a. Expression of bioluminescence was induced through addition of xylose and repressed in the presence of glucose. A method to quantitate bioluminescence directly from the culture is described.
Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. Glucose is transported and concomitantly phosphorylated by the glucose permease (PtsG) of the phosphoenolpyruvate:sugar phosphotransferase system. The phosphate is transferred from enzyme I via HPr and domains IIA and IIB of the glucose permease to the sugar. In this study mutants affected in the putative phosphorylation sites of glucose permease were constructed and the effect on sugar transport and glucose repression tested. Phosphorylation of both domains IIAGlc and IIBGlc is required for efficient glucose transport and repression of beta-xylosidase and the bglPH operon.
The creA genes of two plant pathogenic fungi, the gibberellin-producing rice pathogen Gibberella fujikuroi and the gray mold Botrytis cinerea, were isolated and characterized. The deduced amino acid sequences of both glucose repressors are 64% identical to each other and 59% (G. fujikuroi) and 61% (B. cinerea) identical to the CreA protein of Aspergillus nidulans. The zinc finger regions of the Gibberella and Botrytis CreA proteins shared 98% identity with the corresponding zinc finger region of the A. nidulans protein, and studies by complementation of a creA null mutant of A. nidulans showed that the proteins are functional homologues of A. nidulans CreA. Northern blot analysis revealed that creA transcript levels are independent of the carbon source in both fungi.
The dyatic symmetric element (DSE) present in the alpha-amylase gene promoter region of the thermophilic Streptomyces strain sp. TO1, and the whole alpha-amylase gene (amy TO1) with a 3-bp change in the DSE, were cloned in the high copy number replicative cloning vector pIJ702 giving pLM10 and pLM11 plasmids respectively. In TO1/pLM10 and Streptomyces lividans TK24/pLM11 strains, the expression of alpha-amylase TO1 gene became insensible to the negative effect of glucose and glycerol. These results strongly suggest that, in a high copy number system, the negative transcriptional regulator was titrated out by the DSE and the repression of the expression of amy TO1 gene is abolished.
Saccharomyces cerevisiae is able to use a wide variety of nitrogen sources for growth. Not all nitrogen sources support growth equally well. In order to select the best out of a large diversity of available nitrogen sources, the yeast has developed molecular mechanisms. These mechanisms consist of a sensing mechanism and a regulatory mechanism which includes induction of needed systems, and repression of systems that are not beneficial. The first step in use of most nitrogen sources is its uptake via more or less specific permeases. Hence the first level of regulation is encountered at this level. The next step is the degradation of the nitrogen source to useful building blocks via the nitrogen metabolic pathways. These pathways can be divided into routes that lead to the degradation of the nitrogen source to ammonia and glutamate, and routes that lead to the synthesis of nitrogen containing compounds in which glutamate and glutamine are used as nitrogen donor. Glutamine is synthesized out of ammonia and glutamate. The expression of the specific degradation routes is also regulated depending on the availability of a particular nitrogen source. Ammonia plays a central role as intermediate between degradative and biosynthetic pathways. It not only functions as a metabolite in metabolic reactions but is also involved in regulation of metabolic pathways at several levels. This review describes the central role of ammonia in nitrogen metabolism. This role is illustrated at the level of enzyme activity, translation and transcription.
The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. We show that the ptsN gene product IIA(Ntr) participates in this repression. Crc, involved in yeast extract-dependent repression in batch cultures, did not influence expression when cells were growing in a chemostat with succinate at maximum rate.
beta-Glucosidase activity in Myceliophthora thermophila D-14 (= ATCC 48104) was inducible and was produced in culture filtrate during growth with various inducers, of which PNPG (p-nitrophenyl-beta-d-glucoside) was the most efficient. Induction of beta-glucosidase also occurred when the organism was grown in medium supplemented with different carbon sources. Carboxymethyl cellulose, cellobiose, and Solka-Floc were found effective for induction of enzyme biosynthesis. The addition of glucose to the culture medium severely repressed beta-glucosidase synthesis, which could not be reversed by exogenous cyclic AMP or dibutyryl cyclic AMP.
Vectors which possess a truncated niaD gene encoding nitrate reductase were developed to allow targeted gene integration during transformation of an niaD mutant Penicillium chrysogenum host. The Penicillium genes pcbC and penAB are immediately adjacent to each other and are divergently transcribed, with an intergenic control region serving as their promoters. Gene fusions were constructed with a reporter gene, uidA, which encodes beta-glucuronidase. The pcbC-penAB intergenic region was fused to the uidA gene in both orientations so that regulated expression of each structural gene could be investigated. These fusion genes were targeted to the chromosomal site of the niaD locus of P. chrysogenum, and their expression was examined under different growth conditions. The expression of each of these penicillin biosynthesis genes was found to be regulated by nitrogen repression, glucose repression, and growth stage control.