Induction and Catabolite Repression of Glucosidases in Pseudomonas maltophilia

Intracellular α-and β-glucosidases were induced in cell suspensions of Pseu-domonas maltophilia by maltose or cellobiose, and the synthesis of these enzymes was sensitive to apparent catabolite repression by α-ketoglutarate.     

Regulation of Hfq mRNA and Protein Levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA

Small non-coding RNAs (sRNAs) are important players of gene expression regulation in bacterial pathogens. MtvR is a 136-nucleotide long sRNA previously identified in the human pathogen Burkholderia cenocepacia J2315 and with homologues restricted to bacteria of the Burkholderia cepacia complex. In this work we have investigated the effects of expressing MtvR in Escherichia coli and Pseudomonas aeruginosa. Results are presented showing that MtvR negatively regulates the hfq mRNA levels in both bacterial species. In the case of E. coli, this negative regulation is shown to involve binding of MtvR to the 5′-UTR region of the hfq_Ec mRNA. Results presented also show that expression of MtvR in E. coli and P. aeruginosa originates multiple phenotypes, including reduced resistance to selected stresses, biofilm formation ability, and increased susceptibility to various antibiotics.     

Carbon Catabolite Repression Regulates Glyoxylate Cycle Gene Expression in Cucumber

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.     

Regulation of Aconitase Synthesis in Bacillus subtilis: Induction, Feedback Repression, and Catabolite Repression

The synthesis of aconitase in Bacillus subtilis wild-type and different citric acid cycle mutants has been studied and the influence of various growth conditions examined. Aconitase is induced by citrate and precursors of citrate and repressed by glutamate. Induction and repression counteract each other, and at equimolar concentrations of citrate and glutamate, aconitase synthesis is unaffected. Induction by citrate can partly overcome catabolite repression of aconitase. Isocitrate dehydrogenase show endogenous induction of aconitase due to citrate accumulation. Leaky mutants defective in citrate synthase and aconitase cannot be induced by citrate, which indicates that they carry a regulatory mutation. The complex regulation of aconitase is discussed with reference to the participation of this enzyme in glutamate biosynthesis and energy metabolism.     

Distinct and overlapping binding sites of Pseudomonas aeruginosa Hfq and RsmA proteins on the non-coding RNA RsmY

In Pseudomonas aeruginosa the Rsm system is involved in regulation of quorum-sensing and virulence gene expression. Our recent studies revealed that the stability and abundance of the non-coding RNA RsmY, which antagonizes the translational regulator RsmA, is dependent on Hfq. Here, we show that Hfq and RsmA bind concurrently to RsmY. Enzymatic probing of RsmY RNA in the presence of RsmA and Hfq verified the proposed -GGA- motifs as RsmA binding sites and located Hfq binding sites in single-stranded regions adjacent to stem-loop structures, respectively. We conclude that distinct binding of Hfq and RsmA on RsmY RNA permits RsmY-mediated RsmA titration upon binding to and stabilization of RsmY RNA by Hfq. In addition, we provide evidence that Hfq sequesters RNase E cleavage sites on RsmY, which explains the previously observed dependence of RsmY RNA stability on Hfq.     

Catabolite repression and nitrogen control of allantoin-degrading enzymes in Pseudomonas aeruginosa

The formation of the allantoin-degrading enzymes allantoinase, allantoicase and ureidoglycolase in Pseudomonas aeruginosa was found to be regulated by induction, catabolite repression and nitrogen control. Induction was observed when urate, allantoin or allantoate were included in the growth medium, but not with ureidoglycolate. Tricarboxylic acid cycle intermediates exerted catabolite repression of the synthesis of the three enzymes, while pyruvate and glucose caused less repression. The operation of a nitrogen control mechanism in the regulation of the allantoin-degrading enzymes could be demonstrated with glutamine synthetase-negative mutants, which showed elevated synthesis and escape from catabolite repression when growth was limited for glutamine.     

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.     

Genetic Modification of Carbon Catabolite Repression in Trichoderma reesei for Improved Protein Production

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Δcre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.The filamentous fungus Trichoderma reesei (Hypocrea jecorina) produces large amounts of extracellular enzymes. The majority of the secreted proteins are various plant polymer-degrading enzymes; the most abundant of these enzymes are the cellobiohydrolases and endoglucanases that act synergistically to break down cellulose. This fungus has been used as a production host for various industrial enzymes, including products tailored for textile, feed, food, and pulp and paper applications (6, 10). It has been reported that protein production levels in the industrial T. reesei process exceed 100 g/liter (7).The major cellulase and hemicellulase genes are regulated in a coordinate manner by the carbon source available (2, 9, 14). Cellulose and other plant materials and other substances (for example, lactose) induce the expression of cellulase and hemicellulase genes, while glucose acts as a repressing carbon source. Several genes coding for regulators of cellulase and hemicellulase expression have been isolated. These include CREI mediating carbon catabolite repression, the repressor ACEI, the activator ACEII, the CCAAT binding complex Hap2/3/5 (reviewed in references 2, 17, and 27) and the activator XYRI (29). The CREI protein has sequence similarity with other fungal proteins mediating glucose repression, such as Aspergillus nidulans CREA (8) and Saccharomyces cerevisiae MIG1 and RGR1 (22). In T. reesei, glucose repression has been shown to occur upon binding of CREI to specific sequences in the cbh1 promoter (13). A mutant cre1 gene (cre1-1) encoding a truncated form of CREI has been isolated from the hypercellulolytic T. reesei strain Rut-C30, which is capable of cellulase and hemicellulase production on glucose-containing media. Further evidence for the function of CREI in glucose repression was obtained by complementation of the cre1-1 mutation of Rut-C30 by the wild-type cre1 gene, which restored the glucose-repressed phenotype of the strain (15).In this paper, we wanted to address three questions. (i) What is the effect of cre1 mutations in the wild-type background? (ii) Is cre1-1 a null mutation? (iii) Can enzyme production be further improved by cre1 deletion in an industrial production strain improved greatly by mutagenesis and screening programs? Therefore, we introduced cre1-1 allele and cre1 deletion to the wild-type strain QM6a and the cre1 deletion into the industrial strain VTT-D-80133 and studied the effects of these mutations on enzyme production.