Showing papers on "Catabolite repression published in 1999"

Nitrogen catabolite repression in Saccharomyces cerevisiae

Abstract: In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Dal80, and Deh1. This is known as nitrogen catabolite repression (NCR). They bind to motifs in the promoter region to the consensus sequence 5'GATAA 3'. Gln3 and Gat1 act positively on gene expression whereas Dal80 and Deh1 act negatively. Expression of nitrogen catabolite pathway genes known to be regulated by these four regulators are glutamine, glutamate, proline, urea, arginine. GABA, and allantonie. In addition, the expression of the genes encoding the general amino acid permease and the ammonium permease are also regulated by these four regulatory proteins. Another group of genes whose expression is also regulated by Gln3, Gat1, Dal80, and Deh1 are some proteases, CPS1, PRB1, LAP1, and PEP4, responsible for the degradation of proteins into amino acids thereby providing a nitrogen source to the cell. In this review, all known promoter sequences related to expression of nitrogen catabolite pathways are discussed as well as other regulatory proteins. Overview of metabolic pathways and promotors are presented.

Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory signals.

Abstract: Regulation of the expression of the two major chitinase genes, ech42 (encoding the CHIT42 endochitinase) and nag1 (encoding the CHIT73 N-acetyl-β-d-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungus Trichoderma atroviride (= Trichoderma harzianum P1) was investigated by using a reporter system based on the Aspergillus niger glucose oxidase. Strains harboring fusions of the ech42 or nag1 5′ upstream noncoding sequences with the A. niger goxA gene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the native ech42 and nag1 genes, as assayed by Northern analysis. The expression product of goxA in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay. nag1 gene expression was triggered during growth on fungal (Botrytis cinerea) cell walls and on the chitin degradation product N-acetylglucosamine. N-Acetylglucosamine, di-N-acetylchitobiose, or tri-N-acetylchitotriose also induced nag1 gene expression when added to mycelia pregrown on different carbon sources. ech42 expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant ech42 expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition, ech42 gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the ech42 promoter.

An Alkane-Responsive Expression System for the Production of Fine Chemicals

Abstract: Pseudomonas putida mt-2-derived xylene oxygenase is encoded in the catabolic TOL plasmid pWW0 upper pathway operon, and together with a set of other enzymes it forms a catalytic cluster which degrades toluene and xylene to (substituted) benzoic acids (18, 30, 40). The ability of xylene oxygenase to hydroxylate methyl substituents on substituted benzenes or their heteroaromatic equivalents has made it an important biotechnological enzyme (24, 56). This enzyme consists of a membrane-bound component, XylM, which carries out the oxygenation step (50), and a cytoplasmic NADH:acceptor reductase component, XylA, which supplies reducing equivalents to XylM (45). The potential of this system for biological production of fine chemicals has already been exploited; wild-type cells of P. putida mt-2 are used by Lonza to produce heteroaromatic acids on a commercial scale (24). Furthermore, xylene oxygenase is selective for the si-face of prochiral vinyl functions on aromatic ring systems, which leads to the formation of optically active epoxides, such as (S)-styrene oxide (55) (Fig. ​(Fig.1).1). Escherichia coli recombinants carrying the genes for xylene oxygenase have produced (S)-styrene oxide from inexpensive styrene in a 2-liter reactor (54). Unfortunately, so far the productivities displayed by such recombinants have been insufficient to commercially exploit their synthetic potential (17, 55). A number of observations have indicated that expressing the xylene oxygenase genes via the alk regulatory system of Pseudomonas oleovorans GPo1 might provide suitable biocatalysis strains for two-liquid-phase cultures. P. oleovorans GPo1 degrades medium-chain-length alkanes with a set of enzymes encoded by two alk gene clusters on the catabolic OCT plasmid (Fig. ​(Fig.2A)2A) (51). The first cluster contains the alkBFGHJKL operon, which contains all but one of the structural genes for conversion of alkanes to the corresponding alkanoic acids and coupling of these compounds to coenzyme A (Fig. ​(Fig.2B).2B). The second cluster contains the remaining structural gene, alkT, and the gene which encodes the regulatory protein AlkS (11). Expression of the genes in the first cluster is under control of alkBp, the alk promoter, and is initiated in the presence of functional AlkS and alkanes or other, structurally nonrelated inducers, such as dicyclopropylketone (DCPK) (16, 49). DCPK is water soluble and hence is a convenient inducer in aqueous cultures, while alkanes are useful inducers in two-liquid-phase cultures which contain an organic phase. Expression of the alk genes in E. coli W3110 via the alk regulatory system from the low-copy-number RK2 derivative pGEc47 led to accumulation of membrane-located AlkB until it accounted for up to 10% of the total cell protein (35). This indicated that AlkS, together with its cognate promoter alkBp (26), could be a powerful general system to direct synthesis of recombinant proteins. In addition, the alk regulatory system is not subject to catabolite repression in E. coli (46, 58), which allows convenient utilization of cheap carbon sources, such as glucose, in cultures of recombinant strains. Because of these attractive features of the alk regulatory system we developed its components into an expression system for general use. In this paper we describe this system and its potential for efficient synthesis of xylene oxygenase for biotransformation of styrene to (S)-styrene oxide at high enantiomeric excess in a two-liquid-phase biotransformation system. FIG. 1 Conversion of inexpensive styrene to (S)-styrene oxide by xylene monooxygenase. The chiral carbon atom is indicated by an asterisk. FIG. 2 (A) Organization and regulation of the alk genes on the OCT plasmid in P. oleovorans GPo1. The regulatory protein AlkS is activated by octane or DCPK and induces transcription from the alkBp promoter. The directions of transcription are indicated by arrows ...

Suppression of toxin production in Clostridium difficile VPI 10463 by amino acids.

Abstract: The impact of various growth conditions on the expression of toxins and other proteins by Clostridium difficile VPI 10463 was studied. During non-starved conditions, the rate of toxin synthesis paralleled that of total protein during both exponential growth and stationary phase, and in both defined and complex media. Biotin limitation reduced growth rate and bulk protein synthesis, whereas toxin expression continued, leading to a 50- to 200-fold increase in intracellular toxin levels. Concomitantly, several 22 kDa proteins were up-regulated as revealed by two-dimensional PAGE analysis. The toxin yield was 30-fold higher in peptone yeast extract (PY) than in PY containing glucose (PYG). By contrast glucose limitation reduced toxin yields by 20- to 100-fold in defined media. By elevating the buffering capacity and bicarbonate concentration, toxin yields were increased by 10-fold in PY and PYG. The high toxin production by C. difficile during growth in PY was lowered 100-fold by adding a blend of nine amino acids and several 60-100 kDa proteins were concomitantly down-regulated. It was concluded that toxin expression in C. difficile VPI 10463 was not affected by growth rate, growth phase, catabolite repression or the stringent response. Instead the co-expression of toxins and a few specific additional proteins appeared to be influenced by metabolic pathways involving CO2 assimilation, carboxylation reactions and metabolism of certain amino acids.

Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis.

Abstract: In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.

Regulation of expression of the fructan hydrolase gene of Streptococcus mutans GS-5 by induction and carbon catabolite repression.

Abstract: The polymers of fructose, levan and inulin, as well as sucrose and raffinose, are substrates for the product of the fruA gene of Streptococcus mutans GS-5. The purpose of this study was to characterize the DNA immediately flanking fruA, to explore the regulation of expression of fruA by the carbohydrate source, and to begin to elucidate the molecular basis for differential expression of the gene. Located 3' to fruA was an open reading frame (ORF) with similarity to beta-fructosidases which was cotranscribed with fruA. A transcriptional initiation site, located an appropriate distance from an extended -10-like promoter, was mapped at 165 bp 5' to the fruA structural gene. By the use of computer algorithms, two overlapping, stable stem-loop sequences with the potential to function as rho-independent terminators were found in the 5' untranslated region. Catabolite response elements (CREs), which have been shown to govern carbon catabolite repression (CCR) by functioning as negative cis elements in gram-positive bacteria, were located close to the promoter. The levels of production of fruA mRNA and FruA were elevated in cells growing on levan, inulin, or sucrose as the sole carbohydrate source, and repression was observed when cells were grown on readily metabolizable hexoses. Deletion derivatives containing fusions of fruA promoter regions, lacking sequences 5' or 3' to the promoter, and a promoterless chloramphenicol acetyltransferase gene were used (i) to demonstrate the functionality of the promoter mapped by primer extension, (ii) to demonstrate that CCR of the fru operon requires the CRE that is located 3' to the promoter region, and (iii) to provide preliminary evidence that supports the involvement of an antitermination mechanism in fruA induction.

The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level

Abstract: The creA gene of A. nidulans encodes a wide-domain regulatory protein mediating carbon catabolite repression. Northern blot analysis of creA mRNA revealed a complex expression profile: the addition of monosaccharides to a carbon-starved culture of A. nidulans provoked a strong transient stimulation of creA transcript formation within a few minutes. In the case of repressing carbon sources, creA mRNA levels were subsequently downregulated, whereas the high creA mRNA levels were maintained in a creA mutant strain and in the presence of derepressing monosaccharides. A high creA transcript level is essential to achieve carbon catabolite repression and is dependent on glucose transport and, at least partially, on the creB gene product. Subsequent downregulation of creA mRNA levels, on the other hand, is typical of carbon catabolite repression and requires a functional CreA recognition site in the creA promoter (and thus involves autoregulation) and formation of glucose-6-phosphate. Despite the presence of continuing high transcript levels of creA in the presence of derepressing carbohydrates, EMSA demonstrated the presence of only low levels of a CreA–DNA complex in respective cell-free extracts. Upon transfer of carbon catabolite derepressed mycelia to catabolite-repressing conditions, a CreA–DNA complex is formed, and this process is dependent on de novo protein synthesis.

Inactivation and regulation of the aerobic C(4)-dicarboxylate transport (dctA) gene of Escherichia coli.

Abstract: inactivated by inserting Sp r or Ap r cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was ;10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C4-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C4dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive with respect to C4-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

The bvr locus of Listeria monocytogenes mediates virulence gene repression by beta-glucosides

Abstract: The β-glucoside cellobiose has been reported to specifically repress the PrfA-dependent virulence genes hly and plcA in Listeria monocytogenes NCTC 7973. This led to the hypothesis that β-glucosides, sugars of plant origin, may act as signal molecules, preventing the expression of virulence genes if L. monocytogenes is living in its natural habitat (soil). In three other laboratory strains (EGD, L028, and 10403S), however, the effect of cellobiose was not unique, and all fermentable carbohydrates repressed hly. This suggested that the downregulation of virulence genes by β-glucosides is not a specific phenomenon but, rather, an aspect of a global regulatory mechanism of catabolite repression (CR). We assessed the effect of carbohydrates on virulence gene expression in a panel of wild-type isolates of L. monocytogenes by using the PrfA-dependent phospholipase C gene plcB as a reporter. Utilization of any fermentable sugar caused plcB repression in wild-type L. monocytogenes. However, an EGD variant was identified in which, as in NCTC 7973, plcB was only repressed by β-glucosides. Thus, the regulation of L. monocytogenes virulence genes by sugars appears to be mediated by two separate mechanisms, one presumably involving a CR pathway and another specifically responding to β-glucosides. We have identified in L. monocytogenes a 4-kb operon, bvrABC, encoding an antiterminator of the BglG family (bvrA), a β-glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system (bvrB), and a putative ADP-ribosylglycohydrolase (bvrC). Low-stringency Southern blots showed that this locus is absent from other Listeria spp. Transcription of bvrB was induced by cellobiose and salicin but not by arbutin. Disruption of the bvr operon by replacing part of bvrAB with an interposon abolished the repression by cellobiose and salicin but not that by arbutin. Our data indicate that the bvr locus encodes a β-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found in L. monocytogenes that is involved in environmental regulation of virulence genes.

Identification of genes in an extraintestinal isolate of Escherichia coli with increased expression after exposure to human urine

Abstract: The identification of genes with increased expression in vivo may lead to the identification of novel or unrecognized virulence traits and/or recognition of environmental signals involved in modulating gene expression. Our laboratory is studying an extraintestinal isolate of Escherichia coli as a model pathogen. We had previously used human urine ex vivo to identify the unrecognized urovirulence genes guaA and argC and to establish that arginine and guanine (or derivatives) were limiting in this body fluid (T. A. Russo et al., Mol. Microbiol. 22:217-229, 1996). In this study, we have continued with this approach and identified three additional genes that have increased expression in human urine relative to Luria-Bertani (LB) medium. Expression of ure1 (urine-responsive element) is increased a mean of 47.6-fold in urine but completely suppressed by exogenous glucose. This finding suggests that ure1 is regulated by catabolite repression and that limiting glucose in urine is a regulatory signal. ure1 is present in the E. coli K-12 genome, but its function is unknown. Although disruption of ure1 results in diminished growth in human urine, limiting concentrations of amino acids, nucleosides, or iron (Fe), or changes in osmolarity or pH do not affect the expression of ure1. Therefore, Ure1 appears to have a role independent of the synthesis or uptake of these nutrients and does not appear to be involved in osmoprotection. iroN(E. coli) is a novel E. coli gene with 77% DNA homology to a catecholate siderophore receptor gene recently identified in Salmonella. Its expression is increased a mean of 27.2-fold in urine and is repressed by exogenous Fe and a urinary pH of 5.0. This finding supports the contention that Fe is a limiting element in urine and that alteration of pH can affect gene expression. It is linked to the P-pilus (prs) and F1C fimbrial (foc) gene clusters on a pathogenicity island and appears to have been acquired by IS1230-mediated horizontal transmission. The homologous iroN(E. coli) sequence is significantly more prevalent in urinary tract and blood isolates of E. coli compared to fecal isolates. Last, the expression of ArtJ, an arginine periplasmic binding protein, is increased a mean of 16.6-fold in urine. This finding implicates arginine concentrations as limited in urine and, in combination with previous data demonstrating that argC is important for urovirulence, suggests that the ability of E. coli to synthesize or acquire arginine is important for urovirulence. ure1, iroN(E. coli), and artJ all have increased expression in human blood and ascites relative to LB medium as well. The identification of these genes increases our understanding of regulatory signals present in human urine, blood, and ascites. Ure1, IroN(E. coli), and ArtJ also warrant further evaluation as virulence traits both within and outside the urinary tract.

Relationships between arginine degradation, pH and survival in Lactobacillus sakei

Abstract: Lactobacillus sakei is one of the most important lactic acid bacteria of meat and fermented meat products. It is able to degrade arginine with ammonia and ATP production by the arginine deiminase pathway (ADI). This pathway is composed of three enzymes: arginine deiminase, ornithine transcarbamoylase and carbamate kinase, and an arginine transport system. The transcription of the ADI pathway is induced by arginine and subjected to catabolite repression. In order to understand the physiological role of the degradation of this amino acid we investigated the growth of bacteria under various conditions. We show that arginine degradation is responsible for an enhanced viability during the stationary phase when cells are grown under anaerobiosis. Arginine is necessary for the induction of the ADI pathway but in association with another environmental signal. Using a mutant of the L-lactate dehydrogenase unable to lower the pH we could clearly demonstrate that (i) low pH is not responsible for cell death during the stationary phase, so survival is due to another factor than elevated pH, (ii) neither low pH nor oxygen limitation is responsible for the induction of the ADI pathway together with arginine since the ldhL mutant is able to degrade arginine under aerobiosis.

The Catabolite Control Protein CcpA Controls Ammonium Assimilation in Bacillus subtilis

Abstract: Carbon catabolite repression of several catabolic operons in Bacillus subtilis is mediated by the repressor CcpA. An inactivation of the ccpA gene has two distinct phenotypes: (i) catabolite repression of catabolic operons is lost and (ii) the growth of bacteria on minimal medium is severely impaired. We have analyzed the physiological properties of a ccpA mutant strain and show that the ccpA mutation does not affect sugar transport. We have isolated extragenic suppressors of ccpA that suppress the growth defect (sgd mutants). Catabolite repression of s-xylosidase synthesis was, however, not restored suggesting that the suppressor mutations allow differentiation between the phenotypes of the ccpA mutant. A close inspection of the growth requirements of the ccpA mutant revealed the inability of the mutant to utilize inorganic ammonium as a single source of nitrogen. An intact ccpA gene was found to be required for expression of the gltAB operon encoding glutamate synthase. This enzyme is necessary for the assimilation of ammonium. In a sgd mutant, gltAB operon expression was no longer dependent on ccpA, suggesting that the poor expression of the gltAB operon is involved in the growth defect of the ccpA mutant.

Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

Abstract: In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.

Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation.

Abstract: The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon. LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine-containing protein (HPr). We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr. The phosphoryl group transfer between HPr and LicT is reversible. Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide-urea gels. These bands probably correspond to one-, two- and threefold phosphorylated LicT. After phosphorylation of LicT with [32P]-PEP, enzyme I and HPr, proteolytic digestion of [32P]-P-LicT, separation of the peptides by reverse-phase chromatography, mass spectrometry and N-terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT. These three histidyl residues (His-159, His-207 and His-269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators. Phosphorylation of LicT in the presence of serylphosphorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylation in the presence of HPr. The slower phosphorylation in the presence of P-Ser-HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon.

Carbon catabolite repression of the Aspergillus nidulans xlnA gene

Abstract: Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA, is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creAd30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA.C1, that is responsible for direct CreA repression in vivo. Using the creAd30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.

Phosphorylation of HPr and Crh by HprK, Early Steps in the Catabolite Repression Signalling Pathway for the Bacillus subtilis Levanase Operon

Abstract: Carbon catabolite repression (CCR) of Bacillus subtilis catabolic genes is mediated by CcpA and in part by P-Ser–HPr For certain operons, Crh, an HPr-like protein, is also implicated in CCR In this study we demonstrated that in ptsH1 crh1 and hprK mutants, expression of the lev operon was completely relieved from CCR and that both P-Ser–HPr and P-Ser–Crh stimulated the binding of CcpA to the cre sequence of the lev operon

Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression.

Abstract: In baker's yeast (Saccharomyces cerevisiae) the hexokinases PI (Hxk1) and PII (Hxk2) are required for triggering of the activation of the Ras-cAMP pathway and catabolite repression. Specifically, Hxk2 is essential for the establishment of glucose repression, whereas either Hxk1 or Hxk2 can sustain fructose repression. Previous studies have suggested that the extent of glucose repression is inversely correlated with hexokinase catalytic activity and hence with an adequate elevation of intracellular sugar phosphate levels. However, several lines of evidence indicate that glucose 6-phosphate is not the trigger of catabolite repression in yeast. In the present study we employed site-directed mutagenesis of amino acids important for the binding of sugar and ATP, for efficient phosphoryl transfer and for the closure of the substrate-binding cleft, to obtain an insight into the structural requirements of Hxk2 for sugar-induced signalling. We show that the ATP-binding Lys-111 is not essential for catalysis in vivo or for signal triggering. Substitution of the catalytic-centre Asp-211 caused loss of catalytic activity, but high-affinity sugar binding was retained. However, this was not sufficient to cause cAMP activation nor catabolite repression. Mutation of Ser-158 abrogated glucose-induced, but not fructose-induced, repression. Moreover, 2-deoxyglucose sustained repression despite an extremely low catalytic activity. We conclude that the establishment of catabolite repression is dependent on the onset of the phosphoryl transfer reaction on hexokinase and is probably related to the stable formation of a transition intermediate and concomitant conformational changes within the enzyme. In contrast, the role of Hxk2 in Ras-cAMP activation seems to be directly connected to its catalytic function. The implications of this model are discussed.

Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2.

Abstract: In the yeast Saccharomyces cerevisiae, glucose or fructose represses the expression of a large number of genes. The phosphorylation of glucose or fructose is catalysed by hexokinase PI (Hxk1), hexokinase PII (Hxk2) and a specific glucokinase (Glk1). The authors have shown previously that either Hxk1 or Hxk2 is sufficient for a rapid, sugar-induced disappearance of catabolite-repressible mRNAs (short-term catabolite repression). Hxk2 is specifically required and sufficient for long-term glucose repression and either Hxk1 or Hxk2 is sufficient for long-term repression by fructose. Mutants lacking the TPS1 gene, which encodes trehalose 6-phosphate synthase, can not grow on glucose or fructose. In this study, suppressor mutations of the growth defect of a tps1Δ hxk1Δ double mutant on fructose were isolated and identified as novel HXK2 alleles. All six alleles studied have single amino acid substitutions. The mutations affected glucose and fructose phosphorylation to a different extent, indicating that Hxk2 binds glucose and fructose via distinct mechanisms. The mutations conferred different effects on long- and short-term repression. Two of the mutants showed very similar defects in catabolite repression, despite large differences in residual sugar-phosphorylation activity. The data show that the long- and short-term phases of catabolite repression can be dissected using different hexokinase mutations. The lack of correlation between in vitro catalytic hexokinase activity, in vivo sugar phosphate accumulation and the establishment of catabolite repression suggests that the production of sugar phosphate is not the sole role of hexokinase in repression. Using the set of six hxk2 mutants it was shown that there is a good correlation between the glucose-induced cAMP signal and in vivo hexokinase activity. There was no correlation between the cAMP signal and the short- or long-term repression of SUC2, arguing against an involvement of cAMP in either stage of catabolite repression.

Penicillin and cephalosporin biosynthesis: mechanism of carbon catabolite regulation of penicillin production.

Abstract: Penicillins and cephalosporins are synthesized by a series of enzymatic reactions that form the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and convert this tripeptide into the final penicillin or cephalosporin molecules. One of the enzymes, isopenicillin N synthase has been crystallyzed and its active center identified. The three genes pcbAB, pcbC and penDE involved in penicillin biosynthesis are clustered in Penicillium chrysogenum, Aspergillus nidulans and Penicillium nalgiovense. Carbon catabolite regulation of penicillin biosynthesis is exerted by glucose and other easily utilizable carbon sources but not by lactose. The glucose effect is enhanced by high phosphate concentrations. Glucose represses the biosynthesis of penicillin by preventing the formation of the penicillin biosynthesis enzymes. Transcription of the pcbAB, pcbC and penDE genes of P. chrysogenum is strongly repressed by glucose and the repression is not reversed by alkaline pHs. Carbon catabolite repression of penicillin biosynthesis in A. nidulans is not mediated by CreA and the same appears to be true in P. chrysogenum. The first two genes of the penicillin pathway (pcbAB and pcbC) are expressed from a bidirectional promoter region. Analysis of different DNA fragments of this bidirectional promoter region revealed two important DNA sequences (boxes A and B) for expression and glucose catabolite regulation of the pcbAB gene. Using protein extracts from mycelia grown under carbon catabolite repressing or derepressing conditions DNA-binding proteins that interact with the bidirectional promoter region were purified to near homogeneity.

Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the LicR regulator protein by site-directed mutagenesis.

Abstract: The lic operon of Bacillus subtilis is required for the transport and degradation of oligomeric β-glucosides, which are produced by extracellular enzymes on substrates such as lichenan or barley glucan. The lic operon is transcribed from a ςA-dependent promoter and is inducible by lichenan, lichenan hydrolysate, and cellobiose. Induction of the operon requires a DNA sequence with dyad symmetry located immediately upstream of the licBCAH promoter. Expression of the lic operon is positively controlled by the LicR regulator protein, which contains two potential helix-turn-helix motifs, two phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and a domain similar to PTS enzyme IIA (EIIA). The activity of LicR is stimulated by modification (probably phosphorylation) of both PRD-I and PRD-II by the general PTS components and is negatively regulated by modification (probably phosphorylation) of its EIIA domain by the specific EIILic in the absence of oligomeric β-glucosides. This was shown by the analysis of licR mutants affected in potential phosphorylation sites. Moreover, the lic operon is subject to carbon catabolite repression (CCR). CCR takes place via a CcpA-dependent mechanism and a CcpA-independent mechanism in which the general PTS enzyme HPr is involved.

The PecT Repressor Coregulates Synthesis of Exopolysaccharides and Virulence Factors in Erwinia chrysanthemi

Abstract: Erwinia chrysanthemi 3937 synthesizes an exopolysaccharide (EPS) composed of rhamnose, galactose, and galacturonic acid. Fourteen transcriptional fusions in genes required for EPS synthesis, named eps, were obtained by Tn5-B21 mutagenesis. Eleven of them are clustered on the chromosome and are repressed by PecT, a regulator of pectate lyase synthesis. In addition, expression of these fusions is repressed by the catabolite regulatory protein, CRP, and induced in low osmolarity medium. The three other mutations are located in genes that are not regulated by pecT. A 13-kb DNA fragment containing pecT-regulated eps genes has been cloned. All the genes identified on this fragment are transcribed in the same orientation and could form a large operon. The promoter region of this operon has been sequenced. It contains a JUMP-start sequence, a sequence required for the expression of polysaccharide-associated operons. E. chrysanthemi 3937 produces a systemic soft rot on its host Saintpaulia ionantha. An eps mutant ...

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

Abstract: Summary: 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 K m and V max 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 °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.

The PalkBFGHJKL promoter is under carbon catabolite repression control in Pseudomonas oleovorans but not in Escherichia coli alk+ recombinants.

Abstract: The alk genes are located on the OCT plasmid of Pseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in P. oleovorans and Escherichia coli alk+ recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The alk genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both E. coli and pseudomonads. Pseudomonas putida GPo12 is a P. oleovorans derivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant E. coli HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the alkB gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently in Pseudomonas and in E. coli strains. These results also indicate that PalkBFGHJKL, the Palk promoter, might be useful in attaining high expression levels of heterologous genes in E. coli grown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.

Elements involved in catabolite repression and substrate induction of the lactose operon in

Abstract: ABSTRACT In Lactobacillus casei ATCC 393, the chromosomally encoded lactose operon, lacTEGF, encodes an antiterminator protein (LacT), lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements (LacE and LacF), and a phospho-β-galactosidase. lacT, lacE, andlacF mutant strains were constructed by double crossover. The lacT strain displayed constitutive termination at a ribonucleic antiterminator (RAT) site, whereas lacE andlacF mutants showed an inducer-independent antiterminator activity, as shown analysis of enzyme activity obtained from transcriptional fusions of lac promoter (lacp) and lacpΔRAT with the Escherichia coli gusAgene in the different lac mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis detected a 100-nucleotide transcript starting at the transcription start site and ending a consensus RAT sequence and terminator region. In a ccpA mutant, transcription initiation was derepressed but no elongation through the terminator was observed in the presence of glucose and the inducing sugar, lactose. Full expression oflacTEGF was found only in a man ccpA double mutant, indicating that PTS elements are involved in the CcpA-independent catabolite repression mechanism probably via LacT.

Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

Abstract: Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.