Showing papers on "Catabolite repression published in 1997"

Carbon repression in aspergilli

Abstract: Many microorganisms prefer easily metabolizable carbon sources over alternative, less readily metabolized carbon sources. One of the mechanisms to achieve this is repression of the synthesis of enzymes related to catabolism of the alternative carbon sources, i.e. carbon repression. It is now clear that in Aspergillus nidulans and Aspergillus niger the repressor protein CREA plays a major role in carbon repression. CREA inhibits transcription of many target genes by binding to specific sequences in the promoter of these genes. Unfortunately there is little information on other components of the signalling pathway that triggers repression by CREA. In this review we summarize the current understanding of carbon repression in aspergilli.

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression

Abstract: Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. In this study, we report the discovery of a new B. subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids. Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh. Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate. We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage. In a B. subtilis ptsH1 mutant strain, synthesis of β-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR. Additional disruption of the crh gene caused almost complete relief from CCR. In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of β-xylosidase was also completely relieved from glucose repression. These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46.

Physiology of Carbohydrate to Solvent Conversion by Clostridia

Abstract: The solvent-forming clostridia have attracted interest because of their ability to convert a range of carbohydrates to end-products such as acetone, butanol and ethanol. Polymeric substrates such as cellulose, hemicellulose and starch are degraded by extracellular enzymes. The majority of cellulolytic clostridia, typified by Clostridium thermocellum, produce a multi-enzyme cellulase complex in which the organization of components is critical for activity against the crystalline substrate. A variety of enzymes involved in degradation of hemicellulose and starch have been identified in different strains. The products of degradation, and other soluble substrates, are accumulated via membrane-bound transport systems which are generally poorly characterized. It is clear, however, that the phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in solute uptake in several species. Accumulated substrates are converted by intracellular enzymes to end-products characteristic of the organism, with production of ATP to support growth. The metabolic pathways have been described, but understanding of mechanisms of regulation of metabolism is incomplete. Synthesis of extracellular enzymes and membrane-bound transport systems is commonly subject to catabolite repression in the presence of a readily metabolized source of carbon and energy. While many genes encoding cellulases, xylanases and amylases have been cloned and sequenced, little is known of control of their expression. Although the mechanism of catabolite repression in clostridia is not understood, some recent findings implicate a role for the PTS as in other low G-C Gram-positive bacteria. Emphasis has been placed on describing the mechanisms underlying the switch of C. acetobutylicum fermentations from acidogenic to solventogenic metabolism at the end of the growth phase. Factors involved include a lowered pH and accumulation of undissociated butyric acid, intracellular concentration of ATP and reduced pyridine nucleotides, nutrient limitation, and the interplay between pathways of carbon and electron flow. Genes encoding enzymes of solvent pathways have been cloned and sequenced, and their expression correlated with the pattern of end-product formation in fermentations. There is evidence that the initiation of solvent formation may be subject to control mechanisms similar to other stationary-phase phenomena, including sporulation. The application of recently developed techniques for genetic manipulation of the bacterium is improving understanding of the regulatory circuits, but a complete molecular description of the control of solvent formation remains elusive. Experimental manipulation of the pathways of electron flow in other species has been shown to influence the range and yield of fermentation end-products. Acid-forming clostridia can, under appropriate conditions, be induced to form atypical solvents as products. While the mechanisms of regulation of gene expression are not at all understood, the capacity to adapt in this way further illustrates the metabolic flexibility of clostridial strains.

Mathematical Model of the lac Operon: Inducer Exclusion, Catabolite Repression, and Diauxic Growth on Glucose and Lactose

Abstract: A mathematical model of the lactose (lac) operon was developed to study diauxic growth on glucose and lactose. The model includes catabolite repression, inducer exclusion, lactose hydrolysis to glucose and galactose, and synthesis and degradation of allolactose. Two models for catabolite repression were tested: (i) cyclic AMP (cAMP) synthesis inversely correlated with the external glucose concentration and (ii) synthesis inversely correlated with the glucose transport rate. No significant differences in the two models were observed. In addition to synthesis, degradation and secretion of cAMP were also included in the model. Two models for the phosphorylation of the glucose produced from lactose hydrolysis were also tested: (i) phosphorylation by intracellular hexokinase and (ii) secretion of glucose and subsequent phosphorylation upon transport back into the cell. The latter model resulted in weak catabolite repression when the glucose produced from lactose was transported out of the cell, whereas the former model showed no catabolite repression during growth on lactose. Parameter sensitivity analysis indicates the importance of key parameters to lac operon expression and cell growth: the lactose and allolactose transformation rates by beta-galactosidase and the glucose concentrations that affect catabolite repression and inducer exclusion. Large values of the allolactose hydrolysis rate resulted in low concentrations of allolactose, low-level expression of the lac operon, and slow growth due to limited import and metabolism of lactose; small values resulted in a high concentration of allolactose, high-level expression of the lac operon, and slow growth due to a limiting concentration of glucose 6-phosphate formed from allolactose. Changes in the rates of all beta-galactosidase-catalyzed reactions showed similar behavior, but had more drastic effects on the growth rate. Changes in the glucose concentration that inhibited lactose transport could extend or contract the diauxic growth period during growth in the presence of glucose and lactose. Moreover, changes in the glucose concentration that affected catabolite repression affected the cAMP levels and lac operon expression, but had a lesser effect on the growth rate.

Carbon‐source regulation of virulence gene expression in Listeria monocytogenes

Abstract: All known virulence genes of Listeria monocytogenes are under positive regulation by the transcription factor PrfA. Previous work employing the L. monocytogenes strain NCTC7973 suggested that the disaccharide cellobiose might serve as a specific "signature molecule' which functions to prevent activation of the PrfA-controlled regulon in a soil environment. We have examined three other L. monocytogenes strains, 10403S, LO28 and EGD, all commonly regarded as wild-type isolates, and find that NCTC7973 is anomalous with respect to the effect of carbohydrates on the expression of PrfA-controlled gene expression. In the case of 10403S, LO28 and EGD, several other readily metabolized mono- and disaccharides are as effective as cellobiose in repressing expression of the PrfA-controlled gene hly, indicating that the cellobiose effect is not specific, and suggesting that NCTC7973 may be a partially deregulated variant. Moreover, concentrations of cellobiose and other sugars required for repression of hly expression (> 1 mM) were found to significantly enhance growth of L. monocytogenes cultures, suggesting that the repression phenomenon probably results from a metabolic effect of sugar utilization rather than a signal-sensing response. Thus the previously reported cellobiose effect may reflect an aspect of a more global mechanism of catabolite repression in L. monocytogenes. Although cellobiose represses expression of hly and plcA at the level of transcript accumulation, quantitative Western blot analysis indicates that cellobiose has no effect on PrfA levels. These results are consistent with a model in which PrfA activity is controlled by interaction with a hypothetical cofactor, the synthesis or depletion of which is responsive to the presence of readily metabolized carbohydrates.

Anaerobic citrate metabolism and its regulation in enterobacteria.

Abstract: Several species of enterobacteria are able to utilize citrate as carbon and energy source. Under oxic conditions in the presence of a functional tricarboxylic acid cycle, growth on this compound solely depends on an appropriate transport system. During anaerobiosis, when 2-oxoglutarate dehydrogenase is repressed, some species such as Klebsiella pneumoniae and Salmonella typhimurium, but not Escherichia coli, are capable of growth on citrate by a Na+-dependent pathway forming acetate, formate, and CO2 as products. During the last decade, several novel features associated with this type of fermentation have been discovered in K. pneumoniae. The biotin protein oxaloacetate decarboxylase, one of the key enzymes of the pathway besides citrate lyase, is a Na+ pump. Recently it has been shown that the proton required for the decarboxylation of carboxybiotin is taken up from the side to which Na+ ions are pumped, and a membrane-embedded aspartate residue that is probably involved both in Na+ and in H+ transport was identified. The Na+ gradient established by oxaloacetate decarboxylase drives citrate uptake via CitS, a homodimeric carrier protein with a simultaneous-type reaction mechanism, and NADH formation by reversed electron transfer involving formate dehydrogenase, quinone, and a Na+-dependent NADH:quinone oxidoreductase. All enzymes specifically required for citrate fermentation are induced under anoxic conditions in the presence of citrate and Na+ ions. The corresponding genes form a cluster on the chromosome and are organized as two divergently transcribed operons. Their co-ordinate expression is dependent on a two-component system consisting of the sensor kinase CitA and the response regulator CitB. The citAB genes are part of the cluster and are positively autoregulated. In addition to CitA/CitB, the cAMP receptor protein (Crp) is involved in the regulation of the citrate fermentation enzymes, subjecting them to catabolite repression.

The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

Abstract: The concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli. Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose. Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate:glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase. A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter. To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E. coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes. Responses were followed in batch, dialysis and glucose-limited continuous culture. A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0.3 mM glucose. Likewise, addition of >0.3 mM glucose, but not <0.3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria. There was no striking shift in growth rate or [14C]glucose assimilation in bacteria passing through the 0.5 to 0.3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels. The (IIA/IIBC)GIc PTS is 96-97% saturated at 0.3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation. Aside from the transition in cAMP levels initiated above 0.3 mM, a second shift occurred below 1 μM glucose. Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E. coli K-12 strains.

Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole.

Abstract: When Saccharomyces cerevisiae cells growing on galactose are transferred onto glucose medium containing cycloheximide, an inhibitor of protein synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed. We show that glucose-induced inactivation of Gal2p is due to its degradation. Stabilization of Gal2p in pra1 mutant cells devoid of vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis. In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex. In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway. Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome. Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation.

Glucose repression affects ion homeostasis in yeast through the regulation of the stress‐activated ENA1 gene

Abstract: In this report we show that the ENA1/PMR2A gene is under glucose repression. The SNF1 protein kinase, acting independently from the HOG and calcineurin pathways, is essential to release ENA1 from glucose repression. The transcriptional repressor Ssn6p negatively regulates ENA1 expression and, like other glucose repressible genes, this repression is mediated in part by Mig1p. Deletion of a fragment from the ENA1 promoter that includes two Mig1p consensus binding sites gives a high level of expression in glucose without added salt. We suggest that regulation of ENA1 by the SNF1 pathway could be part of a general mechanism through which yeast cells respond to carbon source starvation by activating protective systems against different types of stress.

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

Abstract: The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria.

The cyclic AMP receptor protein is the main activator of pectinolysis genes in Erwinia chrysanthemi.

Abstract: The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall. Although physiological studies suggested that pectinase production in Erwinia species is subjected to catabolite repression, the direct implication of the cyclic AMP receptor protein (CRP) in this regulation has never been demonstrated. To investigate the role of CRP in pectin catabolism, we cloned the E. chrysanthemi crp gene by complementation of an Escherichia coli crp mutation and then constructed E. chrysanthemi crp mutants by reverse genetics. The carbohydrate fermentation phenotype of the E. chrysanthemi crp mutants is similar to that of an E. coli crp mutant. Furthermore, these mutants are unable to grow on pectin or polygalacturonate as the sole carbon source. Analysis of the nucleotide sequence of the E. chrysanthemi crp gene revealed the presence of a 630-bp open reading frame (ORF) that codes for a protein highly similar to the CRP of E. coli. Using a crp::uidA transcriptional fusion, we demonstrated that the E. chrysanthemi CRP represses its own expression, probably via a mechanism similar to that described for the E. coli crp gene. Moreover, in the E. chrysanthemi crp mutants, expression of pectinase genes (pemA, pelB, pelC, pelD, and pelE) and of genes of the intracellular part of the pectin degradation pathway (ogl, kduI, and kdgT), which are important for inducer formation and transport, is dramatically reduced in induced conditions. In contrast, expression of pelA, which encodes a pectate lyase important for E. chrysanthemi pathogenicity, seems to be negatively regulated by CRP. The E. chrysanthemi crp mutants have greatly decreased maceration capacity in potato tubers, chicory leaves, and celery petioles as well as highly diminished virulence on saintpaulia plants. These findings demonstrate that CRP plays a crucial role in expression of the pectinolysis genes and in the pathogenicity of E. chrysanthemi.

Expression of the SUC2 Gene of Saccharomyces cerevisiae is Induced by Low Levels of Glucose

Abstract: High levels of glucose repress expression of the SUC2 gene in the yeast Saccharomyces cerevisiae. We have discovered that low levels of glucose are required for maximal transcription of SUC2: SUC2 expression is induced about five- to ten-fold in cells growing on low levels of glucose (0.1%) compared to cells growing on galactose or glycerol. Two pieces of evidence suggest that this low-glucose-induced expression is mediated by a repression mechanism that involves an upstream repression site in the SUC2 promoter (URS(SUC2)). First, deletion of the URS(SUC2) results in expression of the SUC2 gene in the absence of glucose, and second the URS(SUC2) mediates a six-fold repression of a reporter gene when inserted into a heterologous promoter. However, this URS(SUC2) mediated repression occurs on all tested carbon sources, suggesting that this URS element acts in concert with all other promoter elements to respond to low concentrations of glucose. This repression requires the general repressor SSn6p. SNF3, which encodes a glucose transporter that appears to be a sensor of low levels of glucose, is also required for low-glucose-induced expression of SUC2.

Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937

Abstract: Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of paeY expression appears to be dependent on the KdgR repressor, which controls all the steps of pectin catabolism, and on the catabolite regulatory protein (CRP), the global activator of sugar catabolism. The contiguous pelD, paeY and pemA genes are transcribed as an operon from a promoter proximal to pelD which allows the regulation by KdgR and CRP. However, transcription can be interrupted at the intra-operon Rho-independent terminator situated between pelD and paeY. The paeY mutant inoculated into Saintpaulia plants was less invasive than the wild-type E. chrysanthemi strain 3937, demonstrating the important role of PaeY in the soft-rot disease.

Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis.

Abstract: A new catabolic system in Bacillus subtilis involved in utilization of beta-glucosidic compounds has been investigated. It consists of five genes encoding phosphotransferase system (PTS) enzyme II (licB and licC) and enzyme IIA (licA), a presumed 6-phospho-beta-glucosidase (licH), as well as a putative regulator protein (licR). The genes map around 334 degrees of the B. subtilis chromosome, and their products are involved in the uptake and utilization of lichenan degradation products. These five genes are organized in two transcriptional units. A weak promoter precedes gene licR, and transcription is obviously terminated at a secondary structure immediately downstream of the reading frame, as shown by Northern RNA blot analysis. Genes licB, licC, licA, and licH constitute an operon. Initiation of transcription at the promoter in front of this operon presumably requires activation by the gene product of licR. The LicR protein shows an unusual domain structure, i.e., similarities to (i) the conserved transcriptional antiterminator BgIG family signature and (ii) PTS enzyme II. Using RNA techniques and transcriptional lacZ fusions, we have shown that the expression of the licBCAH operon is inducible by products of lichenan hydrolysis, lichenan and cellobiose. The presence of excess glucose prevents the induction of this operon, indicating the control by carbon catabolite repression. Moreover, the expression of the operon requires the general PTS components and seems to be negatively controlled by the specific lic PTS enzymes.

Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite‐responsive elements

Abstract: Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-Lacl family of bacterial regulatory proteins, and the seryl-phosphorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the cre in the first gene (gntR) of the gnt operon (credown), this operon contains another cre located in the promoter region (creup). A translational gntR'-'lacZ fusion expressed under the control of various combinations of wild-type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of beta-galactosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P-ser-HPr, and catabolite repression exerted by credown was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose-6-phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.

The D-allose operon of Escherichia coli K-12.

Abstract: Escherichia coli K-12 can utilize D-allose, an all-cis hexose, as a sole carbon source. The operon responsible for D-allose metabolism was localized at 92.8 min of the E. coli linkage map. It consists of six genes, alsRBACEK, which are inducible by D-allose and are under the control of the repressor gene alsR. This operon is also subject to catabolite repression. Three genes, alsB, alsA, and alsC, appear to be necessary for transport of D-allose. D-Allose-binding protein, encoded by alsB, is a periplasmic protein that has an affinity for D-allose, with a Kd of 0.33 microM. As was found for other binding-protein-mediated ABC transporters, the allose transport system includes an ATP-binding component (AlsA) and a transmembrane protein (AlsC). It was found that AlsE (a putative D-allulose-6-phosphate 3-epimerase), but not AlsK (a putative D-allose kinase), is necessary for allose metabolism. During this study, we observed that the D-allose transporter is partially responsible for the low-affinity transport of D-ribose and that strain W3110, an E. coli prototroph, has a defect in the transport of D-allose mediated by the allose permease.

Catabolite repression by glucose 6-phosphate, gluconate and lactose in Escherichia coli.

Abstract: Summary While catabolite repression by glucose has been studied extensively and is understood in large detail in Enterobacteriaceae, catabolite repression by carbohydrates that are not transported by the phosphotransferase system (PTS) has always remained an enigma. Examples of non-PTS carbohydrates that cause catabolite repression in Escherichia coli are gluconate, lactose and glucose 6-phosphate. In this article it is shown that enzyme IIA Glc of the PTS is not involved in catabolite repression by these carbon sources. Carbon sources that caused strong catabolite repression of b-galactosidase lowered the concentration of both cAMP and the cAMP receptor protein (CRP). A strong correlation was found between the amounts of cAMP and CRP and the strength of the repression. The levels of cAMP and CRP were modulated in various ways. Neither overproduction of CRP nor an increased cAMP concentration could completely relieve the repression by glucose 6-phosphate, lactose and gluconate. Simultaneously increasing the cAMP and the CRP levels was lethal for the cells. In a mutant expressing a constant amount of cAMP-independent CRP *protein, catabolite repression was absent. The same was found in a mutant in which lac transcription is independent of cAMP/CRP. These results, combined with the fact that both the cAMP and the CRP levels are lowered by glucose 6-phosphate, lactose and gluconate, lead to the conclusion that the decreased cAMP and CRP levels are the cause of catabolite repression by these nonPTS carbon sources.

Comparative physiology of salt tolerance in Candida tropicalis and Saccharomyces cerevisiae

Abstract: The salt tolerance of the respiratory yeast Candida tropicalis and the fermentative yeast Saccharomyces cerevisiae have been compared in glucose media. C. tropicalis showed a better adaptation to Na+ and Li+ and maintained higher intracellular K+:Na+ and K+:Li+ ratios than S. cerevisiae. However, C. tropicalis showed a poorer adaptation to osmotic stress (produced by KCI and sorbitol) and exhibited reduced glycerol production as compared to S. cerevisiae. In media with the non-repressing sugar galactose as carbon source, S. cerevisiae exhibited reduced glycerol production and increased sensitivity to osmotic stress. Under these conditions, S. cerevisiae, but not C. tropicalis, utilized trehalose as a more important osmolyte than glycerol. These results suggest that the relative tolerance of yeast to the osmotic and cation toxicities of NaCl, and the underlying relative capabilities for osmolyte synthesis and cation transport, are modulated by the general catabolite control exerted by glucose.

The geranylgeranyl diphosphate synthase gene of Gibberella fujikuroi: isolation and expression.

Abstract: The rice pathogen, Gibberella fujikuroi, produces large amounts of gibberellins, a group of natural plant hormones, which induce the superelongation (bakanae) disease of rice. Gibberellins are diterpenoid compounds which are synthesized via the isoprenoid pathway. Here we report the isolation and molecular characterization of the geranylgeranyl diphosphate synthase (ggs) gene from G. fujikuroi. Geranylgeranyl diphosphate synthase is a key enzyme in isoprenoid biosynthesis. Southern blot analysis showed that G. fujikuroi has a single copy of the ggs gene, which is not linked to the farnesyl diphosphate synthase gene. This indicates that the genes of the isoprenoid pathway are not clustered in the fungal genome. The ggs gene is not interrupted by an intron and codes for a polypetide of 418 amino acids. Peptide sequence comparison showed a high degree of similarity to the corresponding Neurospora crassa gene (al-3). However, transcription analyses revealed that the ggs gene, in contrast to the analogous N. crassa gene, is not regulated by blue light. Ammonium and glucose did not affect the transcription of the G. fujikuroi ggs gene, indicating that it is not subject to nitrogen and carbon catabolite repression. The G. fujikuroi gene complements a N. crassa al-3 mutant.

Catabolite inactivation of the yeast maltose transporter requires ubiquitin-ligase npi1/rsp5 and ubiquitin-hydrolase npi2/doa4.

Abstract: The maltose transporter in Saccharomyces cerevisiae is degraded in the vacuole after internalization by endocytosis when protein synthesis is impaired and a fermentable substrate is present. The possible implication of the ubiquitin pathway in this inactivation, known as catabolite inactivation, has been investigated. Using mutants deficient in npi1/rsp5 ubiquitin-protein ligase and npi2/doa4 ubiquitin-protein hydrolase, we have shown that these two enzymes are required for normal endocytosis and degradation of the transporter. These facts indicate that the ubiquitin pathway is involved in catabolite inactivation of the maltose transporter. The results also revealed that both enzymes act in the internalization step of endocytosis.

Construction and properties of aconitase mutants of Escherichia coli.

Abstract: Escherichia coli contains two genes (acnA and acnB) encoding aconitase activities. An acnB mutant was engineered by replacing the chromosomal acnB gene by an internally deleted derivative containing a tetR cassette. An acnB double mutant was then made by transducing a previously constructed acnA::kanR mutation into the acnB::tetR strain. Western blotting confirmed that the AcnA and AcnB proteins were no longer produced by the corresponding mutants and PCR analysis showed that the chromosomal acnB gene had been replaced by the disrupted gene. Aerobic and anaerobic growth in glucose minimal medium were impaired but not abolished by the acnB mutation, indicating that the lesion is partially complemented by the acnA+ gene, and growth was enhanced by glutamate. The acnAB double mutant would not grow on unsupplemented glucose minimal medium and although it responded to glutamate like a typical auxotroph under anaerobic conditions, under aerobic conditions no response to glutamate was observed before it was over-grown by 'revertants' lacking citrate synthase (acnAB gltA). The acnAB double mutant retained a low but significant aconitase activity (< or = 5% of wild-type), designated AcnC. Enzymological and regulatory studies with acn-lacZ fusions indicated that AcnB is the major aconitase, which is synthesized earlier in the growth cycle than AcnA, and subject to catabolite and anaerobic repression.

Aerobic regulation of isocitrate dehydrogenase gene (icd) expression in Escherichia coli by the arcA and fnr gene products.

Abstract: Isocitrate dehydrogenase, the icd gene product, has been studied extensively regarding the regulation of enzymatic activity and its relationship to the metabolic flux between the tricarboxylic acid cycle and the glyoxylate bypass. In this study, the transcriptional regulation of icd gene expression was monitored by using an icd-lacZ gene fusion and shown to vary over a 15-fold range in response to changes in oxygen and carbon availability. Anaerobic cell growth resulted in fivefold-lower icd-lacZ expression than during aerobic growth. This negative control is mediated by the arcA and fnr gene products. When different carbon compounds were used for cell growth, icd-lacZ expression varied threefold. The results of continuous cell culture studies indicated that this control may be due to variations in cell growth rate rather than to catabolite repression. DNase I footprinting at the icd promoter revealed a 42-bp ArcA-phosphate-protected region that overlaps the start site of icd transcription. Phosphorylation of ArcA considerably enhanced its binding to DNA, while ArcA-phosphate exhibited an apparent dissociation value of approximately 0.1 microM. Based on these studies, ArcA appears to function as a classical repressor of transcription by binding at a site overlapping the icd promoter during anaerobic cell growth conditions.

A stable lipase from Candida lipolytica: cultivation conditions and crude enzyme characteristics.

Abstract: Although lipases have been intensively studied, some aspects of enzyme production like substrate uptake, catabolite repression, and enzyme stability under long storage periods are seldom discussed in the literature. This work deals with the production of lipase by a new selected strain of Candida lipolytica. Concerning nutrition, it was observed that inorganic nitrogen sources were not as effective as peptone, and that oleic acid or triacylglycerides (TAG) were essential carbon sources. Repression by glucose and stimulation by oleic acid and long chain TAG (triolein and olive oil) were observed. Extracellular lipase activity was only observed at high levels at late stationary phase, whereas intracellular lipase levels were constant and almost undetectable during the cultivation period, suggesting that the produced enzyme was attached to the cell wall, mainly at the beginning of cultivation. The crude lipase produced by this yeast strain shows the following optima conditions: pH 8.0-10.0, temperature of 55 degrees C. Moreover, this preparation maintains its full activity for at least 370 d at 5 degrees C.

Partial Purification and Characterization of Red Chlorophyll Catabolite Reductase, a Stroma Protein Involved in Chlorophyll Breakdown

Abstract: Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family.

Establishing a model to study the regulation of the lactose operon in Lactobacillus casei

Abstract: The chromosomally encoded lactose-specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) has been investigated in Lactobacillus casei ATCC 393 [pLZ15-] and it was considered an excellent system to study the regulation of the lactose operon. This chromosomal operon has been cloned and sequenced, being 99% homologous to that encoded on the plasmid pLZ64. Expression of the lactose operon in different mutants of L. casei ATCC 393 [pLZ15−] and primer extension analysis revealed that it is subject to a dual regulation: (i) glucose repression possibly mediated by CcpA and PTS elements, and (ii) induction by lactose through transcriptional antitermination.

Influence of Mutations in Hexose‐Transporter Genes on Glucose Repression in Kluyveromyces Lactis

Abstract: The variability of Kluyveromyces lactis strains in sensitivity to glucose is correlated with genetic differences in Kluyveromyces hexose transporter (KHT) genes. The glucose sensitive strain JA6 was shown to contain an additional gene, KHT2, not found in strains that are less sensitive. KHT2 is tandemly arranged with KHT1 which is identical to the low-affinity transporter gene RAG1, except for the C-terminus. Sequence analysis indicated that most of KHT2 had been lost by a recombination event between KHT1 and KHT2 generating the chimeric gene RAG1. Recombination between KHT1 and KHT2 was also found in mutants of JA6 selected as 2-deoxyglucose resistant colonies. These mutants, like khtl kht2 double mutants were unable to grow on glucose when respiration was blocked (Rag− phenotype) and glucose repression was strongly reduced, kht1 or kht2 single mutants of JA6 were Rag+ but still an influence of the kht mutations on glucose repression was detectable. Repression was not affected in a Rag− mutant deleted for the phosphoglucose isomerase gene suggesting that the influence of transporter genes on repression is not caused by a reduction of the glycolytic flux. The data rather suggest that sensitivity to glucose repression is dependent on the rate of glucose uptake.