Regulation of the galactose-inducible lac operon and the histidine utilization operons in pts mutants of Klebsiella aerogenes.

Galactose appears to be the physiological inducer of the chromosomal lac operon in Klebsiella aerogenes. Both lactose and galactose are poor inducers in strains having a functional galactose catabolism (gal) operon, but both are excellent inducers in gal mutants. Thus the slow growth of K. aerogenes on lactose reflects the rapid degradation of the inducer. Several pts mutations were characterized and shown to affect both inducer exclusion and permanent catabolite repression. The beta-galactosidase of pts mutants cannot be induced at all by lactose, and pts mutants appear to have a permanent and constitutive inducer exclusion phenotype. In addition, pts mutants show a reduced rate of glucose metabolism, leading to slower growth on glucose and a reduced degree of glucose-mediated permanent catabolite repression. The crr-type pseudorevertants of pts mutations relieve the constitutive inducer exclusion for lac but do not restore the full level of glucose-mediated permanent catabolite repression and only slightly weaken the glucose-mediated inducer exclusion. Except for weakening the glucose-mediated permanent catabolite repression, pts and crr mutations have no effect on expression of the histidine utilization (hut) operons.     

Catabolite repression of the lac operon: effect of operator-constitutive mutations

Summary We have constructed and tested three lac diploid strains in an attempt to show whether operator-constitutive mutations relieve catabolite repression of the lac operon. Each of these carries a different operator mutation on the chromosome, and all three have the genotype I⁺P⁺O^cZ⁺Y^-polar/Flac I⁺P⁺O⁺Z^delY⁺A⁺. When these strains were grown in medium containing glucose plus gluconate, synthesis of -galactosidase (directed by a gene cis to a mutant operator) and of thiogalactoside transacetylase (directed by a gene cis to an intact operator) suffered equal catabolite repression. We conclude that the operator-constitutive mutations have no effect on catabolite repression. Since it has been shown in analogous experiments that all promoter mutations tested do alleviate catabolite repression, these results are consistent with the view that the operator and promoter are functionally distinct.     

ScoC Mediates Catabolite Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and Ï^H in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucoseReceived: 12 November 2002 / Accepted: 13 December 2002     

Regulation of Arabinose and Xylose Metabolism in Escherichia coli

Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.The transporters and enzymes in many sugar metabolic pathways are conditionally expressed in response to their cognate sugar or a downstream pathway intermediate. While the induction of these pathways in response to a single sugar has been studied extensively (28), far less is known about how these pathways are induced in response to multiple sugars. One notable exception is the phenomenon observed when bacteria are grown in the presence of glucose and another sugar (10, 15). In such mixtures, the bacteria will often consume glucose first before consuming the other sugar, a process known as carbon catabolite repression (27). The classic example of carbon catabolite repression is the diauxic shift seen in the growth of Escherichia coli on mixtures of glucose and lactose, where the cells first consume glucose before consuming lactose. When the cells are consuming glucose, the genes in the lactose metabolic pathway are not induced, thus preventing the sugar from being consumed. A number of molecules participate in this regulation, including the cyclic AMP receptor protein (CRP), adenylate cyclase, cyclic AMP (cAMP), and EIIA from the phosphoenolpyruvate:glucose phosphotransferase system (PTS) (33). In addition to lactose, the metabolic genes for many other sugars are subject to catabolite repression by glucose in E. coli (27). While the preferential utilization of glucose is well known, it is an open question whether additional hierarchies exist among other sugars.Recently, substantial effort has been directed toward developing microorganisms capable of producing chemicals and biofuels from plant biomass (1, 34, 42). After glucose, l-arabinose and d-xylose are the next most abundant sugars found in plant biomass. Therefore, a key step in producing various chemicals and fuels from plant biomass will be the engineering of strains capable of efficiently fermenting these three sugars. However, one challenge concerns catabolite repression, which prevents microorganisms from fermenting these three sugars simultaneously and, as a consequence, may decrease the efficiency of the fermentation process. E. coli cells will first consume glucose before consuming either arabinose or xylose. As in the case of lactose, the genes in the arabinose and xylose metabolic pathways are not expressed when glucose is being consumed. In addition to glucose catabolite repression, a second hierarchy, between arabinose and xylose, appears to exist. Kang and coworkers have observed that the genes in the xylose metabolic pathway were repressed when cells were grown in a mixture of arabinose and xylose (21). Hernandez-Montalvo and coworkers also observed that E. coli utilizes arabinose before xylose (19). While a number of strategies exist for breaking the glucose-mediated repression of arabinose and xylose metabolism (8, 16, 19, 31), none exist for breaking the arabinose-mediated repression of xylose metabolism. Moreover, little is known about this repression beyond the observations made by these researchers.In this work, we investigate how the arabinose and xylose metabolic pathways are jointly regulated. We demonstrate that E. coli will consume arabinose before consuming xylose when it is grown in a mixture of the two sugars. Consistent with a mechanism involving catabolite repression, the genes in the xylose metabolic pathway are repressed in the presence of arabinose. We found that this repression is AraC dependent and is most likely due to binding by arabinose-bound AraC to the xylose promoters, with consequent inhibition of gene expression.     

Catabolite repression mutants of yeast

The mechaṅism of catabolite repression in yeast is not well understood, although it has been established that cAMP does not play a role similar to that found in Escherichia coli. To identify the elements implicated in catabolite repression in yeast, a variety of mutants affected in this process have been isolated by different research groups. A systematic review of the results reported in the literature is presented. The conclusion that can be drawn is that the mechanism of catabolite repression is a complex one, with no single gene controlling all the genes subject to repression. The expression of a given gene or set of genes is controlled by several regulatory genes, but it is not yet known whether these genes act cooperatively or sequentially.     

A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and β-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on β-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, β-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.     

Expression and regulation of lactose genes carried by plasmids.

A number of plasmids carrying the lactose character have been studied. All of the plasmids examined so far code for proteins essential for lactose utilization, i.e., beta-galactosidase and galactoside permease. None of them carries enzymatically or immunologically detectable thiogalactoside transacetylase. The expression of the two enzymes is both negatively and positively controlled: they are inducible by different galactosides and are sensitive to catabolite repression. Since the plasmid-coded lactose systems have many features in common with the Escherichia coli lactose operon, it is suggested that the plasmids could have acquired the lactose genes from an E. coli chromosome.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels

The regulation of the syntheses of two arabinan-degrading extracellular enzymes and several intracellular l-arabinose catabolic enzymes was examined in wild-type and carbon catabolite derepressed mutants of Aspergillus nidulans. α-l-Arabinofuranosidase B, endoarabinase, l-arabinose reductase, l-arabitol dehydrogenase, xylitol dehydrogenase, and l-xylulose reductase were all inducible to varying degrees by l-arabinose and l-arabitol and subject to carbon catabolite repression by d-glucose. With the exception of l-xylulose reductase, all were clearly under the control of creA, a negative-acting wide domain regulatory gene mediating carbon catabolite repression. Measurements of intracellular enzyme activities and of intracellular concentrations of arabitol and xylitol in mycelia grown on d-glucose in the presence of inducer indicated that carbon catabolite repression diminishes, but does not prevent uptake of inducer. Mutations in creA resulted in an apparently, in some instances very marked, elevated inducibility, perhaps reflecting an element of “self” catabolite repression by the inducing substrate. creA mutations also resulted in carbon catabolite derepression to varying degrees. The regulatory effects of a mutation in creB and in creC, two genes whose roles are unclear, but likely to be indirect, were, when observable, more modest. As with previous data showing the effect of creA mutations on structural gene expression, there were striking instances of phenotypic variation amongst creA mutant alleles and this variation followed no discernible pattern, i.e. it was non-hierarchical. This further supports molecular data obtained elsewhere, indicating a direct role for creA in regulating structural gene expression, and extends the range of activities under creA control.     

Regulation of alcR, the positive regulatory gene of the ethanol utilization regulon of Aspergillus nidulans

The alcR positive control gene is necessary for the expression of both alcA (coding for alcohol dehydro-genase ADH I.) and aldA (coding for aldehyde dehydro-genase, AldDH) in Aspergillus nidulans. Using a cloned alcR probe and Northern blots analysis we show that: (1)alcR itself is inducible; (2)alcR inducibility depends on the expression of the alcR gene Itself; and (3) alcR is subject to carbon catabolite repression and its expression Is controlled by the negatively acting creA wide specificity gene. The repression of alcR is sufficient to explain the cariaon catabolite repression of ADH I and AldDH.     

The role of mitochondria in carbon catabolite repression in yeast

Summary The role of mitochondria in carbon catabolite repression in Saccharomyces cerevisiae was investigated by comparing normal, respiratory competent (RHO) strains with their mitochondrially inherited, respiratory deficient mutant derivatives (rho). Formation of maltase and invertase was used as an indicator system for the effect of carbon catabolite repression on carbon catabolic reactions. Fermentation rates for glucose, maltose and sucrose were the same in RHO and rho strains. Specific activities of maltase and invertase were usually higher in the rho-mutants. A very pronounced difference in invertase levels was observed when cells were grown on maltose; rho-mutants had around 30 times more invertase than their RHO parent strains.The fact that rho-mutants were much less sensitive to carbon catabolite repression of invertase synthesis than their RHO parents was used to search for the mitochondrial factor(s) or function(s) involved in carbon catabolite repression. A possible metabolic influence of mitochondria on this system of regulation was tested after growth of RHO strains under anaerobic conditions (no respiration nor oxidative phosphorylation), in the presence of KCN (respiration inhibited), dinitrophenol (uncoupling of oxidative phosphorylation) and of both inhibitors anaerobic conditions and dinitrophenol had no effect on the extent of invertase repression. KCN reduced the degree of repression but not to the level found in rho-mutants. A combination of both inhibitors gave the same results as with KCN alone. Erythromycin and chloramphenicol were used as specific inhibitors of mitochondrial protein synthesis. Erythromycin prevented the formation of mitochondrial respiratory systems but did not induce rho-mutants under the conditions used. However, repression of invertase was as strong as in the absence of the inhibitor. Chloramphenicol led only to a slight reduction of the respiratory systems and did not affect invertase levels. A combination of both antibiotics had about the same effect as growth in the presence of KCN.The results showed that mitochondria are involved in carbon catabolite repression and they cause an increase in the degree of repression. These effects cannot be due to mere metabolic activities nor to factors made on the mitochondrial protein synthesizing machinery. This regulatory role of mitochondria is observed as long as an intact mitochondrial genome is maintained.     

Induction and Repression of β-Galactosidase Synthesis by Verticillum albo-atrum

Verticillium albo-atrum grew on lactose-containing culture media only after a prolonged lag phase. The intracellular specific activity of β-galactosidase [EC 3.2.1.23] increased 40–200 times during he lag phase. The β-galactosidase was induced by lactose and to a lesser degree by galactose. The appearance of the enzyme in lactose cultures was decreased by cycloheximide. Glucose and other readily metabolized carbon sources were effective repressors of β-galactosidase production. The production of β-galactosidase therefore appeared under control by lactose induction and catabolite repression.