Glucose transport in crabtree-positive and crabtree-negative yeasts

The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0.1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.     

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5–6.0 for E. fibuligera and 5.5–7.0 for S. castellii.     

Glucose kinase has a regulatory role in carbon catabolite repression in Streptomyces coelicolor.

A glucose kinase (glkA) mutant of Streptomyces coelicolor A3(2) M145 was selected by the ability to grow in the presence of the nonmetabolizable glucose analog 2-deoxyglucose. In this glkA mutant, carbon catabolite repression of glycerol kinase and agarase was relieved on several carbon sources tested, even though most of these carbon sources are not metabolized via glucose kinase. This suggests that catabolite repression is not regulated by the flux through glucose kinase and that the protein itself has a regulatory role in carbon catabolite repression. A 10-fold overproduction of glucose kinase also results in relief of catabolite repression, suggesting that excess glucose kinase can titrate the repressing signal away. This could be achieved directly by competition of excess glucose kinase with its repressing form for binding sites on DNA promoter regions or indirectly by competition for binding of another regulatory protein.     

Nitrogen catabolite repression in Saccharomyces cerevisiae

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 allantoine. 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 protease, 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.     

AbrB modulates expression and catabolite repression of a Bacillus subtilis ribose transport operon.

A Bacillus subtilis ribose transport operon (rbs) was shown to be subject to AbrB-mediated control through direct AbrB-DNA binding interactions in the vicinity of the promoter. Overproduction of AbrB was shown to relieve catabolite repression of rbs during growth in the presence of poorer carbon sources such as arabinose but had much less effect when cells were grown in the presence of glucose, a rapidly metabolizable carbon source. A ccpA mutation relieved catabolite repression of rbs under all conditions tested. One of the AbrB-binding sites on the rbs promoter contains the putative site of action for the B. subtilis catabolite repressor protein CcpA, suggesting that competition for binding to this site could be at least partly responsible for modulating rbs expression during carbon-limited growth.     

CcpA-independent carbon catabolite repression in Bacillus subtilis

The past decade has witnessed an exiting unveiling of numerous molecular mechanisms that characterize signal transduction by protein-protein interaction. The recent findings encouraged an increasing effort to understand the sequential metabolism of different sugars available as energy sources at the same time. It seems probable that at least three principle mechanisms which act together or separately, mediate carbon catabolite repression (CCR) depending on the system which is under metabolic control: i) by the main signal transducing chain via the ATP-dependent HPr-kinase, HPr(Ser46-P) or alternatively Crh via the central component CcpA and its interaction with cre, ii) by signals sensed from the specific regulators directly or via phosphorylation by HPr, iii) by inducer exclusion based on the concurrence of the enzyme IIA(Glc) domain of the glucose permease, and other PTS-dependent permeases composed only of the B and C domains and lacking the enzyme IIA domain.     

Carbon catabolite repression by the catabolite control protein CcpA in Staphylococcus xylosus

Carbon catabolic repression (CR) by the catabolite control protein CcpA has been analyzed in Staphylococcus xylosus. Genes encoding components needed to utilize lactose, sucrose, and maltose were found to be repressed by CcpA. In addition, the ccpA gene is under negative autogenous control. Among several tested sugars, glucose caused strongest CcpA-dependent repression. Glucose can enter S. xylosus in nonphosphorylated form via the glucose uptake protein GlcU. Internal glucose is then phosphorylated by the glucose kinase GlkA. Alternatively, glucose can be transported and concomitantly phosphorylated by glucose-specific permease(s) of the phosphotransferase system (PTS). S. xylosus mutant strains deficient in GlcU or GlkA showed partial relief of glucose-specific, CcpA-dependent repression. Likewise, blocking PTS activity completely by inactivation of the gene encoding the general PTS protein enzyme I resulted in diminished glucose-mediated repression. Thus, both glucose entry routes contribute to glucose-specific CR in S. xylosus. The sugar transport activity of the PTS is not required to trigger glucose-specific repression. The phosphocarrier protein HPr however, is absolutely essential for CcpA activity. Inactivation of the HPr gene led to a complete loss of CR. Repression is also abolished upon inactivation of the HPr kinase gene or by replacing serine at position 46 of HPr by alanine. These results clearly show that HPr kinase provides the signal, seryl-phosphorylated HPr, to activate CcpA in S. xylosus.     

Nitrogen catabolite repression in Saccharomyces cerevisiae.

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.     

Altered end-product patterns and catabolite repression in Escherichia coli

Dobrogosz, Walter J. (North Carolina State University, Raleigh). Altered end-product patterns and catabolite repression in Escherichia coli. J. Bacteriol. 91:2263-2269. 1966.-End products formed during growth of Escherichia coli ML30 on glucose were examined under various conditions known to promote or prevent catabolite repression of the inducible beta-galactosidase system in this organism. Cultures were grown under these conditions in the presence of C(14)-glucose or C(14)-pyruvate. The products formed were assayed isotopically after separation on columns of silicic acid. Under conditions known to promote catabolite repression, glucose was degraded primarily to acetate and CO(2). When repression was turned off by anaerobic shock, glucose metabolism was characterized by the accumulation of ethyl alcohol in addition to acetate and CO(2). The results presented in this report indicate that oxidative decarboxylation of pyruvate may markedly affect the amount of energy that can be derived from glucose catabolism. In turn, the amount of energy derived from catabolic processes may play a key role in the mechanism of catabolite repression.     

Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for beta-galactosidase in the presence of glucose, although repression of beta-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.     

Phosphorylation of 3-O-methyl-D-glucose and catabolite repression in yeast

The glucose analog, 3-O-methyl-D-glucose, inhibited growth of yeast on non-fermentable carbon sources. The sugar was phosphorylated by the yeast and also in vitro by a commercial preparation of yeast hexokinase. The chromatographic behaviour of the phosphorylated product was identical in both cases. This suggests that 3-O-methyl-D-glucose is phosphorylated to form 3-O-methyl-D-glucose 6-phosphate. The inhibition of the growth appears to be due to interference with the derepression of several enzymes necessary to grow on non-fermentable carbon sources. Spontaneous mutants whose growth was unaffected by 3-O-methyl-D-glucose were isolated. In these mutants there was no significant accumulation of the phosphorylated ester and the derepression of the enzymes tested was not affected by the glucose analog.