Glucose repression in Streptomyces coelicolor A3(2): a likely regulatory role for glucose kinase

The glucose kinase gene (glkA-ORF3) of Streptomyces coelicolor A3(2) plays an essential role in glucose utilisation and in glucose repression of a variety of genes involved in the utilisation of alternative carbon sources. These genes include dagA, which encodes an extracellular agarase that permits agar utilisation. Suppressor mutants of glkA-ORF3 deletion strains capable of utilising glucose (Glc⁺) arise at a frequency of about 10^?5 on prolonged incubation. The Glc⁺ phenotype of the mutants is reversible (at a frequency of about 10^?3) and reflects either the activation of a normally silent glucose kinase gene or the modification of an existing sugar kinase. Although the level of glucose kinase activity in the Glc⁺ supressor mutants is similar to that in the glkA ⁺ parental strain, glucose repression of dagA remains defective. Expression of the glucose kinase gene of Zymomonas mobilis in glkA-ORF3 mutants restored glucose utilisation, but not glucose repression of dagA. Over-expression of glkA-ORF3 on a high-copy-number plasmid failed to restore glucose repression of dagA in glkA-ORF3 mutants and led to loss of glucose repression of dagA in a glkA ⁺ strain. These results suggest that glucose phosphorylation itself is not sufficient for glucose repression and that glkA-ORF3 plays a specific regulatory role in triggering glucose repression in S. coelicolor A3(2).     

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.     

Genetic mapping, cloning and physiological aspects of the glucose kinase gene of Streptomyces coelicolor

Summary Glucose kinase in Streptomyces coelicolor has a molecular weight of about 110,000. In crude extracts, the enzyme exhibited apparent K_m values of 0.20 mM for ATP, 0.27 mM for glucose, and 2.2 mM for the glucose analogue 2-deoxyglucose. Mutations (glk) to 2-deoxyglucose-resistance, which greatly reduce glucose kinase activity and result in relief of glucose repression of utilisation of various carbon sources, were mapped between proA and hisA in the S. coelicolor linkage map. Glucose kinase activity, 2-deoxyglucose-sensitivity, glucose utilisation and glucose repression, were all restored to glk mutants by a 3.5 kb DNA fragment cloned from S. coelicolor into a phage vector (C31 KC515), and by larger (10–30 kb) fragments cloned into a low copy number plasmid vector (pIJ916). The glk gene was further localised to a 2.9 kb BclI fragment of the cloned DNA by sub-cloning. Part or all of this fragment was present in each of five primary plasmid clones tested.     

Cloning and sequencing of two genes fromStaphylococcus carnosus coding for glucose-specific PTS and their expression inEscherichia coliK-12

Phosphoenolpyruvate (PEP)-dependent phosphorylation experiments have indicated that the grampositive bacteriumStaphylococcus carnosus possesses an EIICBA fusion protein specific for glucose. Here we report the cloning of a 7 kb genomic DNA fragment containing two genes,glcA andglcB, coding for the glucose-specific PTS transporters EII^Glc1 and EII^Glc2 which are 69% identical. The translation products derived from the nucleotide sequence consist of 675 and 692 amino acid residues and have calculated molecular weights of 73 025 and 75 256, respectively. Both genes can be stably maintained inEscherichia coli cells and restore the ability to ferment glucose toptsG deletion mutants ofE. coli. This demonstrates the ability of the PTS proteins HPr and/or EIIA^Glc of a gram-negative organism (E. coli) to phosphorylate an EIICBA^Glc from a gram-positive organism (S. carnosus).     

Identification of glucose kinase‐dependent and ‐independent pathways for carbon control of primary metabolism,development and antibiotic production in Streptomyces coelicolor by quantitative proteomics

Members of the soil‐dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient availability is a major determinant for the switch to development and antibiotic production in streptomycetes. Carbon catabolite repression (CCR), a main signalling pathway underlying this phenomenon, was so far considered fully dependent on the glycolytic enzyme glucose kinase (Glk). Here we provide evidence of a novel Glk‐independent pathway in Streptomyces coelicolor, using advanced proteomics that allowed the comparison of the expression of some 2000 proteins, including virtually all enzymes for central metabolism. While CCR and inducer exclusion of enzymes for primary and secondary metabolism and precursor supply for natural products is mostly mediated via Glk, enzymes for the urea cycle, as well as for biosynthesis of the γ‐butyrolactone Scb1 and the responsive cryptic polyketide Cpk are subject to Glk‐independent CCR. Deletion of glkA led to strong downregulation of biosynthetic proteins for prodigionins and calcium‐dependent antibiotic (CDA) in mannitol‐grown cultures. Repression of bldB, bldN, and its target bldM may explain the poor development of S. coelicolor on solid‐grown cultures containing glucose. A new model for carbon catabolite repression in streptomycetes is presented.     

Sugar uptake and utilisation in Streptomyces coelicolor: a PTS view to the genome

Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation. We have surveyed the public available S. coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues (http://www.sanger.ac.uk/Projects/S_coelicolor/). Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIA^Crr (crr; IIA^Glc-homologue) and six genes encoding homologues of sugar-specific PTS components were identified. The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family. A model is presented, in which possible functions of the novel described PTS homologues are discussed.     

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme II^Glc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme II^Glc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme II^Glc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y _Glc values. Assuming equal Y _ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme II^Glc.Abbreviations PTS phosphoenolpyruvate: carbohydrate phosphotransferase system - HPr histidine-containing phosphocarrier protein - GalP galactose permease     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

Phosphoenolpyruvate: Sugar Phosphotransferase Systems and Sugar Metabolism in Brevibacterium flavum

Brevibacterium flavum mutants defective in the phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system (PTS) were selected with high frequency by 2-deoxyglucose-resistance. Most of them (DOG^r) still had the fructose-PTS and grew not only on fructose but also on glucose like the wild-type strain. A mutant having 1 /8th the fructose-PTS activity of the wild strain but normal glucose-PTS activity was isolated as a xylitol-resistant mutant. It grew on glucose but not on fructose. The glucose-PTS was active on glucose, glucosamine, 2-deoxyglucose and mannose, and slightly on methyl-a-glucoside and N-acetylglucosamine, but not on fructose or xylitol. The fructose-PTS acted on fructose and xylitol, and to some extent on glucose but not on glucosamine or 2-deoxyglucose. Mutants unable to grow on glucose (DOG^rGlc^-) derived from a DOG^r mutant were all defective in the fructose-PTS. All revertants able to grow on glucose derived from a DOG^rGlc“ mutant had the fructose-PTS. The glucokinase activity was about 2/3rds the glucose activity of the fructose-PTS. All the DOG^rGlc^- mutants had normal levels of glucokinase. One of these mutants grew on maltose and sucrose, which were hydrolyzed to glucose. Thus, glucokinase seems to contribute to the phosphorylation of glucose liberated inside the cell. The fructose-PTS was induced by fructose and repressed by glucose. The glucose repression was not observed in a mutant defective in the glucose-PTS.     

Protein phosphatase type-1 regulatory subunits Reg1p and Reg2p act as signal transducers in the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae

The REG1 gene encodes a regulatory subunit of the type-1 protein phosphatase (PP1) Glc7 in Saccharomyces cerevisiae, which directs the catalytic subunit to substrates involved in glucose repression. Loss of REG1 relieves glucose repression of many genes, including the MAL structural genes that encode the maltose fermentation enzymes. In this report, we explore the role of Reg1p and its homolog Reg2p in glucose-induced inactivation of maltose permease. Glucose stimulates the proteolysis of maltose permease and very rapid loss of maltose transport activity – more rapid than can be explained by loss of the permease protein alone. In a reg1Δ strain we observe a significantly reduced rate of glucose-induced proteolysis of maltose permease, and the rapid loss of maltose transport activity does not occur. Instead, surprisingly, the slow rate of proteolysis of maltose permease is accompanied by an increase in maltose transport activity. Loss of Reg2p modestly reduces the rates of both glucose-induced proteolysis of maltose permease and inactivation of maltose transport activity. Overexpression of Reg2p in a reg1Δ strain suppresses the effect on maltose permease proteolysis and partially restores the inactivation of maltose transport activity, but does not affect the insensitivity of MAL gene expression to repression by glucose observed in this strain. Thus, protein phosphatase type-1 (Glc7p-Reg1p and Glc7p-Reg2p) plays a role in transduction of the glucose signal during glucose-induced proteolysis of maltose permease, but only Glc7p-Reg1p is involved in glucose-induced inactivation of maltose transport activity and glucose repression of MAL gene expression. Overexpression of REG1 partially restores proteolysis of maltose permease in a grr1Δ strain, which lacks glucose signaling, but does not rescue rapid inactivation of maltose transport activity or sensitivity to glucose repression. A model for the role of Reg1p and Reg2p in glucose signaling pathways is discussed. We also uncovered a previously unrecognized G2/M delay in the grr1Δ but not the reg1Δ strains, and this delay is suppressed by REG1 overexpression. The G1/S delay seen in grr1Δ mutants is slightly suppressed as well, but REG1 overexpression does not suppress other grr1Δ phenotypes such as insensitivity to glucose repression. Received: 21 October 1999 / Accepted: 28 December 1999     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.     

Energetics of glucose uptake in Salmonella typhimurium

We have studied the energetics of glucose uptake in Salmonella typhimurium. Strain PP418 transprots glucose via the phosphoenolpyruvate: glucose phosphotransferase system, while strain PP1705 lacks this system and can only use the galactose permease for glucose uptake. These two strains were cultured anaerobically in glucose-limited chemostats. Both strains produced ethanol and acetate in equimolar amounts but a significant difference was observed in the molar growth yield on glucose (Y _Glc). It is suggested that this difference is due to a difference in the energetics of the glucose uptake systems in the two strains.Assuming an equal Y _ATP for both strains, we could calculate that uptake of 1 mole of glucose via the galactose permease consumes the equivalent of 0.5 mole of ATP. With the additional assumption that one proton is transported in symport with one glucose molecule, these results imply a stoichiometry of two protons per ATP hydrolysed.Abbreviations PTS Phosphoenolpyruvate: carbohydrate phosphotransferase system - D dilution rate (h^-1 - DW dry weight - GalP galactose permease - EtOH ethanol - HAc acetate - Lact lactate - Suc succinate - HFo formate - Glc Glucose - Y _Glc, Y _ATP yield of cells per glucose or ATP - q specific production rate