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Favier A Brutscher B Blackledge M Galinier A Deutscher J Penin F Marion D 《Journal of molecular biology》2002,317(1):131-144
The solution structure and dynamics of the Bacillus subtilis HPr-like protein, Crh, have been investigated using NMR spectroscopy. Crh exhibits high sequence identity (45 %) to the histidine-containing protein (HPr), a phospho-carrier protein of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system, but contains no catalytic His15, the site of PEP-dependent phosphorylation in HPr. Crh also forms a mixture of monomers and dimers in solution whereas HPr is known to be monomeric. Complete backbone and side-chain assignments were obtained for the monomeric form, and 60 % of the dimer backbone resonances; allowing the identification of the Crh dimer interface from chemical-shift mapping. The conformation of Crh was determined to a precision of 0.46(+/-0.06) A for the backbone atoms, and 1.01(+/-0.08) A for the heavy atoms. The monomer structure is similar to that of known HPr 2.67(+/-0.22) A (C(alpha) rmsd), but has a few notable differences, including a change in the orientation of one of the helices (B), and a two-residue shift in beta-sheet pairing of the N-terminal strand with the beta4 strand. This shift results in a shortening of the surface loop present in HPr and consequently provides a flatter surface in the region of dimerisation contact, which may be related to the different oligomeric nature of these two proteins. A binding site of phospho-serine(P-Ser)-Crh with catabolite control protein A (CcpA) is proposed on the basis of highly conserved surface side-chains between Crh and HPr. This binding site is consistent with the model of a dimer-dimer interaction between P-Ser-Crh and CcpA. (15)N relaxation measured in the monomeric form also identified differential local mobility in the helix B which is located in the vicinity of this site. 相似文献
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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 sigmaH 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 glucose. 相似文献
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Chastukhina IB Sharipova MR Gabdrakhmanova LA Balaban NP Kostrov SV Rudenskaia GN Leshchinskaia IB 《Mikrobiologiia》2005,74(1):39-47
We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different. 相似文献
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RNA synthesis was studied in Bacillus subtilis Cgr4 grown in the mineral sporulation medium enriched with glucose up to 2% and amino acids up to 1%. To study mRNA synthesis, a method of transfer of the 3H-uridine pulse-labeled culture to the supernatant of physiologically identical, not labeled culture, followed by further incubation was used, the amount of 3H-uridine in the supernatant as well as in cells being measured. RNA was also analysed electrophoretically and distribution of the label among the fractions was determined. It is shown that mRNA synthesized in the logarithmic phase degrades up to 12% on the 2nd hour of growth during 10 min; the mRNA in the stationary phase is stable on the 7th hour of growth; no degradation is observed in the course of 2-3 hours. The beginning of degradation coincides in time with secondary induction of the synthesis of serine proteases and with the onset of sharp decrease in incorporation of 3H-uridine in RNA as well as with induction of spore morphogenesis. On the basis of electrophoretical analysis of pulse-labeled RNA, it was demonstrated that, prior to the transfer, labeled uridine was included and preserved in RNA fraction for 2-3 hours after the transfer, this fraction corresponding in mobility with mRNA in polyacrylamide gel. The following conclusion may be drawn: stable mRNAs are synthesized in the stationary phase and may be used for the translation of extracellular serine protease. 相似文献
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Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression. 总被引:24,自引:20,他引:4
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Strain SF22, a glutamine-requiring (Gln-) mutant of Bacillus subtilis SMY, is likely to have a mutation in the structural gene for glutamine synthetase, since this strain synthesized 22 to 55% as much glutamine synthetase antigen as did wild-type cells in a 10-min period but had less than 3% of wild-type glutamine synthetase enzymatic activity. The expression of several genes subject to glucose catabolite repression was altered in the Gln- mutant. The induced levels of alpha-glucosidase, histidase, and aconitase were 3.5- to 4-fold higher in SF22 cells than in wild-type cells grown in glucose-glutamine medium, and citrate synthase levels were 8-fold higher in the Gln- mutant than in wild-type cells. The relief of glucose catabolite repression in the Gln- mutant may result from poor utilization of glucose. Examination of the intracellular metabolite pools of cells grown in glucose-glutamine medium showed that the glucose-6-phosphate pool was 2.5-fold lower, the pyruvate pool was 4-fold lower, and the 2-ketoglutarate pool was 2.5-fold lower in the Gln- cells than they were in wild-type cells. Intracellular levels of glutamine were sixfold higher in the Gln- mutant than in wild-type cells. Measurements of enzymes involved in glutamine transport and utilization showed that the elevated pools of glutamine in the Gln- mutant resulted from a threefold increase in glutamine permease and a fivefold decrease in glutamate synthase. The pleiotropic effect of the gln-22 mutation on the expression of several genes suggests that either the glutamine synthetase protein or its enzymatic product, glutamine, is involved in the regulation of several metabolic pathways in B. subtilis. 相似文献
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Isolation of Bacillus subtilis mutants pleiotropically insensitive to glucose catabolite repression. 总被引:9,自引:8,他引:1
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A pleiotropic mutant of Bacillus subtilis was isolated which overproduced in the presence of glucose several enzymes whose synthesis is subject to glucose catabolite repression. Examination of intracellular metabolites suggested that the mutation may have resulted in a defect in glycolysis, increasing phosphoenolpyruvate and decreasing pyruvate, 2-ketoglutarate, and oxaloacetate. 相似文献
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Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus. 总被引:7,自引:0,他引:7
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The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus. 相似文献
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Role of sugar uptake and metabolic intermediates on catabolite repression in Bacillus subtilis.
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Many phosphorylated intermediates exert catabolite repression on the enzyme acetoin dehydrogenase in Bacillus subtilis. This was shown with strains that are blocked at different positions in central metabolism when they receive sugars that cannot be metabolized past enzymatic block(s). In the case of sorbitol, transport events were not involved in catabolite repression, for this sugar cannot repress acetoin dehydrogenase in a strain lacking sorbitol dehydrogenase but otherwise able to take up sorbitol. The presence of glucose did not markedly influence the uptake of acetoin. 相似文献
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trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis
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In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR. 相似文献
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AbrB modulates expression and catabolite repression of a Bacillus subtilis ribose transport operon. 总被引:2,自引:2,他引:0
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M A Strauch 《Journal of bacteriology》1995,177(23):6727-6731
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. 相似文献
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The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation. 总被引:5,自引:2,他引:3
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Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins. 相似文献
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Experiments performed in batch fermentation under phosphate-limited growth conditions showed that the citric acid yield was inversely related to the excess nitrogen concentration in the medium. Results from chemostat culture confirmed a negative relationship between the citric acid yield and both the specific growth rate and the nitrogen consumption rate. This is evidence for nitrogen catabolite repression. A fed-batch fermentation performed under dual phosphate/nitrogen limitation produced results very similar to those from a culture limited by nitrogen alone. There is no advantage in maintaining an excess of phosphate during citric acid production and the process will therefore be more economic when operated under dual limitation conditions. 相似文献
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The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon. 总被引:3,自引:3,他引:0
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J Stülke I Martin-Verstraete V Charrier A Klier J Deutscher G Rapoport 《Journal of bacteriology》1995,177(23):6928-6936
The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS. 相似文献