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991.
The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export. 总被引:21,自引:16,他引:5
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flaFIX, the structural gene for the periplasmic P ring of the flagellar basal body of Salmonella typhimurium, was cloned. Two gene products with apparent molecular weights of 38,000 and 40,000 were identified by minicell analysis. Data from pulse-chase and membrane fractionation experiments and data on the inhibitory effect of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone all indicated that the 40-kilodalton protein was a precursor form which, after export across the cytoplasmic membrane accompanied by cleavage of a signal peptide, gave rise to the mature protein in the periplasm. The N-terminal amino acid sequence of the FlaFIX protein, predicted from the DNA sequence, conformed well to known signal peptide sequences. The results indicate that the P-ring protein of the basal body (unlike flagellin and possible some other external flagellar components) crosses the cytoplasmic membrane in a conventional signal peptide-dependent manner. 相似文献
992.
Characterization of the specific pyruvate transport system in Escherichia coli K-12. 总被引:1,自引:0,他引:1
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A mutant of Escherichia coli K-12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study the transport of pyruvate by whole cells. Uptake of pyruvate was maximal in mid-log phase cells, with a Michaelis constant for transport of 20 microM. Pretreatment of the cells with respiratory chain poisons or uncouplers, except for arsenate, inhibited transport up to 95%. Lactate and alanine were competitive inhibitors, but at nonphysiological concentrations. The synthetic analogs 3-bromopyruvate and pyruvic acid methyl ester inhibited competitively. The uptake of pyruvate was also characterized in membrane vesicles from wild-type E. coli K-12. Transport required an artificial electron donor system, phenazine methosulfate and sodium ascorbate. Pyruvate was concentrated in vesicles 7- to 10-fold over the external concentration, with a Michaelis constant of 15 microM. Energy poisons, except arsenate, inhibited the transport of pyruvate. Synthetic analogs such as 3-bromopyruvate were competitive inhibitors of transport. Lactate initially appeared to be a competitive inhibitor of pyruvate transport in vesicles, but this was a result of oxidation of lactate to pyruvate. The results indicate that uptake of pyruvate in E. coli is via a specific active transport system. 相似文献
993.
Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13. 总被引:11,自引:5,他引:6
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The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases. 相似文献
994.
995.
Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development. 总被引:24,自引:20,他引:4
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R W Carlson S Kalembasa D Turowski P Pachori K D Noel 《Journal of bacteriology》1987,169(11):4923-4928
The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain. 相似文献
996.
Streptococcus faecium mutants that are temperature sensitive for cell growth and show alterations in penicillin-binding proteins. 总被引:9,自引:2,他引:7
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The penicillin-binding proteins (PBPs) of 209 cell division (or growth) temperature-sensitive mutants of Streptococcus faecium were analyzed in this study. A total of nine strains showed either constitutive or temperature-sensitive conditional damage in the PBPs. Analysis of these nine strains yielded the following results: one carried a PBP 1 constitutively showing a lower molecular weight; one constitutively lacked PBP 2; two lacked PBP 3 at 42 degrees C, but not at 30 degrees C; one was normal at 30 degrees C but at 42 degrees C lacked PBP 3 and overproduced PBP 5; two were normal at 42 degrees C and lacked PBP 5 at 30 degrees C; one constitutively lacked PBP 5; and one carried a PBP 6 constitutively split in two bands. The mutant lacking PBP 3 and overproducing PBP 5 continued to grow at 42 degrees C for 150 min and then lysed. Revertants selected for growth capability at 42 degrees C from the mutants altered in PBPs 5 and 6 maintained the same PBP alterations, while those isolated from the strains with altered PBP 1 or lacking PBP 2 or PBP 3 showed a normal PBP pattern. Penicillin-resistant derivatives were isolated at 30 degrees C from the mutants lacking PBP 2 and from that lacking PBP 3. All these derivatives continued to show the same PBP damage as the parents, but overproduced PBP 5 and grew at 42 degrees C. These findings indicate that high-molecular-weight, but not low-molecular-weight, PBPs are essential for cell growth in S. faecium. This is in complete agreement with previous findings obtained with a different experimental system. On the basis of both previous and present data it is suggested that PBPs 1, 2, and 3 appear necessary for cell growth at optimal temperature (and at maximal rate), but not for cell growth at a submaximal one (or at a reduced rate), and an overproduced PBP 5 is capable of taking over the function of PBPs 1, 2, and 3. 相似文献
997.
Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris. 总被引:24,自引:18,他引:6
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In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force. 相似文献
998.
999.
Involvement of a low-molecular-weight substance in in vitro activation of the molybdoenzyme respiratory nitrate reductase from a chlB mutant of Escherichia coli. 总被引:2,自引:1,他引:1
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The soluble subcellular fraction of a chlB mutant contains an inactive precursor form of the molybdoenzyme nitrate reductase, which can be activated by the addition to the soluble fraction of protein FA, which is thought to be the active product of the chlB locus. Dialysis or desalting of the chlB soluble fraction leads to the loss of nitrate reductase activation, indicating that some low-molecular-weight material is required for the activation. The protein FA-dependent activation of nitrate reductase can be restored to the desalted chlB soluble fraction by the addition of a clarified extract obtained after heating the chlB soluble fraction at 100 degrees C for 8 min. The heat-stable substance present in this preparation has a molecular weight of approximately 1,000. This substance is distinct from the active molybdenum cofactor since its activity is unimpaired in heat-treated extracts prepared from the organism grown in the presence of tungstate, which leads to loss of cofactor activity. Mutations at the chlA or chlE locus, which are required for molybdenum cofactor biosynthesis, similarly do not affect the activity of the heat-treated extract in the in vitro activation process. Moreover, the active material can be separated from the molybdenum cofactor activity by gel filtration. None of the other known pleiotropic chlorate resistance loci (chlD, chlG) are required for the expression of its activity. Magnesium ATP appears to have a role in the formation of the active substance. We conclude that a low-molecular-weight substance, distinct from the active molybdenum cofactor, is required to bestow activity on the molybdoenzyme nitrate reductase during its biosynthesis. 相似文献
1000.
Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing. 总被引:19,自引:9,他引:10
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The colicin A lysis protein (Cal) is required for the release of colicin A to the medium by producing bacteria. This protein is produced in a precursor form that contains a cysteine at the cleavage site (-Leu-Ala-Ala-Cys). The precursor must be modified by the addition of lipid before it can be processed. The maturation is prevented by globomycin, an inhibitor of signal peptidase II. Using oligonucleotide-directed mutagenesis, the alanine and cystein residues in the -1 and +1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs. Both substitutions prevented the normal modification and cleavage of the protein. The marked activation of the outer membrane detergent-resistant phospholipase A observed with wild-type Cal was not observed with the Cal mutants. Both Cal mutants were also defective for the secretion of colicin A. In one mutant, the signal peptide appeared to be cleaved off by an alternative pathway involving signal peptidase I. Electron microscope studies with immunogold labeling of colicin A on cryosections of pldA and cal mutant cells indicated that the colicin remains in the cytoplasm and is not transferred to the periplasmic space. These results demonstrate that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of colicin A. 相似文献