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1.
Deletions in the argD, crp, cysG genes (73-74 min of the Escherichia coli genetic map) were obtained by heat induction of the phage lambda c1857 b221 rex::Tn5 integrated previously into the cysG gene by homologous recombination in the cysG::Tn5 mutant. Properties of the deletions obtained suggest the gene order: argD-crp-cysG. 相似文献
2.
High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor. 总被引:5,自引:2,他引:3
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The flavoprotein and hemoprotein components of Escherichia coli B NADPH-sulfite reductase are encoded by cysJ and cysI, respectively. Plasmids containing these two genes overexpressed flavoprotein catalytic activity and apohemoprotein by 13- to 35-fold, but NADPH-sulfite reductase holoenzyme activity was increased only 3-fold. Maximum overexpression of holoenzyme activity was achieved by the inclusion in such plasmids of Salmonella typhimurium cysG, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, a cofactor for the hemoprotein. Thus, cofactor deficiency, in this case siroheme, can limit overexpression of a cloned enzyme. Catalytically active holoenzyme accounted for 10% of total soluble protein in a host containing cloned cysJ, cysI, and cysG. A 5.3-kb DNA fragment containing S. typhimurium cysG was sequenced, and the open reading frame corresponding to cysG was identified by subcloning and by identifying plasmid-encoded peptides in maxicells. Comparison with the sequence reported for the E. coli cysG region (J. A. Cole, unpublished data; GenBank sequence ECONIRBC) indicates a gene order of nirB-nirC-cysG in the cloned S. typhimurium fragment. In addition, two open reading frames of unknown identity were found immediately downstream of cysG. One of these contains 11 direct repeats of 33 nucleotides each, which correspond to the consensus amino acid sequence Asp-Asp-Val-Thr-Pro-Pro-Asp-Asp-Ser-Gly-Asp. 相似文献
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4.
Biochemical and genetic characterization of nirB mutants of Escherichia coli K 12 pleiotropically defective in nitrite and sulphite reduction 总被引:6,自引:0,他引:6
Mutants of Escherichia coli K12 defective in the nirB gene lack NADH-dependent nitrite reductase activity and reduce nitrite slowly during anaerobic growth. With one exception these mutants require cysteine for growth. Cytochrome C552 synthesis and the assimilation of ammonia are unaffected by the nirB mutation. The defective gene is located between the crp and aroB genes at minute 73 on the E. coli chromosome. Mapping and reversion studies indicate the nirB is identical to the previously described cysG gene. It is suggested that the product of the cysG+ (nirB+)?gene is an enzyme required for the synthesis of sirohaem, a prosthetic group of enzymes which catalyse the six-electron reduction of nitrite to ammonia and sulphite to sulphide. 相似文献
5.
Mutants have been isolated which lack NADH-dependent nitrite reductase activity but retain NADPH-dependent sulphite reductase and formate hydrogenlyase activities. These NirB- strains synthesize cytochrome c552 and grow normally on anaerobic glycerol-fumarate plates. The defects map in a gene, nirB, which is extremely close to cysG, the gene order being crp, nirB, cysG, aroB. Complementation studies established that nirB+ and cysG+ can be expressed independently. The data strongly suggest that nirB is the structural gene for the 88 kDal NADH-dependent nitrite oxidoreductase apoprotein (EC 1.6.6.4). The nirB gene is apparently defective in the previously described nirD mutant, LCB82. The nirH mutant, LCB197, was unable to use formate as electron donor for nitrite reduction, but NADH-dependent nitrite reductase was extremely active in this strain and a normal content of cytochrome c552 was detected. Strains carrying a nirE, nirF or nirG mutation gave normal rates of nitrite reduction by glucose, formate or NADH. 相似文献
6.
Gene cloning, sequencing, and biochemical characterization of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI. 总被引:2,自引:1,他引:2
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The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli. A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA. An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa. xynA from T. saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases. The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively. The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan. The cloned xylanase was an endo-acting enzyme. 相似文献
7.
Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui. 总被引:2,自引:0,他引:2
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The 6.2-kbp DNA fragment encoding the enzymes in the porphyrin synthesis pathway of a cellulolytic anaerobe, Clostridium josui, was cloned into Escherichia coli and sequenced. This fragment contained four hem genes, hemA, hemC, hemD, and hemB, in order, which were homologous to the corresponding genes from E. coli and Bacillus subtilis. A typical promoter sequence was found only upstream of hemA, suggesting that these four genes were under the control of this promoter as an operon. The hemA and hemD genes cloned from C. josui were able to complement the hemA and hemD mutations, respectively, of E. coli. The COOH-terminal region of C. josui HemA and the NH2-terminal region of C. josui HemD were homologous to E. coli CysG (Met-1 to Leu-151) and to E. coli CysG (Asp-213 to Phe-454) and Pseudomonas denitrificans CobA, respectively. Furthermore, the cloned 6.2-kbp DNA fragment complemented E. coli cysG mutants. These results suggested that both C. josui hemA and hemD encode bifunctional enzymes. 相似文献
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Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes 总被引:2,自引:0,他引:2
C. Clepet F. Borne V. Krishnapillai C. Baird J. C. Patte B. Cami 《Molecular microbiology》1992,6(21):3109-3119
Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrc genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed-field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species. 相似文献
10.
Mutants of bacteria belonging the genus Erwinia (Erwinia chrysanthemi and Erwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI+ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that the ptsI and ptsH genes of E. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas the ptsH gene is located at 175 min. Sequencing of a portion of the E. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I. 相似文献
11.
Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii). 总被引:1,自引:1,他引:0
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I Sattler C A Roessner N J Stolowich S H Hardin L W Harris-Haller N T Yokubaitis Y Murooka Y Hashimoto A I Scott 《Journal of bacteriology》1995,177(6):1564-1569
We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon. 相似文献
12.
chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100. 总被引:7,自引:3,他引:4
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The pem locus is responsible for stable maintenance of plasmid R100 and consists of two genes, pemI and pemK. The pemK gene product is a growth inhibitor, while the pemI gene product is a suppressor of this inhibitory function. We found that the PemI amino acid sequence is homologous to two open reading frames from Escherichia coli called mazE and orf-83, which are located at 60 and 100 min on the chromosome, respectively. We cloned and sequenced these loci and found additional open reading frames, one downstream of each pemI homolog, both of which encode proteins homologous to PemK. The pem locus homolog at 60 min was named chpA and consists of two genes, chpAI and chpAK; the other, at 100 min, was named chpB and consists of two genes, chpBI and chpBK. The distal portion of chpBK was found to be adjacent to the ppa gene that encodes pyrophosphatase, whose map position had not been previously determined. We then demonstrated that the chpAK and chpBK genes encode growth inhibitors, while the chpAI and chpBI genes encode suppressors for the inhibitory function of the ChpAK and ChpBK proteins, respectively. These E. coli pem locus homologs may be involved in regulation of cell growth. 相似文献
13.
Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli. 总被引:25,自引:18,他引:7
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M Wachi M Doi S Tamaki W Park S Nakajima-Iijima M Matsuhashi 《Journal of bacteriology》1987,169(11):4935-4940
A chromosomal region of Escherichia coli contiguous to the fabE gene at 71 min on the chromosomal map contains multiple genes that are responsible for determination of the rod shape and sensitivity to the amidinopenicillin mecillinam. The so-called mre region was cloned and analyzed by complementation of two closely related but distinct E. coli mutants characterized, respectively, by the mutations mre-129 and mre-678, that showed a rounded to irregular cell shape and altered sensitivities to mecillinam; the mre-129 mutant was supersensitive to mecillinam at 30 degrees C, but the mre-678 mutant was resistant. The mre-678 mutation also caused simultaneous overproduction of penicillin-binding proteins 1Bs and 3. A chromosomal region of the wild-type DNA containing the total mre region and the fabE gene was first cloned on a lambda phage; a 7-kilobase (kb) fragment containing the whole mre region, but not the fabE gene, was then recloned on a mini F plasmid, pLG339; and finally, a 2.8-kb fragment complementing only mre-129 was also cloned on this low-copy-number plasmid. The whole 7-kb fragment was required for complementing the mre-678 mutant phenotypes. Fragments containing fabE but not the mre-129 region could be cloned on a high-copy-number plasmid. Southern blot hybridization indicated that the mre-678 mutant had a large deletion of 5.25 kb in its DNA, covering at least part of the mre-129 gene. 相似文献
14.
Genetic studies of the phs locus of Escherichia coli, a mutation causing pleiotropic lesions in metabolism and pH homeostasis 总被引:5,自引:0,他引:5
In Escherichia coli a pleiotropic mutation, phs, has been reported to affect Na+-linked metabolic functions and pH homeostasis. The phs mutation was previously mapped by its proximity to a met marker, presumed to be metB at 89 min. We have shown that a second mutation to auxotrophy, cymX, which is satisfied by either methionine or cysteine, is closely linked to phs. The cymX and phs lesions map close to trkB and rpsL at 73.5 min and we postulate that they are alleles of cysG and crp, respectively. The basis of the pH sensitivity of DZ3 is discussed in the light of this new information. 相似文献
15.
Koyama M Katayama S Kaji M Taniguchi Y Matsushita O Minami J Morita S Okabe A 《Microbiology and immunology》1999,43(10):947-957
The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin. 相似文献
16.
Rapid and precise mapping of the Escherichia coli release factor genes by two physical approaches. 总被引:11,自引:7,他引:4
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C C Lee Y Kohara K Akiyama C L Smith W J Craigen C T Caskey 《Journal of bacteriology》1988,170(10):4537-4541
The termination of protein synthesis in Escherichia coli requires two codon-specific factors termed RF1 and RF2. RF1 mediates UAA- and UAG-directed termination, while RF2 mediates UAA- and UGA-directed termination. The genes encoding these factors have been isolated and sequenced, and RF2 was found to be encoded in two separate reading frames. The map position of RF1 has been reported as 27 min on the E. coli chromosome, while the RF2 map position has not yet been identified. In this study, two new and independent methods for gene mapping, using pulsed field gel electrophoresis and an ordered bacteriophage library spanning the entire chromosome, were used to localize the map position of the RF2 gene. In addition, the location of the RF1 gene was more precisely defined. The RF2 gene is located at 62.3 min on the chromosome, while the RF1 gene is located at 26.7 min. This approach to mapping cloned genes promises to be a rapid and simple means for determining the gene order of the genome. 相似文献
17.
A gene cluster ORFabcd from a Sinorhizobium fredii HN01 mutant strain HSMRalpha was isolated. We showed that it was an operon involved in sulfur metabolism. Functional studies revealed that, except for ORFb, the three genes ORFa, ORFc and ORFd were involved in sulfite reduction. ORFa and ORFc were similar to the cysG and cysI from Sinorhizobium meliloti 1021 and Rhizobium etli CFN 42, respectively. ORFd encodes a conserved hypothetical protein in other bacteria. We demonstrate here, for the first time, that it was a new locus involved in sulfate assimilation in S. fredii HN01 and we designated it as cysII. 相似文献
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Murakami S Hayashi T Maeda T Takenaka S Aoki K 《Bioscience, biotechnology, and biochemistry》2003,67(11):2351-2358
Genes encoding an aniline dioxygenase of Frateuria sp. ANA-18, which metabolizes aniline via the ortho-cleavage pathway of catechol, were cloned and named tdn genes. The tdn genes were located on the chromosomal DNA of this bacterium and weren't clustered with catechol-degrading gene clusters. These results show that the ANA-18 aniline-degrading gene cluster is constructionally different from Pseudomonas tdn and Acinetobacter atd gene clusters, which degrade aniline via the meta-cleavage pathway of catechol and organize catechol-metabolic genes in the gene clusters. When cloned tdnQTA1A2B genes were expressed in Eschherichia coli, aniline dioxygenase activity was observed. Southern blot analysis revealed that homologues of the tdnA1A2B genes didn't exist in strain ANA-18. Disruption of the tdnA1A2 genes gave the parent strain ANA-18 a defect in aniline metabolism. On the basis of these results, we concluded that only the cloned tdn genes function as genes encoding aniline dioxygenase in strain ANA-18 although this bacterium had two catechol-degrading gene clusters. 相似文献
20.
A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis.
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The role of cbiK, a gene found encoded within the Salmonella typhimurium cob operon, has been investigated by studying its in vivo function in Escherichia coli. First, it was found that cbiK is not required for cobalamin biosynthesis in the presence of a genomic cysG gene (encoding siroheme synthase) background. Second, in the absence of a genomic cysG gene, cobalamin biosynthesis in E. coli was found to be dependent upon the presence of cobA(P. denitrificans) (encoding the uroporphyrinogen III methyltransferase from Pseudomonas denitrificans) and cbiK. Third, complementation of the cysteine auxotrophy of the E. coli cysG deletion strain 302delta a could be attained by the combined presence of cobA(P. denitrificans) and the S. typhimurium cbiK gene. Collectively these results suggest that CbiK can function in fashion analogous to that of the N-terminal domain of CysG (CysG(B)), which catalyzes the final two steps in siroheme synthesis, i.e., NAD-dependent dehydrogenation of precorrin-2 to sirohydrochlorin and ferrochelation. Thus, phenotypically CysG(B) and CbiK have very similar properties in vivo, although the two proteins do not have any sequence similarity. In comparison to CysG, CbiK appears to have a greater affinity for Co2+ than for Fe2+, and it is likely that cbiK encodes an enzyme whose primary role is that of a cobalt chelatase in corrin biosynthesis. 相似文献