共查询到20条相似文献,搜索用时 31 毫秒
1.
The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria. 相似文献
2.
3.
4.
Expression of the cobalamin (Cbl) biosynthetic cob operon in Salmonella typhimurium is repressed by the end-product. This regulation is conferred mainly at the translational level and involves a cobalamin-induced folding of an RNA hairpin that sequesters the ribosomal binding site (RBS) of the cob mRNA and prevents translation initiation. A combined structural and mutational analysis shows that a cis-acting translational enhancer (TE) element, located 83 nucleotides upstream of the Shine-Dalgarno sequence in the 5'-untranslated region (5'-UTR) of the cob mRNA, is required to unfold the inhibitory RBS hairpin in the absence of cobalamin. The TE element, which consists of 5 nucleotides, is proposed to confer its enhancer function in the absence of cobalamin by interacting with nucleotides in the stem of the RBS hairpin. This interaction destabilizes the RNA hairpin and allows ribosome binding. In the presence of cobalamin, the enhancer function is inhibited. As a result, the RBS hairpin forms and prevents translation initiation. Several additional RNA hairpins in the 5'-UTR were also identified and are suggested to be important for repression. The above data suggest that normal cobalamin repression of the cob operon requires that the 5'-UTR has a defined secondary and tertiary structure. 相似文献
5.
Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. 总被引:18,自引:16,他引:2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J R Roth J G Lawrence M Rubenfield S Kieffer-Higgins G M Church 《Journal of bacteriology》1993,175(11):3303-3316
Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic conditions. Of the 30 cobalamin synthetic genes, 25 are clustered in one operon, cob, and are arranged in three groups, each group encoding enzymes for a biochemically distinct portion of the biosynthetic pathway. We have determined the DNA sequence for the promoter region and the proximal 17.1 kb of the cob operon. This sequence includes 20 translationally coupled genes that encode the enzymes involved in parts I and III of the cobalamin biosynthetic pathway. A comparison of these genes with the cobalamin synthetic genes from Pseudomonas denitrificans allows assignment of likely functions to 12 of the 20 sequenced Salmonella genes. Three additional Salmonella genes encode proteins likely to be involved in the transport of cobalt, a component of vitamin B12. However, not all Salmonella and Pseudomonas cobalamin synthetic genes have apparent homologs in the other species. These differences suggest that the cobalamin biosynthetic pathways differ between the two organisms. The evolution of these genes and their chromosomal positions is discussed. 相似文献
6.
We have examined the distribution of cobalamin (coenzyme B(12)) synthetic ability and cobalamin-dependent metabolism among enteric bacteria. Most species of enteric bacteria tested synthesize cobalamin under both aerobic and anaerobic conditions and ferment glycerol in a cobalamin-dependent fashion. The group of species including Escherichia coli and Salmonella typhimurium cannot ferment glycerol. E. coli strains cannot synthesize cobalamin de novo, and Salmonella spp. synthesize cobalamin only under anaerobic conditions. In addition, the cobalamin synthetic genes of Salmonella spp. (cob) show a regulatory pattern different from that of other enteric taxa tested. We propose that the cobalamin synthetic genes, as well as genes providing cobalamin-dependent diol dehydratase, were lost by a common ancestor of E. coli and Salmonella spp. and were reintroduced as a single fragment into the Salmonella lineage from an exogenous source. Consistent with this hypothesis, the S. typhimurium cob genes do not hybridize with the genomes of other enteric species. The Salmonella cob operon may represent a class of genes characterized by periodic loss and reacquisition by host genomes. This process may be an important aspect of bacterial population genetics and evolution. 相似文献
7.
PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment. 相似文献
8.
Recent studies have begun to reveal that numerous fundamental metabolic pathways in bacteria are regulated by riboswitches residing within certain messenger RNAs. These riboswitches selectively bind metabolites and modulate gene expression in response to changing ligand concentrations. Previously, we provided evidence that the btuB mRNAs of Escherichia coli and Salmonella typhimurium each carry a coenzyme B12-dependent riboswitch that causes repressed translation of the encoded cobalamin-transport protein at elevated coenzyme concentrations. Herein, we use a phylogenetic analysis to define a consensus sequence and secondary structure model for the ligand- binding domain of this riboswitch class. RNA structures that conform to this model are widespread in both Gram-positive and Gram-negative organisms. In addition, we find that the 5'-untranslated region (5'-UTR) of the cobalamin biosynthesis (cob) operon of S.typhimurium carries an RNA motif that matches this consensus sequence. Biochemical and genetic characterization of this motif confirms that the RNA directly binds coenzyme B12, and that it likely serves as a genetic control element for regulating expression of the 25-gene operon for cobalamin production in this pathogen. 相似文献
9.
In Rhodobacter capsulatus, cobalamin biosynthesis has been shown to occur when the bacteria are grown either aerobically or anaerobically. However, a comparison of the main cobalamin biosynthetic operon found within R. capsulatus would suggest that the encoded proteins belong to the oxygen-dependent pathway for cobalamin biosynthesis, although, significantly, no homologue of the essential mono-oxygenase CobG has yet been detected. Nonetheless, within this main cob operon is found a large open reading frame termed orf663 that is not found in any other cobalamin biosynthetic operon. When overproduced in Escherichia coli, orf663 was found to encode a 90 kDa integral membrane protein. Some of this protein is cleaved within E. coli to give a soluble N-terminal region that can easily be purified and yields a 50 kDa flavoprotein. When expressed in harness with the genes for precorrin-3a synthesis, ORF663 appears to mediate the transformation of precorrin-3a into a new chromophoric compound. Another open reading frame in close proximity to orf663 is termed orf647, and was found to encode a 2Fe-2S ferredoxin-like protein. We suggest that these two proteins may provide an alternative oxygen-independent mechanism for ring contraction within R. capsulatus. 相似文献
10.
Involvement of the Arc system in redox regulation of the Cob operon in Salmonella typhimurium 总被引:6,自引:0,他引:6
Dan I. Andersson 《Molecular microbiology》1992,6(11):1491-1494
11.
A Salmonella typhimurium cobalamin-deficient mutant blocked in 1-amino-2-propanol synthesis.
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Salmonella typhimurium synthesizes cobalamin (vitamin B12) when grown under anaerobic conditions. All but one of the biosynthetic genes (cob) are located in a single operon which includes genes required for the production of cobinamide and dimethylbenzimidazole, as well as the genes needed to form cobalamin from these precursors. We isolated strains carrying mutations (cobD) which are unlinked to any of the previously described B12 biosynthetic genes. Mutations in cobD are recessive and map at minute 14 of the linkage map, far from the major cluster of B12 genes at minute 41. The cobD mutants appear to be defective in the synthesis of 1-amino-2-propanol, because they can synthesize B12 when this compound is provided exogenously. Labeling studies in other organisms have shown that aminopropanol, derived from threonine, is the precursor of the chain linking dimethylbenzimidazole to the corrinoid ring of B12. Previously, a three-step pathway has been proposed for the synthesis of aminopropanol from threonine, including two enzymatic steps and a spontaneous nonenzymatic decarboxylation. We assayed the two enzymatic steps of the hypothetical pathway; cobD mutants are not defective in either. Furthermore, mutants blocked in one step of the proposed pathway continue to make B12. We conclude that the aminopropanol for B12 synthesis is not made by this pathway. Expression of a lac operon fused to the cobD promoter is unaffected by vitamin B12 or oxygen, both of which are known to repress the main cob operon, suggesting that the cobD gene is not regulated. 相似文献
12.
13.
Maria A. Soto Dhwani Desai Catherine Bannon Julie LaRoche Erin M. Bertrand 《Environmental microbiology》2023,25(7):1300-1313
Cobalamin availability can influence primary productivity and ecological interactions in marine microbial communities. The characterization of cobalamin sources and sinks is a first step in investigating cobalamin dynamics and its impact on productivity. Here, we identify potential cobalamin sources and sinks on the Scotian Shelf and Slope in the Northwest Atlantic Ocean. Functional and taxonomic annotation of bulk metagenomic reads, combined with analysis of genome bins, were used to identify potential cobalamin sources and sinks. Cobalamin synthesis potential was mainly attributed to Rhodobacteraceae, Thaumarchaeota, and cyanobacteria (Synechococcus and Prochlorococcus). Cobalamin remodelling potential was mainly attributed to Alteromonadales, Pseudomonadales, Rhizobiales, Oceanospirilalles, Rhodobacteraceae, and Verrucomicrobia, while potential cobalamin consumers include Flavobacteriaceae, Actinobacteria, Porticoccaceae, Methylophiliaceae, and Thermoplasmatota. These complementary approaches identified taxa with the potential to be involved in cobalamin cycling on the Scotian Shelf and revealed genomic information required for further characterization. The Cob operon of Rhodobacterales bacterium HTCC2255, a strain with known importance in cobalamin cycling, was similar to a major cobalamin producer bin, suggesting that a related strain may represent a critical cobalamin source in this region. These results enable future inquiries that will enhance our understanding of how cobalamin shapes microbial interdependencies and productivity in this region. 相似文献
14.
R M Jeter 《Journal of general microbiology》1990,136(5):887-896
The enteric bacterium Salmonella typhimurium utilizes 1,2-propanediol as a sole carbon and energy source during aerobic growth, but only when the cells are also provided with cobalamin as a nutritional supplement. This metabolism is mediated by the cobalamin-dependent propanediol dehydratase enzyme pathway. Thirty-three insertion mutants were isolated that lacked the ability to utilize propanediol, but retained the ability to degrade propionate. This phenotype is consistent with specific blocks in one or more steps of the propanediol dehydratase pathway. Enzyme assays confirmed that propanediol dehydratase activity was absent in some of the mutants. Thus, the affected genes were designated pdu (for defects in propanediol utilization). Seventeen mutants carried pdu::lac operon fusions, and these fusions were induced by propanediol in the culture medium. All of the pdu mutations were located in a single region (41 map units) on the S. typhimurium chromosome between the his (histidine biosynthesis) and branch I cob (cobalamin biosynthesis) operons. They were shown to be P22-cotransducible with a branch I cob marker at a mean frequency of 12%. Mutants that carried deletions of the genetic material between his and cob also failed to utilize propanediol as a sole carbon source. Based upon the formation of duplications and deletions between different pairs of his::MudA and pdu::MudA insertions, the pdu genes were transcribed in a clockwise direction relative to the S. typhimurium genetic map. 相似文献
15.
Reaction of alkylcobalamins with thiols 总被引:2,自引:0,他引:2
Carbon-13 NMR spectroscopy and phosphorus-31 NMR spectroscopy have been used to study the reaction of several alkylcobalamins with 2-mercaptoethanol. At alkaline pH, when the thiol is deprotonated, the alkyl-transfer reactions involve a nucleophilic attack of the thiolate anion on the Co-methylene carbon of the cobalamins, yielding alkyl thioethers and cob(II)alamin. In these nucleophilic displacement reactions cob(I)alamin is presumably formed as an intermediate. The higher alkylcobalamins react more slowly than methylcobalamin. The lower reactivity of ethyl- and propylcobalamin is probably the basis of the inhibition of the corrinoid-dependent methyl-transfer systems by propyl iodide. The transfer of the upper nucleoside ligand of adenosylcobalamin to 2-mercaptoethanol is a very slow process; S-adenosyl-mercaptoethanol and cob(II)alamin are the final products of the reaction. The dealkylation of (carboxymethyl)cobalamin is a much more facile reaction. At alkaline pH S-(carboxymethyl)mercaptoethanol and cob(II)alamin are produced, while at pH values below 8 the carbon-cobalt bond is cleaved reductively to acetate and cob(II)alamin. The reductive cleavage of the carbon-cobalt bond of (carboxymethyl)cobalamin by 2-mercaptoethanol is extremely fast when the cobalamin is in the "base-off" form. Because we have been unable to detect trans coordination of 2-mercaptoethanol, we favor a mechanism that involves a hydride attack on the Co-methylene carbon of (carboxymethyl)cobalamin rather than a trans attack of the thiol on the cobalt atom. 相似文献
16.
Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a large, modular enzyme that uses a cobalamin prosthetic group as a donor or acceptor in three separate methyl transfer reactions. The prosthetic group alternates between methylcobalamin and cob(I)alamin during catalysis as homocysteine is converted to methionine using a methyl group derived from methyltetrahydrofolate. Occasional oxidation of cob(I)alamin to cob(II)alamin inactivates the enzyme. Reductive methylation with flavodoxin and adenosylmethionine returns the enzyme to an active methylcobalamin state. At different points during the reaction cycle, the coordination state of the cobalt of the cobalamin changes. The imidazole side chain of His759 coordinates to cobalamin in a "His-on" state and dissociates to produce a "His-off" state. The His-off state has been associated with a conformation of MetH that is poised for reactivation of cobalamin by reductive methylation rather than catalysis. Our studies on cob(III)alamins bound to MetH, specifically aqua-, methyl-, and n-propylcobalamin, show a correlation between the accessibility of the reactivation conformation and the order of the established ligand trans influence. The trans influence also controls the affinity of MetH in the cob(III)alamin form for flavodoxin. Flavodoxin, which acts to shift the conformational equilibrium toward the reactivation conformation, binds less tightly to MetH when the cob(III)alamin has a strong trans ligand and therefore has less positive charge on cobalt. These results are compared to those for cob(II)alamin MetH, illustrating that access to the reactivation conformation is governed by the net charge on the cobalt as well as the trans influence in cob(III)alamins. 相似文献
17.
The pdu operon encodes proteins for the catabolism of 1,2-propanediol; the nearby cob operon encodes enzymes for the biosynthesis of adenosyl-cobalamin (vitamin B12), a cofactor required for the use of propanediol. These operons are transcribed divergently from distinct promoters separated by several kilobases. The regulation of the two operons is tightly integrated in that both require the positive activator protein PocR and both are subject to global control by the Crp and ArcA proteins. We have determined the DNA nucleotide sequences of the promoter-proximal portion of the pdu operon and the region between the pdu and cob operons. Four open reading frames have been identified, pduB, pduA, pduF, and pocR. The pduA and pduB genes are the first two genes of the pdu operon (transcribed clockwise). The pduA gene encodes a hydrophobic protein with 56% amino acid identity to a 10.9-kDa protein which serves as a component of the carboxysomes of several photosynthetic bacteria. The pduF gene encodes a hydrophobic protein with a strong similarity to the GlpF protein of Escherichia coli, which facilitates the diffusion of glycerol. The N-terminal end of the PduF protein includes a motif for a membrane lipoprotein-lipid attachment site as well as a motif characteristic of the MIP (major intrinsic protein) family of transmembrane channel proteins. We presume that the PduF protein facilitates the diffusion of propanediol. The pocR gene encodes the positive regulatory protein of the cob and pdu operons and shares the helix-turn-helix DNA binding motif of the AraC family of regulatory proteins. The mutations cobR4 and cobR58 cause constitutive, pocR-independent expression of the cob operon under both aerobic and anaerobic conditions. Evidence that each mutation is a deletion creating a new promoter near the normal promoter site of the cob operon is presented. 相似文献
18.
19.
Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica serovar typhimurium LT2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH(2) dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH(2) occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH(2) in the reaction mixture. FMNH(2) failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a fre mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that S. enterica serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases. 相似文献
20.
Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modular enzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptor in three separate methyl transfer reactions. At different points during the reaction cycle, the coordination to the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinates to the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinct species of the cob(II)alamin-bound His759Gly variant have been identified and separated. Limited proteolysis with trypsin was employed to demonstrate that the two species differ in protein conformation. Magnetic circular dichroism and electron paramagnetic resonance spectroscopies were used to show that the two species also differ with respect to the axial coordination to the central cobalt ion of the cobalamin cofactor. One form appears to be in a conformation poised for reductive methylation with adenosylmethionine; this form was readily reduced to cob(I)alamin and subsequently methylated [albeit yielding a unique, five-coordinate methylcob(III)alamin species]. Our spectroscopic data revealed that this form contains a five-coordinate cob(II)alamin species, with a water molecule as an axial ligand to the cobalt. The other form appears to be in a catalytic conformation and could not be reduced to cob(I)alamin under any of the conditions tested, which precluded conversion to the methylcob(III)alamin state. This form was found to possess an effectively four-coordinate cob(II)alamin species that has neither water nor histidine coordinated to the cobalt center. The formation of this four-coordinate cob(II)alamin "dead-end" species in the His759Gly variant illustrates the importance of the His759 residue in governing the equilibria between the different conformations of MetH. 相似文献