Proposed nomenclature for the genes involved in molybdenum metabolism in Escherichia coli and Salmonella typhimurium.
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Coenzymes↗
Biomedical subjects
M Chippaux
Publications and source records attributed to M Chippaux.
Nucleotide sequences of the arb genes, which control beta-glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new beta-glycohydrolase family including enzymes from eubacteria, archeabacteria, and humans.
The phytopathogenic bacterium Erwinia chrysanthemi, unlike other members of the family Enterobacteriaceae, is able to metabolize the beta-glucosides, arbutin, and salicin. A previous genetic analysis of the E. chrysanthemi arb genes, which mediate beta-glucoside metabolism, suggested that they were homologous to the Escherichia coli K-12 bgl genes. We have now determined the nucleotide sequence of a 5,065-bp DNA fragment containing three genes, arbG, arbF, and arbB. Deletion analysis, expression in minicell systems, and comparison with sequences of other proteins suggest that arbF and arbB encode a beta-glucoside-specific phosphotransferase system-dependent permease and a phospho-beta-glucosidase, respectively. The ArbF amino acid sequence shares 55% identity with that of the E. coli BglF permease and contains most residues thought to be important for a phosphotransferase. One change, however, was noted, since BglF Arg-625, presumably involved in phosphoryl transfer, was replaced by a Cys residue in ArbF. An analysis of the ArbB sequence led to the definition of a protein family which contained enzymes classified as phospho-beta-glucosidases, phospho-beta-galactosidases, beta-glucosidases, and beta-galactosidases and originating from gram-positive and gram-negative bacteria, archebacteria, and mammals, including humans. An analysis of this family allowed us (i) to speculate on the ways that these enzymes evolved, (ii) to identify a glutamate residue likely to be a key amino acid in the catalytic activity of each protein, and (iii) to predict that domain II of the human lactate-phlorizin hydrolase, which is involved in lactose intolerance, is catalytically nonactive. A comparison between the untranslated regions of the E. chrysanthemi arb cluster and the E. coli bgl operon revealed the conservation of two regions which, in the latter, are known to terminate transcription under noninducing conditions and be the target of the BglG transcriptional antiterminator under inducing conditions. ArbG was found to share a high level of similarity with the BglG antiterminator as well as with Bacillus subtilis SacT and SacY antiterminators, suggesting that ArbG functions as an antiterminator in regulating the expression of the E. chrysanthemi arb genes.
Amino Acid Sequence↗
Possible involvement of a L-delta 1-pyrroline-5-carboxylate (P5C) reductase in the synthesis of proline in Desulfovibrio desulfuricans Norway.
A L-delta 1-pyrroline-5-carboxylate reductase activity has been detected in crude extracts of Desulfovibrio desulfuricans Norway. This P5C reductase activity is also found when a 2.5 kb D. desulfuricans DNA fragment is introduced into an Escherichia coli proC mutant. Although it restores growth of the proC mutant, the ProDd enzyme might be detrimental to the E. coli host since the plasmid carrying the cognate proDd gene is segregated at high rate by the cells but is stabilized by small deletions which lead to a loss of the P5C reductase activity.
DNA, Bacterial↗
A regulatory mutant of the trimethylamine N-oxide reductase of Escherichia coli K12.
A mutant of Escherichia coli was isolated in which the trimethylamine N-oxide (TMAO) reductase activity is lost and the inducible TMAO reductase protein is absent. The lack of the gene product specifically prevents the expression of torA::lacZ fusion indicating a transcriptional control of torA, the TMAO reductase structural gene. The gene designated torR was mapped at 22 min on the chromosome.
Chromosome Mapping↗
Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis.
Biochemical, genetic and primary sequence analyses of the Erwinia chrysanthemi endoglucanase EGZ allowed us to identify two functional domains and to locate their boundaries. The catalytic domain extends from residue 1 to 288, while a domain required for EGZ to bind to microcrystalline cellulose lies from residues 324 to 385. Each domain was found capable of functioning in the absence of the other. A region rich in Pro, Thr, and Ser residues links both domains and appeared to be susceptible to proteolytic attack. Based upon predictions derived from a method developed to compare sequences sharing a low level of similarity, e.g. hydrophobic cluster analysis (HCA), we analysed the importance of either residue His98 or Glu133 in EGZ catalytic activity. Two EGZ-derived proteins were engineered in which either His98 or Glu133 amino acid was converted to an Ala residue. Characterization of the purified proteins showed that no enzymatic activity could be detected, by using carboxymethylcellulose (CMC) or paranitrophenyl-cellobioside (pNPC) as substrates, while both mutated proteins retained the capacity to bind to microcrystalline cellulose. These studies, which to date constitute the first experimental testing of HCA-derived predictions, allowed us to identify two particular amino acids involved in cellulolytic activity. By taking into account data from chemical modification studies of other cellulases, we speculate that the His98 residue is involved in the folding of the catalytic domain while the Glu133 residue intervenes directly in the beta, 1-4 glycosidic bond cleavage.
Amino Acid Sequence↗
Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans.
A strain of Desulfovibrio fructosovorans deleted from the hydN [NiFe]hydrogenase structural gene was constructed. A plasmid carrying a 7 kb DNA fragment on which the hydN gene had been replaced by the npt reporter gene (kanamycin-resistant, KnR) was introduced into D. fructosovorans by electroporation. Southern analysis of one KnR clone demonstrated that the hydN gene had been eliminated by marker exchange. This mutant, which was devoid of the [NiFe]hydrogenase gene, still showed a 10% residual hydrogenase activity. Its ability to grow efficiently with H2 as sole energy source is discussed. This is the first report, in a member of the sulphate-reducing bacteria, of a successful transformation and concomitant homologous recombination leading to a fully controlled genotype.
Blotting, Southern↗
Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon.
The structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced. They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV. The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism. The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/NarI pair. The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E. coli and their relative order is the same. The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex. Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COOH-terminal end of NarK. The ORFA-narZ intergenic region, however, is about 80 nucleotides long and does not contain the cis-acting elements, NarL and Fnr boxes, nor the terC4 terminator sequence present in the 500 nucleotide narK-narG intergenic region. This might explain why the narZYWV and the narGHJI operons are regulated differently. Our results tend to support the hypothesis that a DNA fragment larger than that encompassing the narGHJI genes has been duplicated.
Amino Acid Sequence↗
Analysis of the Erwinia chrysanthemi arb genes, which mediate metabolism of aromatic beta-glucosides.
Erwinia chrysanthemi is one of the few members of the family Enterobacteriaceae that is capable of metabolizing most of the naturally occurring beta-glucosides. We previously isolated the clb genes, which allow the use of the disaccharide cellobiose as well as the aromatic beta-glucosides arbutin and salicin. We report here the isolation of the arb genes, which permit fermentation of the aromatic beta-glucosides only. Establishment of a functional Arb system in Escherichia coli depended on the presence of the phosphotransferase system and on the activation by the cyclic AMP-cyclic AMP receptor protein complex. Strains carrying mini-Mu-induced LacZ fusions to the arb genes were used to analyze arb genes organization and function. Three arb genes (arbG, arbF, and arbB) were identified and organized in this order. Genetic and structural evidence allowed us to assign a phospho-beta-glucosidase and a permease activity to the ArbB and ArbF proteins, respectively. Several Lac+ arb-lacZ insertions were introduced into the E. chrysanthemi chromosome. Both ArbG- and ArbF- strains were unable to ferment the aromatic beta-glucosides, whereas ArbB- strains were impaired only in salicin fermentation. None of the mutations in the arb genes affected cellobiose metabolism. The expression of the arb genes was substrate inducible and required the ArbF permease and, possibly, the ArbG protein. Collectively, our results underline the resemblance between the naturally expressed E. chrysanthemi arbGFB and the cryptic E. coli bglGFB operons, yet the arbG gene product seemed unable to activate E. coli bgl operon expression.
Cloning, Molecular↗
Nitrate reductase of Escherichia coli: completion of the nucleotide sequence of the nar operon and reassessment of the role of the alpha and beta subunits in iron binding and electron transfer.
The nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed. It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively. The analysis of deduced amino acid sequence failed to reveal any structure capable of binding iron within the NarG polypeptide. In contrast, cysteine arrangements typical of iron-sulfur centers were found in the NarH polypeptide. This suggested that the latter is an electron transfer unit of the nitrate reductase complex. Such a view is opposite to the current description of the nitrate reductase. The findings allowed us to propose a model for the electron transfer steps that occur during nitrate reduction. The NarG polypeptide was found to display a high degree of homology with numerous E. coli molybdoproteins. Moreover, the same genetic and functional organizations as well as the presence of highly conserved stretches of amino acids were noted between both NarG/NarH and DmsA/DmsB (encoding the dimethyl sulfoxide reductase) pairs.
Amino Acid Sequence↗
The beta-glucosides metabolism in Erwinia chrysanthemi: preliminary analysis and comparison to Escherichia coli systems.
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Erwinia↗
Mapping and regulation of the cel genes in Erwinia chrysanthemi.
Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the fusions, (iii) recombinational exchange between the plasmidic mutated and the wild-type chromosomal alleles. These mutations allowed mapping of celZ between ura and pan and celY between xyl and met on the linkage map of E. chrysanthemi. The beta-galactosidase activity of these strains indicated that celZ is expressed in the late exponential and stationary growth phases, while celY expression is almost undetectable.
Bacteriophage mu↗
Aerobic expression of the nar operon of Escherichia coli in a fnr mutant.
Mutations allowing aerobic expression of the anaerobically controlled nar operon have been located in the autoregulated fnr gene. Cloning and sequencing of the mutant fnrd20 allele, and fnr mRNA quantitation by dot blot assay, revealed that the mutation was the result of an IS5 insertion into the control region of fnr that enhanced transcription of the fnr gene at least ten-fold. Examination of the regulatory region of the negatively autoregulated fnr gene indicated that it shared homologous sequences with the positively Fnr-controlled frd and nar operons. The increase in fnr transcription in the fnrd20 mutated allele could be partly the result of loss of autoregulation, since the IS5 separated the Fnr target site from the '-35' region of the promoter.
Alleles↗
Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway.
A library of Desulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidly lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridizations, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.
Cloning, Molecular↗
Presence in the 'silent' terminus region of the Escherichia coli K12 chromosome of cryptic gene(s) encoding a new nitrate reductase.
A cosmid complementing narG mutants defective in nitrate reductase activity was isolated from a genomic library of Escherichia coli. The restriction map of the insert differed from that of the narGHI operon. The new enzyme, termed NarZ, required molybdenum for activity. The expression of narZ was not affected by the factors controlling narGHI. Insertion mutations indicated that the narZ locus covered about 8 kb of DNA; narZ is located at 32.5 U on the chromosome, in the cotransduction gap near the replication terminus. Southern blot experiments under stringent conditions using narGHI or narZ DNA as probes revealed a large extent of homology, with a small area of very high homology. We propose that narZ and narGHI have descended from a common ancestor by gene duplication.
Chromosome Mapping↗
Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme.
Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities.
Electrophoresis, Polyacrylamide Gel↗
Autoregulation of the nar operon encoding nitrate reductase in Escherichia coli.
Nitrate reductase is demonstrated to exert an autogenous control on its own synthesis. This effect requires the participation of the molybdenum cofactor. Use of strains in which the control region of the nar operon is mutated reveals two loci in this region: one, affected in strain LCB94, is common to both autoregulation and induction by nitrate while the other, mutated in strain LCB188, is specific for the induction by nitrate. It is proposed that the autogenous control prevents the unnecessary accumulation of the nitrate reductase subunits in the cytoplasm.
Aerobiosis↗
Alteration by mutation of the control by oxygen of the nar operon in Escherichia coli.
A nar-lac operon fusion was used to isolate a mutant in which the expression of the nar operon was no longer repressed by oxygen. The nard mutation, located upstream of the nar structural genes, was found to be cis dominant; it led to independence from the Fnr protein which, in the wild-type strain, exerts a strict positive control on the nar operon. Both other known controls, nitrate induction and autoregulation, were unaffected. It is proposed that molecular oxygen controls the expression of nar via Fnr and that the nard mutation affects the Fnr binding site of the narGHI control region.
Aerobiosis↗
Influence of gyrA mutation on expression of Erwinia chrysanthemi clb genes cloned in Escherichia coli.
Erwinia chrysanthemi clb genes cloned into Nals Escherichia coli allowed growth on cellobiose, arbutin, or salicin. In contrast, Nalr isogenic strains grew only on cellobiose. It is proposed that expression of cloned E. chrysanthemi clb genes is reduced by the E. coli chromosomal gyrA (Nalr) mutation, resulting in apparent segregation of the Clb and Arb Sal characters.
Cellobiose↗