SummaryThe Helicobacter pylori genome contains a gene ( hp1338 or nikR ) that encodes a nickel-dependent regulator that is homologous to the Escherichia coli nickel-responsive regulator, NikR. The H. pylori nikR product acts as a pleiotropic metal-dependent regulator. We constructed a non-polar isogenic mutant deleted for the nikR gene. NikR was essential for the survival of H. pylori in the presence of high nickel and cobalt ion concentrations in vitro . We screened a DNA macroarray for genes that were differentially expressed in parental and nikR-deficient H. pylori strains grown in the presence of excess nickel. We found that H. pylori NikR mediates the expression of nickel-activated and -repressed genes. In the presence of excess nickel, NikR activated the transcription of ureA-ureB ( hp72-73 ) , nixA ( hp1077 ) , copA2 ( hp1072 ) , hpn ( hp1427 ) and hpn-like ( hp1432 ) genes and repressed the expression of genes encoding proteins involved in ferric iron uptake and storage [ pfr ( hp0653 ) , fur ( hp1027 ), frpB4 ( hp1512 ), exbB/ exbD ( hp1339-1340 ), ceuE ( bgalactosidase assays with the region between nikR and the exbB/exbD divergent operon, and the study of exbB gene expression using a gentamicin/ apramycin reporter gene in H. pylori indicated that NikR is an autorepressor that binds to this intergenic region and also controls the expression of the exbB/ exbD/tonB operon, which provides energy for ferric iron uptake. Thus, as previously suggested for Fur in H. pylori , NikR appears to be a global regulator of the metabolism of some divalent cations within a highly complex regulated network.

The complete nucleotide sequence of the Escherichia coli nik locus, which has been suggested to encode the specific transport system for nickel, has been determined. It was found to contain five overlapping open reading frames that form a single transcription unit. Deduced amino acid sequence of the nik operon shows that its five gene products, NikA to NikE, are highly homologous to components of oligopeptide- and dipeptide-binding protein-dependent transport systems from several Gram-negative and Gram-positive species. NikA represents the periplasmic binding protein, NikB and NikC are similar to integral membrane components of periplasmic permeases, and NikD and NikE possess typical ATP-binding domains that suggest their energy coupling role to the transport process. Insertion mutations in nik genes totally abolished the nickel-containing hydrogenase activity under nickel limitation and markedly altered the rate of nickel transport. Taken together, these data support the notion that the nik operon encodes a typical periplasmic binding-protein-dependent transport system for nickel.

The biological requirement of the trace element selenium was recognized 40 years ago. Selenium is incorporated into several enzymes and transfer RNA species of both prokaryotic and eukaryotic origin. In enzymes which contain a selenopolypeptide, selenium is present as covalently bound selenocysteine which participates in the catalytic reaction. Sequence analysis of the genes coding for two selenoproteins, formate dehydrogenase H from Escherichia coli and glutathione peroxidase from mouse and man, demonstrated that an in-frame UGA opal nonsense codon directs the incorporation of selenocysteine. In the case of formate dehydrogenase incorporation occurs cotranslationally. Recently, we identified four genes whose products are required for selenocysteine incorporation in E. coli. We report here that one of these genes codes for a tRNA species with unique properties. It possesses an anticodon complementary to UGA and deviates in several positions from sequences, until now, considered invariant in all tRNA species. This tRNA is aminoacylated with L-serine by the seryl-tRNA ligase which also charges cognate tRNASer. Selenocysteine, therefore, is synthesized from a serine residue bound to a natural suppressor tRNA which recognizes UGA.

We report here on the isolation and primary characterization of the yohM gene of Escherichia coli. We show that yohM encodes a membrane-bound polypeptide conferring increased nickel and cobalt resistance in E. coli. yohM was specifically induced by nickel or cobalt but not by cadmium, zinc, or copper. Mutation of yohM increased the accumulation of nickel inside the cell, whereas cells harboring yohM in multicopy displayed reduced intracellular nickel content. Our data support the hypothesis that YohM is the first described efflux system for nickel and cobalt in E. coli. We propose rcnA (resistance to cobalt and nickel) as the new denomination of yohM.Nickel and cobalt are both required as trace elements in prokaryotes to fulfill a variety of metabolic functions, but high intracellular concentrations of these transition metals are toxic. One of the strategies evolved by bacteria to prevent damage is to export excess metal by efflux systems. Plasmid-borne determinants responsible for nickel and/or cobalt resistance have been described for the heavy-metal-resistant bacterium Ralstonia metallidurans (11,15), among which are members of the resistance-nodulation-cell division superfamily: the best-characterized CzcCBA (cobalt-zinc-cadmium) three-component cation antiporter (14) and the homologous CnrCBA (cobaltnickel resistance) (10) and NccCBA (nickel-cobalt-cadmium resistance) (18) efflux systems. Moreover, cobalt can be extruded from the cytoplasm by the cation diffusion facilitator CzcD of R. metallidurans at the expense of the proton motive force or a potassium gradient (15). Cobalt may also be a substrate of Zn-CPx-type ATPases, as in Helicobacter pylori (8). There is no evidence for the transport of nickel by one of these two modes of efflux. Instead, this metal can be transported outside the cytoplasm by NreB from R. metallidurans (7) or NrsD from Synechocystis sp. strain PCC 6803 (6), which are members of the major facilitator superfamily and which each exhibit 12 putative transmembrane helices and a histidine-rich carboxy terminus contributing to nickel resistance.In Escherichia coli, anaerobic hydrogenase isoenzymes and urease (in ureolytic strains) require incorporation of nickel to become active (12). Complex assembly processes involve accessory proteins, namely, HypB, implicated in nickel insertion into hydrogenase, and UreE, which delivers nickel to urease. HypB and UreE are well conserved among bacteria apart from a terminal histidine-rich stretch whose function would be nickel storage and which is absent in E. coli proteins (3, 5). In order to gain insights into nickel trafficking and, more precisely, to find proteins that would be involved in nickel resistance, we searched the E. coli genome database with a query based on a consensus alignment of the UreE and HypB histidine-rich variants. The best returned hit was yohM, whose product bears a histidine-rich domain in its center. The aim of the present work is to demonstrate the implication of yohM in nickel and cobalt trafficking in E. coli.Inactivati...

The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H-NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56,613 Da (CaiT), 42,564 Da (CaiA), 59,311 Da (CaiC), 32,329 Da (CaiD) and 21,930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium.

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Hydrogenase activity and other hydrogenase-related functions can be restored to hydC mutants by the specific addition of nickel salts to the growth medium. These mutants are defective in all three hydrogenase isoenzymes and the restoration is dependent upon protein synthesis. The cellular nickel content of the mutant when grown in LB medium is less than 1% of that of the parental strain. Partial suppression of the hydrogenase phenotype of hydC mutants occurs when growth takes place in a different medium. This correlates with an increased cellular nickel content. The phenotype of the mutant is also fully suppressed by growth in media of very low magnesium content. Such media facilitate nickel uptake via the magnesium transport system, which leads to the acquisition of a normal cellular nickel content. Mutations in the fnr gene, which encodes a transcriptional regulator for several anaerobically expressed enzymes, abolishes hydC expression and gives rise to a defective hydrogenase phenotype. The hydrogenase phenotype of fnr is closely similar to that of hydC in all respects examined. The hydrogenase activity of fnr strains can be restored by the presence of a functional hydC gene on a multicopy plasmid. The hydrogenase phenotype of fnr strains therefore arises indirectly via suppression of hydC, which leads to a low cellular nickel content. Nickel has no influence on fumarate reductase or nitrate reductase activities in fnr strains. The hydrogen-metabolism phenotype of fnr strains is, therefore, dependent upon their ability to acquire nickel from growth media. It is likely that hydC encodes a specific transport system for nickel.

Mutants of Escherichia coli were isolated which were affected in the formation of both formate dehydrogenase N (phenazine methosulfate reducing) (FDHN) and formate dehydrogenase H (benzylviologen reducing) (FDHH). They were analyzed, together with previously characterized pleiotropicfdh mutants (fdhA, fdhB, and fdhC), for their ability to incorporate selenium into the selenopolypeptide subunits of FDHN and FDHH. Eight of the isolated strains, along with the fdhA and fdhC mutants, maintained the ability to selenylate tRNA, but were unable to insert selenocysteine into the two selenopolypeptides. ThefdhB mutant tested had lost the ability to incorporate selenium into both protein and tRNA. fdhF, which is the gene coding for the 80-kilodalton selenopolypeptide of FDHH, was expressed from the T7 promoter-polymerase system in the pleiotropic fdh mutants. A truncated polypeptide of 15 kilodaltons was formed; but no full-length (80-kilodalton) gene product was detected, indicating that translation terminates at the UGA codon directing the insertion of selenocysteine. A mutant fdhF gene in which the UGA was changed to UCA expressed the 80-kilodalton gene product exclusively. This strongly supports the notion that the pleiotropicfdh mutants analyzed possess a lesion in the gene(s) encoding the biosynthesis or the incorporation of selenocysteine. The gene complementing the defect in one of the isolated mutants was cloned from a cosmid library. Subclones were tested for complementation of other pleiotropic fdh mutants. The results revealed that the mutations in the eight isolates fell into two complementation groups, one of them containing the fdhA mutation. fdhB, fdhC, and two of the new fdh isolates do not belong to these complementation groups. A new nomenclature (set) is proposed for pleiotropic fdh mutations affecting selenium metabolism. Four genes have been identified so far: selA and selB (at thefdhA locus), selC (previously fdhC), and selD (previously fdhB).Several enzymes from procaryotic and eucaryotic organisms and a number of tRNA species contain selenium in a covalently bound form (21). For bacteria, the first indication for a biological role of this trace element dates back to 1954, when Pinsent detected that gas production by anaerobic cultures of Escherichia coli depended upon the presence of selenium in the medium (20); enzymological studies revealed that it was the formate dehydrogenase component of the formate-hydrogen-lyase complex which specifically required selenium for activity (14,20). By using [75Se]selenite, it was subsequently demonstrated that the isotope was incorporated into just two proteins in E. coli: an 80-kilodalton polypeptide, which is a constituent component of formate dehydrogenase H (FDHH) and is involved in gas formation, and a 110-kilodalton polypeptide, which is part of formate dehydrogenase N (FDHN) and delivers the electrons from formate to nitrate reductase (8,19). FDHH is formed under anaerobic conditions in the absence of external electron acceptors, whereas synthesis of FDH...