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Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A. 总被引:3,自引:1,他引:2
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The nickel-cobalt-cadmium resistance genes carried by plasmid pTOM9 of Alcaligenes xylosoxidans 31A are located on a 14.5-kb BamHI fragment. By random Tn5 insertion mutagenesis, the fragment was shown to contain two distinct nickel resistance loci, ncc and nre. The ncc locus causes a high-level combined nickel, cobalt, and cadmium resistance in strain AE104, which is a cured derivative of the metal-resistant bacterium Alcaligenes eutrophus CH34. ncc is not expressed in Escherichia coli. The nre locus causes low-level nickel resistance in both Alcaligenes and E. coli strains. The nucleotide sequence of the ncc locus revealed seven open reading frames designated nccYXHCBAN. The corresponding predicted proteins share strong similarities with proteins encoded by the metal resistance loci cnr (cnrYXHCBA) and czc (czcRCBAD) of A. eutrophus CH34. When different DNA fragments carrying ncc genes were heterologously expressed under the control of the bacteriophage T7 promoter, five protein bands representing NccA (116 kDa), NccB (40 kDa), NccC (46 kDa), NccN (23.5 kDa), and NccX (16.5 kDa) were detected. 相似文献
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Nickel-Resistant Bacteria from Anthropogenically Nickel-Polluted and Naturally Nickel-Percolated Ecosystems 总被引:8,自引:3,他引:5
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DNA fragments harboring the nickel resistance determinants from bacteria isolated from anthropogenically polluted ecosystems in Europe and Zaire were compared with those harboring the nickel resistance determinants from bacteria isolated from naturally nickel-percolated soils from New Caledonia by DNA-DNA hybridization. The biotinylated DNA probes were derived from the previously described Alcaligenes eutrophus CH34, Alcaligenes xylosoxidans 31A, Alcaligenes denitrificans 4a-2, and Klebsiella oxytoca CCUG 15788 and four new nickel resistance-determining fragments cloned from strains isolated from soils under nickel-hyperaccumulating trees. Nine probes were hybridized with endonuclease-cleaved plasmid and total DNA samples from 56 nickel-resistant strains. Some of the New Caledonian strains were tentatively identified as Acinetobacter, Pseudomonas mendocina, Comamonas, Hafnia alvei, Burkholderia, Arthrobacter aurescens, and Arthrobacter ramosus strains. The DNA of most strains showed homologies to one or several of the following nickel resistance determinants: the cnr and ncc operons of the strains A. eutrophus CH34 and A. xylosoxidans 31A, respectively, the nre operon of strain 31A, and the nickel resistance determinants of K. oxytoca. On the basis of their hybridization reactions the nickel resistance determinants of the strains could be assigned to four groups: (i) cnr/ncc type, (ii) cnr/ncc/nre type, (iii) K. oxytoca type, and (iv) others. The majority of the strains were assigned to the known groups. Among the strains from Belgium and Zaire, exclusively the cnr/ncc and the cnr/ncc/nre types were found. Among the New Caledonian strains all four types were represented. Homologies to the nre operon were found only in combination with the cnr/ncc operon. The homologies to the cnr/ncc operon were the most abundant and were detected alone or together with homologies to the nre operon. Only the DNA of the strains isolated from soil in Scotland and the United States and that of five of the New Caledonian strains did not show any detectable homologies to any of our probes. The nickel resistance fragment isolated from Burkholderia strain 32W-2 was studied in some detail. This 15-kb BamHI fragment conferred resistance to 1 to 5 mM NiCl(inf2) to Escherichia coli and resistance to up to 25 mM NiCl(inf2) to A. eutrophus. It showed strong homologies to both the cnr/ncc operon and the nre operon and conferred strictly regulated (inducible) nickel resistance to A. eutrophus. 相似文献
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Tian J Wu N Li J Liu Y Guo J Yao B Fan Y 《Applied and environmental microbiology》2007,73(7):2364-2368
Leptospirillum ferriphilum strain UBK03 isolated from a mine in Jiangxi, China, is resistant to Ni(2+) (30 to 40 mM). A four-gene nickel resistance cluster was identified and, when transformed into Escherichia coli, enabled growth in 6 mM nickel. Mutation experiments revealed that the genes ncrA, ncrB, and ncrC could confer nickel resistance in Escherichia coli, whereas the gene ncrY could have a negative effect on nickel resistance. 相似文献
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Klebsiella oxytoca CCUG 15788 is resistant to Ni2+ at a concentration of 10 mM and grows in an inducible manner when exposed to lower concentrations of Ni2+. The complete genomic sequence of a 4.2-kb HindIII-digested fragment of this strain was determined from genomic DNA. It was shown to contain four nickel resistance genes (nirA, nirB, nirC, and nirD) encoding transporter and transmembrane proteins for nickel resistance. When the plasmid pKOHI4, encoding nirABCD, was transformed into Escherichia coli JM109, the cells were able to grow in Tris-buffered mineral medium containing 3 mM nickel. TnphoA'-1 insertion mutants in the four nickel genes nirA, nirB, nirC, and nirD showed nickel sensitivity. The nir genes were heterogeneously expressed in E. coli, suggesting functional roles of these genes in nickel resistance. 相似文献
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Molecular mechanisms underlying inducible cobalt and nickel resistance of a bacterial strain isolated from a Cuban serpentine
deposit were investigated. This strain C-1 was assigned to Serratia marcescens by 16S rDNA analysis and DNA/DNA hybridization. Genes involved in metal resistance were identified by transposon mutagenesis
followed by selection for cobalt- and nickel-sensitive derivatives. The transposon insertion causing the highest decrease
in metal resistance was located in the ncrABC determinant. The predicted NcrA product was a NreB ortholog of the major facilitator protein superfamily and central for
cobalt/nickel resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in Escherichia coli and caused decreased accumulation of Co(II) and Ni(II) in this heterologous host. NcrB may be a regulatory protein. NcrC
was a protein of the nickel–cobalt transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased
accumulation of Ni(II) and Co(II). Because the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium
of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute
to nickel and cobalt resistance in this bacterium. 相似文献
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The NreB–NreC two-component system of Staphylococcus carnosus for O2 sensing cooperates with the accessory nitrate sensor NreA in the NreA–NreB–NreC system for coordinated sensing and regulation of nitrate respiration by O2 and nitrate. ApoNreA (NreA in the absence of nitrate) interacts with NreB and inhibits NreB autophosphorylation (and activation). NreB contains the phosphatase motif DxxxQ. The present study shows that NreB on its own was inactive for the dephosphorylation of the phosphorylated response regulator NreC (NreC-P), but co-incubation with NreB and NreA stimulated NreC-P dephosphorylation. Either the presence of instead of apoNreA or mutation of the phosphatase motif (D160 or Q164) of NreB abrogated phosphatase activity of NreB. Phosphatase activity was observed for anoxic (active) NreB as well as oxic NreB, therefore the functional state of NreB is not relevant for phosphatase activity. Thus, NreB is a bifunctional sensor kinase with an integral cryptic phosphatase activity. Activation of phosphatase activity and dephosphorylation of NreC-P requires NreA as a cofactor. Accordingly, NreA and nitrate have major and dual roles in NreA–NreB–NreC regulation by (i) inhibiting NreB phosphorylation and (ii) triggering a kinase/phosphatase switch of NreB when present as apoNreA. 相似文献
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基因工程菌大肠杆菌JM109富集废水中镍离子的研究 总被引:4,自引:2,他引:4
利用通过基因工程技术所构建的在细胞内同时表达出高特异性镍转运蛋白和金属硫蛋白的基因工程菌富集水体中的镍离子。菌体细胞对Ni2+的富集速率很快,富集过程满足Langmuir等温线模型。与原始宿主菌相比,经基因改造的基因工程菌不仅最大镍富集容量增加了5倍多,而且对pH值、离子强度的变化及其它共存重金属离子的影响都呈现出更强的适应性。相比而言,Na+、Ca2+、Cd2+、Pb2+的影响较小,但Mg2+、Hg2+和Cu2+所引起的负面效应较大。进一步的实验表明基因工程菌对Ni2+的富集行为不需要外加营养物质。 相似文献
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Nickel is toxic to all forms of life, but the mechanisms of cell damage are unknown. Indeed, environmentally relevant nickel levels (8 μM) inhibit wild-type Escherichia coli growth on glucose minimal medium. The same concentration of nickel also inhibits growth on fructose, but not succinate, lactate or glycerol; these results suggest that fructose-1,6-bisphosphate aldolase (FbaA) is a target of nickel toxicity. Cells stressed by 8 μM Ni(II) for 20 min lost 75% of their FbaA activity, demonstrating that FbaA is inactivated during nickel stress. Furthermore, overexpression of fbaA restored growth of an rcnA mutant in glucose minimal medium supplemented with 4 μM Ni(II), thus confirming that FbaA is a primary target of nickel toxicity. This class II aldolase has an active site zinc and a non-catalytic zinc nearby. Purified FbaA lost 80 % of its activity within 2 min when challenged with 8 μM Ni(II). Nickel-challenged FbaA lost 0.8 zinc and gained 0.8 nickel per inactivated monomer. FbaA mutants (D144A and E174A) affecting the non-catalytic zinc were resistant to nickel inhibition. These results define the primary site of nickel toxicity in E. coli as the class II aldolase FbaA through binding to the non-catalytic zinc site. 相似文献
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High-Level Nickel Resistance in Alcaligenes xylosoxydans 31A and Alcaligenes eutrophus KTO2 总被引:2,自引:0,他引:2
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Two new nickel-resistant strains of Alcaligenes species were selected from a large number (about 400) of strains isolated from ecosystems polluted by heavy metals and were studied on the physiological and molecular level. Alcaligenes xylosoxydans 31A is a heterotrophic bacterium, and Alcaligenes eutrophus KTO2 is an autotrophic aerobic hydrogen-oxidizing bacterium. Both strains carry—among other plasmids—a megaplasmid determining resistance to 20 to 50 mM NiCl2 and 20 mM CoCl2 (when growing in defined Tris-buffered media). Megaplasmids pTOM8, pTOM9 from strain 31A, and pGOE2 from strain KTO2 confer nickel resistance to the same degree to transconjugants of all strains of A. eutrophus tested but were not transferred to Escherichia coli. However, DNA fragments carrying the nickel resistance genes, cloned into broad-hostrange vector pVDZ'2, confer resistance to A. eutrophus derivatives as well as E. coli. The DNA fragments of both bacteria, TBA8, TBA9, and GBA (14.5-kb BamHI fragments), appear to be identical. They share equal size, restriction maps, and strong DNA homology but are largely different from fragment HKI of nickel-cobalt resistance plasmid pMOL28 of A. eutrophus CH34. 相似文献
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Kasuga NC Sekino K Koumo C Shimada N Ishikawa M Nomiya K 《Journal of inorganic biochemistry》2001,84(1-2):55-65
To investigate the relationship between antimicrobial activities and the molecular structures of nickel(II) complexes with thiosemicarbazone and semicarbazone ligands, nickel(II) complexes with ligands Hmtsc, Hatsc, Hasc and H2dmtsc, were prepared and characterized by elemental analysis, FT-IR, 1H and 13C NMR spectroscopies, magnetic susceptibility measurements, UV-Vis absorption spectra, TG/DTA and single-crystal X-ray analysis. Their antimicrobial activities were evaluated by the MIC against four bacteria (B. subtilis, S. aureus, E. coli and P. aeruginosa), two yeasts (C. albicans and S. cerevisiae) and two molds (A. niger and P. citrinum). The 4-coordinate, diamagnetic nickel(II) complexes showed antimicrobial activities which were different from those of free ligands or the starting nickel(II) compounds; [Ni(mtsc)(OAc)] 1 showed selective and effective antimicrobial activities against two Gram-positive bacteria (B. subtilis and S. aureus) and modest activities against a yeast (S. cerevisiae), [Ni(mtsc)Cl] 3 exhibited moderate activities against a Gram-positive bacterium (S. aureus), and [Ni(atsc)(OAc)] 5 showed modest activities against two Gram-positive bacteria (B. subtilis and S. aureus). On the other hand, the 6-coordinate, paramagnetic nickel(II) complexes with two protonated or deprotonated ligands ([Ni(mtsc)2] 2, [Ni(atsc)(mtsc)] 4, [Ni(atsc)2] 6, [Ni(Hatsc)2](NO3)(2)7, [Ni(Hatsc)2]Cl(2)8 and [Ni(Hasc)2](OAc)(2)9) and the sterically crowded 4-coordinate, diamagnetic nickel(II) complex ([Ni(dmtsc)] 10) did not inhibit the growth of the test organisms. The structure-activity correlation in this series of nickel(II) complexes was discussed based on their ligand-replacement abilities. 相似文献
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Stephanie Nilkens Mareike Koch‐Singenstreu Volker Niemann Friedrich Götz Thilo Stehle Gottfried Unden 《Molecular microbiology》2014,91(2):381-393
In Staphylococci maximal induction of nitrate reductase (narGHJI genes) requires anaerobic conditions, the presence of nitrate, and the NreABC regulatory system. Aerobic regulation is effected by the NreB/NreC two‐component system. The role of the nitrate receptor NreA in nitrate induction and its relation to aerobic regulation was analysed in Staphylococcus carnosus. Nitrate induction of a narG‐lip reporter gene required presence of NreB/NreC. When nreA was deleted, nitrate was no longer required for maximal induction, suggesting that NreA is a nitrate regulated inhibitor of NreB/NreC. In vitro, NreA and mutant NreA(Y95A) decreased NreB phosphorylation in part or completely, which was due to the inhibition of the autophosphorylating activity rather than an increase of phosphatase activity. Inhibition of phosphorylation was relieved completely when the nitrate‐bound NreA was used instead of the nitrate‐free form. In the bacterial two‐hybrid BACTH system and HPINE interaction assays, NreA interacted with NreB, but not with NreC, and the interaction was diminished by nitrate. In summary, NreA interacts with NreB and controls its phosphorylation level in a nitrate dependent manner. In this way nitrate and NreA modulate the function of the oxygen sensor NreB, resulting in nitrate/oxygen co‐sensing by an NreA/NreB sensor unit as part of the NreABC‐system. 相似文献
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Achromobacter xylosoxidans strain A8 was isolated from soil contaminated with polychlorinated biphenyls. It can use 2-chlorobenzoate and 2,5-dichlorobenzoate as sole sources of carbon and energy. This property makes it a good starting microorganism for further development toward a bioremediation tool. The genome of A. xylosoxidans consists of a 7-Mb chromosome and two large plasmids (98 kb and 248 kb). Besides genes for the utilization of xenobiotic organic substrates, it contains genes associated with pathogenesis, toxin production, and resistance. Here, we report the complete genome sequence. 相似文献
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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. 相似文献
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Inducible and constitutive expression of pMOL28-encoded nickel resistance in Alcaligenes eutrophus N9A. 总被引:10,自引:7,他引:3
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The nickel and cobalt resistance plasmid pMOL28 was transferred by conjugation from its natural host Alcaligenes eutrophus CH34 to the susceptible A. eutrophus N9A. Strain N9A and its pMOL28-containing transconjugant M220 were studied in detail. At a concentration of 3.0 mM NiCl2, the wild-type N9A did not grow, while M220 started to grow at its maximum exponential growth rate after a lag of 12 to 24 h. When grown in the presence of subinhibitory concentrations (0.5 mM) of nickel salt, M220 grew actively at 3 mM NiCl2 without a lag, indicating that nickel resistance is an inducible property. Expression of nickel resistance required active growth in the presence of nickel salts at a concentration higher than 0.05 mM. Two mutants of M220 were isolated which expressed nickel resistance constitutively. When the plasmids, pMOL28.1 and pMOL28.2, carried by the mutants were transferred to strains H16 and CH34, the transconjugants expressed constitutive nickel resistance. This indicates that the mutation is plasmid located. Both mutants expressed constitutive resistance to nickel and cobalt. Physiological studies revealed the following differences between strain N9A and its pMOL28.1-harboring mutant derivatives. (i) The uptake of 63NiCl2 occurred more rapidly in the susceptible strain and reached a 30- to 60-fold-higher amount that in the pMOL28.1-harboring mutant; (ii) in intact cells of the susceptible strain N9A, the cytoplasmic hydrogenase was inhibited by 1 to 5 nM NiCl2, whereas 10 mM Ni2+ was needed to inhibit the hydrogenase of mutant cells; (iii) the minimal concentration of nickel chloride for the derepressed synthesis of cytoplasmic hydrogenase was lower in strain N9A (1 to 3 microM) than in the constitutive mutant (8 to 10 microM). 相似文献
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