Nickel Resistance Determinant Research Articles

Response of CnrX from Cupriavidus metallidurans CH34 to nickel binding.

Resistance to high concentration of nickel ions is mediated in Cupriavidus metallidurans by the CnrCBA transenvelope efflux complex. Expression of the cnrCBA genes is regulated by the transmembrane signal transduction complex CnrYXH. Together, the metal sensor CnrX and the transmembrane antisigma factor CnrY control the availability of the extracytoplasmic function sigma factor CnrH. Release of CnrH from sequestration by CnrY at the cytoplasmic side of the membrane depends essentially on the binding of the agonist metal ion Ni(ii) to the periplasmic metal sensor domain of CnrX. CnrH availability leads to transcription initiation at the promoters cnrYp and cnrCp and to the expression of the genes in the cnrYXHCBA nickel resistance determinant. The first steps of signal propagation by CnrX rely on subtle metal-dependent allosteric modifications. To study the nickel-mediated triggering process by CnrX, we have altered selected residues, F66, M123, and Y135, and explored the physiological consequences of these changes with respect to metal resistance, expression of a cnrCBA-lacZ reporter fusion and protein production. M123C- and Y135F-CnrXs have been further characterized in vitro by metal affinity measurements and crystallographic structure analysis. Atomic-resolution structures of metal-bound M123C- and Y135F-CnrXs showed that Ni(ii) binds two of the three canonical conformations identified and that Ni(ii) sensing likely proceeds by conformation selection.

Nickel-resistance determinants in Acidiphilium sp. PM identified by genome-wide functional screening.

Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY). This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms.

Identification of the Transcriptional Regulator NcrB in the Nickel Resistance Determinant of Leptospirillum ferriphilum UBK03

The nickel resistance determinant ncrABCY was identified in Leptospirillum ferriphilum UBK03. Within this operon, ncrA and ncrC encode two membrane proteins that form an efflux system, and ncrB encodes NcrB, which belongs to an uncharacterized family (DUF156) of proteins. How this determinant is regulated remains unknown. Our data indicate that expression of the nickel resistance determinant is induced by nickel. The promoter of ncrA, designated pncrA, was cloned into the promoter probe vector pPR9TT, and co-transformed with either a wild-type or mutant nickel resistance determinant. The results revealed that ncrB encoded a transcriptional regulator that could regulate the expression of ncrA, ncrB, and ncrC. A GC-rich inverted repeat sequence was identified in the promoter pncrA. Electrophoretic mobility shift assays (EMSAs) and footprinting assays showed that purified NcrB could specifically bind to the inverted repeat sequence of pncrA in vitro; this was confirmed by bacterial one-hybrid analysis. Moreover, this binding was inhibited in the presence of nickel ions. Thus, we classified NcrB as a transcriptional regulator that recognizes the inverted repeat sequence binding motif to regulate the expression of the key nickel resistance gene, ncrA.

Enterobacter nickelidurans sp. nov., a novel nickel tolerant enterobacteria isolated from Torsa river water of India

A Gram-negative, rod-shaped, nickel-resistant bacterium, designated as strain NiVas 114', was isolated from waters of Torsa River in Hasimara, West Bengal, India. The strain NiVasl 14 possessing inducible nickel resistance can tolerate maximally 10mM nickel chloride. Southern blot assays of genomic DNA of NiVas 114 using probe(s) generated from known nickel resistance determinants (cnr/ ncc/ nrel nerí nir). under conditions of low stringency, produced no detectable signal except for cnrA gene of Ralstonia metallidurans CH34 (formerly Alcaligenes eutropha CH34) in which weak hybridization signal occurred. Based on 16S rRNA gene sequence similarity, strain NiVas 114 was identified as a member of y Proteobacteria, and the nearest phylogenetic relatives are Enterobacter hormaechei (98.6-98.5%) and Enterobacter cloacae (97.5-98.2 %). In the phylogenetic trees constructed with nucleotide sequence of 16S rRNA gene, hsp60 gene and rpoB gene, strain NiVas 114" clustered with the subspecies of Enterobacter hormaechei. The strain showed DNA-DNA relatedness of 60 -63% with Enterobacter hormaechei subsp hormaechei, Enterobacter hormaechei subsp. oharae, Enterobacter hormaechei subsp. steigerwalti Enterobacter cloacae subsp. cloacae and Enterobacter cloacae subsp. dissobvens. The isolate NiVas 114 differed from the nearest phylogenetic relatives in terms of number of phenotypic characteristics. The G+C content of the genomic DNA of the isolate was 59.5 +0.4 mol %. The predominant cellular faty acids of the isolate are C160, Summed feature 3 (comprises C16wTel 15 io 201) and C 18 w7ei hydroxy fatty acids are found in minor quantities. Thus, on the basis of biochemical characteristics, fatty acid profiles, DNA-DNA relatedness and phylogenetic analysis, the isolate was recognized as a novel species of Enterobacter. for which the name Enterobacter nickellidurans sp. nov. is proposed with the type strain NiVas 114' (= LMG 23000 = CcUGSO594"= JCM13045").

Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage.

Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.

Isolation of a novel plasmid, pNi15, fromEnterobactersp. Ni15 containing a nickel resistance gene

We isolated nickel-resistant bacterium from soil in order to identify a novel nickel resistance determinant. Using 16S rRNA gene sequencing, an isolate was identified as Enterobacter sp. Ni15. This species showed a medium-level (resistant to up to 10 mM) nickel resistance in nutrient-rich media. Enterobacter sp. Ni15 has a novel plasmid, pNi15, and an increased nickel resistance to Escherichia coli DH5alphain trans. To isolate the nickel resistance gene from pNi15, the plasmid was digested with XbaI and its fragments were cloned into pBluescriptIISK(+). The clones were transferred into E. coli DH5alpha. The nickel resistance of the clones was then assayed. From these results, a pNi15100 isolate containing a 5,328 bp XbaI fragment of pNi15 was identified and sequenced. The E. coli DH5alpha harboring the pNi15100 showed a resistance to up to 7 mM nickel. Using a subcloning analysis, we were able to identify the novel nickel resistance determinant: the nrp gene encoding the putative proteins NrpA and NrpB.

Inducible nickel resistance in a river isolate of India phylogenetically ascertained as a novel strain of Acinetobacter junii

A gram negative, motile, short rod-shaped, and nickel resistant (tolerating 6.5 mM Ni2+) bacterium, strain BB1A, was isolated from the waters of the River Torsa in Hashimara, Jalpaiguri district, West Bengal, India. The isolate BB1A was identified as a strain of Acinetobacter junii following detailed analysis of morphological, physio-biochemical and 16S rRNA gene sequence. The expression of nickel resistance in BB1A was inducible by exposure to nickel chloride at a concentration as low as 50 μM Ni2+. The other metal ions, Cu2+, Zn2+, or Pb2+ at a concentration range of 20–30 μM, also induced the nickel resistance system in this bacterium. Southern hybridizations of BB1A genomic DNA with digoxigenin-dUTP labeled DNA probes specific for well known nickel resistance determinants, cnr, ncc or nre, resulted in no detectable signal, but nir specific probe yielded weak hybridization signal with restricted genomic DNA of BB1A. The isolate BB1A, therefore, carries out a novel induction phenomenon of nickel resistance and presumably with a nickel resistance genetic system different from that previously characterized in other bacteria.

Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5.

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+). The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+). A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+). By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A.

Regulation of the cnr cobalt and nickel resistance determinant from Ralstonia sp. strain CH34.

Ralstonia sp. strain CH34 is resistant to nickel and cobalt cations. Resistance is mediated by the cnr determinant located on plasmid pMOL28. The cnr genes are organized in two clusters, cnrYXH and cnrCBA. As revealed by reverse transcriptase PCR and primer extension, transcription from these operons is initiated from promoters located upstream of the cnrY and cnrC genes. These two promoters exhibit conserved sequences at the -10 (CCGTATA) and -35 (CRAGGGGRAG) regions. The CnrH gene product, which is required for expression of both operons, is a sigma factor belonging to the sigma L family, whose activity seems to be governed by the membrane-bound CnrY and CnrX gene products in response to Ni(2+). Half-maximal activation from the cnrCBA operon was determined by using appropriate lacZ gene fusions and was shown to occur at an Ni(2+) concentration of about 50 microM.

Horizontal transfer of bacterial heavy metal resistance genes and its applications in activated sludge systems

The bacterial nickel (Ni) resistance determinant ncc-nre of Alcaligenes 31A strain cloned to an IncQ broad-host-range plasmid pKT240 gave rise to pMOL222. The plasmid was subsequently mobilized into various Eubacteria and found to confer an increased Ni resistance on these recipients. An increase of Ni resistance was also observed after the transfer of pMOL222 into activated sludge bacteria by plate mating. The dissemination of pMOL222 into an activated sludge pilot stabilized the system during a heavy metal shock loading with 0.25 mM Ni.

Horizontal transfer of bacterial heavy metal resistance genes and its applications in activated sludge systems

The bacterial nickel (Ni) resistance determinant ncc-nre of Alcaligenes 31A strain cloned to an IncQ broadhost-range plasmid pKT240 gave rise to pMOL222. The plasmid was subsequently mobilized into various Eubacteria and found to confer an increased Ni resistance on these recipients. An increase of Ni resistance was also observed after the transfer of pMOL222 into activated sludge bacteria by plate mating. The dissemination of pMOL222 into an activated sludge pilot stabilized the system during a heavy metal shock loading with 0.25 mM Ni.

A Novel Nickel Resistance Determinant Found in Sewage-Associated Bacteria

Nickel-resistant bacteria were isolated from effluent discharged from a sewage treatment outfall over an 18-month period. One of these strains, Enterobacter cloacae FBA30, was found to harbor a narrow host range conjugative plasmid, designated pFBA30, which confers nickel resistance on its host. A 10.2-kb SstI restriction fragment was cloned from pFBA30 and was shown to specify inducible nickel resistance, both in its original host and in laboratory strains of Escherichia coli. This DNA fragment, and a 1.75-kb SmaI fragment derived from it, were used as probes to examine other bacterial strains isolated during the study. Homologous nickel resistance genes were detected in enteric bacteria emerging directly from the treatment plant, but not in strains isolated from sediments downstream of the outfall, or in strains harboring well-characterized determinants such as ncc/nre, cnr, or Klebsiella oxytoca type. Thus, this element constitutes a new nickel resistance determinant and was named nrf. Nickel-resistant strains were sorted into two groups, based on the sampling date and the size of restriction fragments homologous to probes developed from pFBA30. Group A isolates all carried a homologous 6.5-kb PvuII restriction fragment and expressed nickel resistance constitutively. Group B strains, isolated independently from the same outfall, were different from group A in that the nickel resistance was associated with a 4.7-kb PvuII fragment, and group B strains displayed a slower growth rate on nickel salts media. Each group contained a range of enteric bacterial species, including K. oxytoca, Citrobacter freundii, and Enterobacter spp. Localized genetic exchange probably occurs within wastewater treatment effluents, and, once effluents are discharged into the environment, enteric bacteria carrying pFBA30-like determinants do not persist downstream.

Nickel-resistant bacteria from anthropogenically nickel-polluted and naturally nickel-percolated ecosystems

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.

The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer, expression, regulation and DNA homologies to various nickel-resistant bacteria

Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mM NiCl2 in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mM NiCl2. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mM Ni2+) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5-5) isolated from nickel-rich soil in New Caledonia.