Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus.

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.     

Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.     

Contribution of dppA to urease activity in Helicobacter pylori 26695

BACKGROUND: The gastric pathogen Helicobacter pylori produces urease in amounts up to 10% of its cell protein. This enzyme, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide, protects the bacterium from gastric acid. Urease, a nickel metalloenzyme, requires active uptake of nickel ions from the environment to maintain its activity. NixA is a nickel transport protein that resides in the cytoplasmic membrane. Mutation of nixA significantly reduces but does not abolish urease activity, strongly suggesting the presence of a second transporter. We postulated that the dipeptide permease (dpp) genes that are homologous to the nik operon of Escherichia coli could be a second nickel transporter. The predicted Dpp polypeptides DppA, DppC, and DppD of H. pylori share approximately 40%, 53%, and 56% amino acid sequence identity with their respective E. coli homologs. METHODS: A mutation in dppA, constructed by insertional inactivation with a chloramphenicol resistance cassette, was introduced by allelic exchange into H. pylori strain 26695. RESULTS: When compared to the parental strain, urease activity was not decreased in a dppA mutant. CONCLUSIONS: DppA does not contribute to the synthesis of catalytically active urease in H. pylori 26695 and is likely not a nickel importer in H. pylori.     

Activities of Urease and Nickel Uptake of Helicobacter pylori Proteins are Media- and Host-Dependent

Background:  Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity.
Aim:  To compare nickel uptake and urease activity on an inter- and intraspecies level.
Results:  In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli . On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media.
Conclusion:  H. pylori took nickel much more efficiently up than E. coli . The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.     

Substrate specificity of nickel/cobalt permeases: insights from mutants altered in transmembrane domains I and II

HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. Two histidine residues in a Ni2+ binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity. Replacement of the corresponding His residues in NhlF affected both Co2+ and Ni2+ uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations. Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF). Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important. The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+ uptake significantly. Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+ and Co2+ with low capacity. The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+ uptake capacity and conferred to the variant the ability to transport Co2+. NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.     

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.     

Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity.

Helicobacter pylori urease requires nickel ions in the enzyme active site for catalytic activity. Nickel ions must, therefore, be actively acquired by the bacterium. NixA (high-affinity nickel transport protein)-deficient mutants of H. pylori retain significant urease activity, suggesting the presence of alternate nickel transporters. Analysis of the nucleotide sequence of the H. pylori genome revealed a homolog of NikD, a component of an ATP-dependent nickel transport system in Escherichia coli. Based on this sequence, a 378-bp DNA fragment was PCR amplified from H. pylori genomic DNA and used as a probe to identify an H. pylori lambda ZAPII genomic library clone that carried these sequences. Four open reading frames of 621, 273, 984, and 642 bp (abcABCD) were revealed by sequencing and predicted polypeptides of 22.7, 9.9, 36.6, and 22.8 kDa, respectively. The 36.6-kDa polypeptide (AbcC) has significant homology (56% amino acid sequence identity) to an E. coli ATP-binding protein component of an ABC transport system, while none of the other putative proteins are significantly homologous to polypeptides in the available databases. To determine the possible contribution of these genes to urease activity, abcC and abcD were each insertionally inactivated with a kanamycin resistance (aphA) cassette and allelic exchange mutants of each gene were constructed in H. pylori UMAB41. Mutation of abcD resulted in an 88% decrease in urease activity to 27 +/- 31 mumol of NH3/min/mg of protein (P < 0.0001), and a double mutant of nixA and abcC resulted in the near abolishment of urease activity (1.1 +/- 1.4 mumol of NH3/min/mg of protein in the double mutant versus 228 +/- 92 mumol of NH3/min/mg of protein in the parent [P < 0.0001]). Synthesis of urease apoenzyme, however, was unaffected by mutations in any of the abc genes. We conclude that the abc gene cluster, in addition to nixA, is involved in production of a catalytically active urease.     

Heterologous production and characterization of bacterial nickel/cobalt permeases

Nickel/cobalt permeases (NiCoTs, TC 2.A.52) are a rapidly growing family of structurally related membrane transporters whose members are found in Gram-negative and Gram-positive bacteria, in thermoacidophilic archaea, and in fungi. Previous studies have predicted two subclasses represented by HoxN of Ralstonia eutropha, a selective nickel transporter, and by NhlF of Rhodococcus rhodochrous, a nickel and cobalt transporter that displays a preference for the Co ion. In the present study, NiCoT genes of five Gram-negative bacteria and one Gram-positive bacterium were cloned and heterologously expressed in Escherichia coli. Based on substrate preference in metal-accumulation assays with the recombinant strains, two of the novel NiCoTs were assigned to the NhlF class. The remaining four NiCoTs belong to a yet unrecognized, third class. They transport both the nickel and the cobalt ion but have a significantly higher capacity for nickel. The observed substrate preferences correlate in many cases with the genomic localization of NiCoT genes adjacent to regions encoding nickel- or cobalt-dependent enzymes or enzymes involved in cobalamin biosynthesis. Alignment of 23 full-length NiCoT sequences and comparison with the available experimental data predict that substrate specificity of NiCoTs is an adaptation to specific transition metal requirements in various organisms from different taxa.     

Genes encoding specific nickel transport systems flank the chromosomal urease locus of pathogenic yersiniae

The transition metal nickel is an essential cofactor for a number of bacterial enzymes, one of which is urease. Prior to its incorporation into metalloenzyme active sites, nickel must be imported into the cell. Here, we report identification of two loci corresponding to nickel-specific transport systems in the gram-negative, ureolytic bacterium Yersinia pseudotuberculosis. The loci are located on each side of the chromosomal urease gene cluster ureABCEFGD and have the same orientation as the latter. The yntABCDE locus upstream of the ure genes encodes five predicted products with sequence homology to ATP-binding cassette nickel permeases present in several gram-negative bacteria. The ureH gene, located downstream of ure, encodes a single-component carrier which displays homology to polypeptides of the nickel-cobalt transporter family. Transporters with homology to these two classes are also present (again in proximity to the urease locus) in the other two pathogenic yersiniae, Y. pestis and Y. enterocolitica. An Escherichia coli nikA insertion mutant recovered nickel uptake ability following heterologous complementation with either the ynt or the ureH plasmid-borne gene of Y. pseudotuberculosis, demonstrating that each carrier is necessary and sufficient for nickel transport. Deletion of ynt in Y. pseudotuberculosis almost completely abolished bacterial urease activity, whereas deletion of ureH had no effect. Nevertheless, rates of nickel transport were significantly altered in both ynt and ureH mutants. Furthermore, the ynt ureH double mutant was totally devoid of nickel uptake ability, thus indicating that Ynt and UreH constitute the only routes for nickel entry. Both Ynt and UreH show selectivity for Ni(2+) ions. This is the first reported identification of genes coding for both kinds of nickel-specific permeases situated adjacent to the urease gene cluster in the genome of a microorganism.     

Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.     

A topological model for the high-affinity nickel transporter of Alcaligenes eutrophus

The gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy-dependent uptake of nickel ions. Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis. To distinguish between these possibilities varying segments of the amino-terminal end of the transporter were fused to the Escherichia coli enzymes aikaline phosphatase (PhoA) or β-galactosidase (LacZ). The enzymatic activity of 16 HoxN-PhoA and 15 HoxN-LacZ fusions was determined. On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two-dimensional model for the nickel transporter was developed. This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices. The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.     

Ynt is the primary nickel import system used by Proteus mirabilis and specifically contributes to fitness by supplying nickel for urease activity

Proteus mirabilis is a Gram-negative uropathogen and frequent cause of catheter-associated urinary tract infection (CAUTI). One important virulence factor is its urease enzyme, which requires nickel to be catalytically active. It is, therefore, hypothesized that nickel import is critical for P. mirabilis urease activity and pathogenesis during infection. P. mirabilis strain HI4320 encodes two putative nickel import systems, designated Nik and Ynt. By disrupting the substrate-binding proteins from each import system (nikA and yntA), we show that Ynt is the primary nickel importer, while Nik only compensates for loss of Ynt at high nickel concentrations. We further demonstrate that these are the only binding proteins capable of importing nickel for incorporation into the urease enzyme. Loss of either nickel-binding protein results in a significant fitness defect in a murine model of CAUTI, but YntA is more crucial as the yntA mutant was significantly outcompeted by the nikA mutant. Furthermore, despite the importance of nickel transport for hydrogenase activity, the sole contribution of yntA and nikA to virulence is due to their role in urease activity, as neither mutant exhibited a fitness defect when disrupted in a urease-negative background.     

Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery

Nickel is a cofactor for various microbial enzymes, yet as a trace element, its scavenging is challenging. In the case of the pathogen Helicobacter pylori, nickel is essential for the survival in the human stomach, because it is the cofactor of the important virulence factor urease. While nickel transport across the cytoplasmic membrane is accomplished by the nickel permease NixA, the mechanism by which nickel traverses the outer membrane (OM) of this Gram-negative bacterium is unknown. Import of iron-siderophores and cobalamin through the bacterial OM is carried out by specific receptors energized by the TonB/ExbB/ExbD machinery. In this study, we show for the first time that H. pylori utilizes TonB/ExbB/ExbD for nickel uptake in addition to iron acquisition. We have identified the nickel-regulated protein FrpB4, homologous to TonB-dependent proteins, as an OM receptor involved in nickel uptake. We demonstrate that ExbB/ExbD/TonB and FrpB4 deficient bacteria are unable to efficiently scavenge nickel at low pH. This condition mimics those encountered by H. pylori during stomach colonization, under which nickel supply and full urease activity are essential to combat acidity. We anticipate that this nickel scavenging system is not restricted to H. pylori, but will be represented more largely among Gram-negative bacteria.     

Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein

The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Molecular cloning of gltS and gltP, which encode glutamate carriers of Escherichia coli B.

Two genes encoding distinct glutamate carrier proteins of Escherichia coli B were cloned into an E. coli K-12 strain by using a cosmid vector, pHC79. One of them was the gltS gene coding for a glutamate carrier of an Na+-dependent, binding protein-independent, and glutamate-specific transport system. The content of the glutamate carrier was amplified about 25-fold in the cytoplasmic membranes from a gltS-amplified strain. The gltS gene was located in a 3.2-kilobase EcoRI-MluI fragment, and the gene product was identified as a membrane protein with an apparent Mr of 35,000 in a minicell system. A gene designated gltP was also cloned. The transport activity of the gltP system in cytoplasmic membrane vesicles from a gltP-amplified strain was driven by respiratory substrates and was independent of the concentrations of Na+, K+, and Li+. An uncoupler, carbonylcyanide m-chlorophenylhydrazone, completely inhibited the transport activities of both systems, whereas an ionophore, monensin, inhibited only that of the gltS system. The Kt value for glutamate was 11 microM in the gltP system and 3.5 microM in the gltS system. L-Aspartate inhibited the glutamate transport of the gltP system but not that of the gltS system. Aspartate was taken up actively by membrane vesicles from the gltP-amplified strain, although no aspartate uptake activity was detected in membrane vesicles from a wild-type E. coli strain. These results suggest that gltP is a structural gene for a carrier protein of an Na+-independent, binding protein-independent glutamate-aspartate transport system.     

Regulation of urease activity in Rhizobium meliloti

Abstract Using an ureC-lacZ fusion, the expression of urease structural genes of the soil bacterium Rhizobium meliloti strain AK631 was studied in response to different nitrogen sources and nickel contents in the growth medium. The expression of urease genes is repressed by ammonia and is not inducible by urea. Urease activity depends on the nickel concentration of the medium. Nickel uptake is repressed in medium containing ammonia and is not affected by the genes located in the urease operon investigated.     

Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl₂ excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.