Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

HoxN, an integral membrane protein with seven transmembrane helices and a molecular mass of 33.1 kDa, is involved in high-affinity nickel transport in Alcaligenes eutrophus H16. From genetic analyses, it has been concluded that HoxN is a single-component ion carrier. To investigate this assumption, hoxN was introduced into Escherichia coli. The recombinant strain showed significantly enhanced nickel uptake in a short-interval assay. Likewise, growth in the presence of 63 NiCl 2 yielded a more than 15-fold-increased cellular nickel content. The HoxN-based nickel transport activity could also be demonstrated in a physiological assay: an E. coli strain coexpressing hoxN and the urease operon of Klebsiella aerogenes exhibited urease activity 10-fold greater than that in the strain lacking a functional hoxN. These results strongly suggest that HoxN is sufficient to operate as a nickel permease. Multiple sequence alignment of HoxN and four other bacterial membrane proteins implicated in nickel metabolism revealed two conserved signatures which may play a role in the nickel translocation process.Nickel is an essential trace element for at least four biological processes: (i) oxidation and evolution of molecular hydrogen, (ii) hydrolysis of urea, (iii) carbon monoxide dehydrogenase-mediated acetate metabolism in methanogens and in homoacetogenic bacteria, and (iv) reduction of methyl coenzyme M to methane in methanogenic archaea (6). Uptake of nickel ions is a prerequisite for those organisms catalyzing nickel-dependent reactions. The microbial transport of the divalent Ni 2ϩ cation, the most commonly occurring oxidation state of nickel, has been investigated with regard to physiology, ion specificity, and very recently, the molecular structure of the transport proteins (3, 18). Nickel uptake is mediated by nonspecific Mg 2ϩ transport systems and by high-affinity systems specific for the transport of nickel (for a review, see reference 7).In Alcaligenes eutrophus, a gram-negative aquatic and soil bacterium which can utilize molecular hydrogen as an energy source, nickel uptake occurs by Mg 2ϩ transporters and by a high-affinity nickel transporter, the product of gene hoxN (2, 11). The phenotype of a HoxN-negative mutant manifested itself in the inability to grow on hydrogen as the energy source under nickel limitation (below 100 nM) in the presence of 0.8 mM Mg 2ϩ . As expected for a mutant lacking the specific nickel transporter, this nickel deficiency was physiologically compensated for by either increasing the Ni 2ϩ concentration to 1 M or decreasing the Mg 2ϩ concentration (1, 2). The nucleotide sequence of hoxN has been determined, and the two-dimensional membrane topology of the HoxN nickel transport protein has been investigated. While computer analyses of potential membrane-spanning segments gave ambiguous results, the construction of a set of fusions to alkaline phosphatase and ␤-galactosidase clearly allowed the identification of segments located in the periplasm and in the cytoplasm, respectively. Using these...

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. NixA was identified as a specific nickel uptake system in this organism. Addition of small amounts of nickel to media strongly stimulates urea hydrolysis. On the other hand, high nickel concentrations are deleterious to cell growth. As a possible protective reaction, nickel uptake seems to be reduced in H. pylori grown in nickel-rich media. These observations led to investigations of regulation of the expression of the nickel permease NixA. We found that increasing the nickel concentration in media reduced the amount of NixA. In order to address the question of whether this phenomenon was subject to transcriptional or translational regulation, we quantified nixA mRNA from H. pylori by real-time PCR. The amount of nixA mRNA was gradually reduced five-to sevenfold in a time-and concentration-dependent manner. Repression could be measured as soon as 5 min after nickel addition, and the maximum repression occurred after 20 to 30 min. The maximum repression was obtained with an external nickel concentration of 100 M. The observed nickel repression of NixA was dependent on nikR encoding the nickel-responsive regulatory protein NikR. In conclusion, we demonstrated that synthesis of the NixA nickel permease of H. pylori shows nickel-responsive regulation mediated by NikR to maintain the balance between effective nickel acquisition and a toxic overload.

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Amino acid exchanges in the Alcaligenes eutrophus nickel permease (HoxN) were constructed by site-directed mutagenesis, and their effects on nickel ion uptake were investigated. Mutant hoxN alleles were expressed in Escherichia coli, and activity of the altered permeases was examined via a recently described physiological assay (Wolfram, L., Friedrich, B., and Eitinger, T. (1995) J. Bacteriol. 177, 1840 -1843). Replacement of Cys-37, Cys-256, or Cys-318 by alanine did not severely affect nickel ion uptake. This activity of a C331A mutant was diminished by 60%, and a similar phenotype was obtained with a cysteine-less mutant harboring four Cys to Ala exchanges. Alterations in a histidine-containing sequence motif (His-62, Asp-67, His-68), which is conserved in microbial nickel transport proteins, strongly affected or completely abolished transport activity in the E. coli system. The analysis of HoxN alkaline phosphatase fusion proteins implied that His-62, Asp-67, and His-68 exchanges did not interfere with overall membrane topology or stability of the nickel permease. These mutations were reintroduced into the A. eutrophus wildtype strain. Analyses of the resulting HoxN mutants indicated that exchanges in the histidine motif led to a clearly decreased affinity of the permease for nickel ion.Alcaligenes eutrophus H16, a member of the ␤ division of the proteobacteria, can utilize various organic compounds or molecular hydrogen as energy sources. Hydrogen oxidation is catalyzed by two metalloenzymes: a cytoplasmic NAD ϩ -reducing hydrogenase and a membrane-bound, electron transport-coupled hydrogenase. Both enzymes belong to the family of [NiFe] hydrogenases (reviewed in Ref. 1). A nickel-dependent urease allows the organism to grow on urea as a nitrogen source.Uptake of nickel ion in sufficient amounts is a prerequisite for the synthesis of nickel-containing enzymes. Microbial nickel uptake is mediated by nonspecific transport systems for divalent cations and by specific systems with a high affinity for Ni 2ϩ . In natural environments the concentration of Ni 2ϩ is generally very low compared with other divalent cations like Mg 2ϩ . Since nonspecific nickel transport is competitively inhibited by a number of divalent cations, this mode of uptake is not suited for meeting the cellular nickel requirements (reviewed in Refs. 2 and 3). Molecular analyses of Ni 2ϩ -specific transport systems of a few bacteria containing [NiFe] hydrogenases and/or ureases showed that two different types of membrane transporters are responsible for high affinity uptake of the transition metal (reviewed in Ref. 3). The best studied Ni 2ϩ