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

Abstract

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.

FIG. 1.

Alignment of the amino acid sequence of the UreH protein from Y. pseudotuberculosis 32777 with those of nickel transporters from R. eutropha (HoxN), B. japonicum (HupN), M. tuberculosis (NicT), and H. pylori (NixA). Asterisks, colons, and periods indicate identical, similar, and related amino acids, respectively. Dashes correspond to gaps introduced to optimize homology between sequences. Motifs critical for biological activity are overlined.

FIG. 2.

Genetic organization of the immediate environment of the chromosomal urease (ure) locus and genotype analysis of ynt- and ureH-deficient mutants of Y. pseudotuberculosis 32777. (A) KpnI (K) and EcoRI (E) restriction map of the chromosome of wild-type strain 32777 and isogenic mutants MYUH (ureH) and MYNT (ynt). (B) (Left) Southern blot of KpnI-digested DNA from wild-type strain 32777 and the yntABCDE-deficient mutant hybridized with probe 1 (0.5 kb, corresponding to the downstream region of yntE) and probe 2 (1.4 kb, detecting the chloramphenicol resistance gene cat). (Right) Southern blot of EcoRI-digested DNA from wild-type strain 32777 and the ureH-deficient mutant hybridized with probe 3 (0.6 kb, corresponding to the upstream region of ureH) and probe 4 (1.3 kb, detecting the kanamycin resistance gene aphA-1a). Primers N1 to N12, K1, K2, C1, and C2 were used both for creating mutants and for checking their genotype. Numbers in parentheses indicate the DNA sequence coordinates.

FIG. 3.

Ureolytic activity of wild-type strain 32777 (gray bars) and the MYUH ureH mutant (black bars) following growth in LB broth containing increasing concentrations of the nickel chelator nitrilotriacetic acid (NTA) (A) or NiCl₂ (B). Each bar is the mean value of three independent experiments ± the standard deviation.

FIG. 4.

Roles of UreH and Ynt in nickel entry into bacteria. Bacterial suspensions were incubated in the presence of 150 nM ⁶³NiCl₂ and 10 mM MgCl₂. Nickel uptake was assessed at regular intervals. Three separate experiments gave similar results. Representative data are shown. (A) Nickel uptake into nikA-deficient E. coli strain trans-complemented with ureH (pFS45) or yntABCDE (pFS78) genes from Y. pseudotuberculosis and nikABCDE genes (pLW21) from E. coli. (B) Nickel uptake into Y. pseudotuberculosis ureH, yntABCDE, and ureH yntABCDE mutants derived from the wild-type strain.