Nickel transporters play a critical role in the biosynthesis of nickel-dependent enzymes such as hydrogenases and ureases (12
). For the first time, we demonstrate the presence of two transporters specific for nickel in a prokaryote. The human and animal pathogen Y. pseudotuberculosis
possesses (i) an ABC transporter encoded by the yntABCDE
gene cluster, which shows some degree of similarity to the Nik system in several gram-negative bacteria (2
), and (ii) a single-component carrier encoded by the ureH
gene, displaying homology to polypeptides of the nickel-cobalt transporter family exemplified by HoxN (13
). Complete suppression of nickel transport in the ynt ureH
double mutant indicated that, apart from Ynt and UreH, there are no other Ni2+
-specific transport systems in Y. pseudotuberculosis
. H. pylori
also has two transport systems for nickel acquisition: the high-affinity nickel carrier protein NixA (22
) and an ABC transporter (17
). However, the latter has not been proven to be specific for nickel.
Database searches revealed that genes homologous to ureH and ynt are present in the genome of the other two pathogenic Yersinia species, Y. pestis (designated by “p”) and Y. enterocolitica (designated by “ent”). Deduced amino acid sequences from ureHp and ureHent are 99 and 93% identical, respectively, to that of the ureH product from Y. pseudotuberculosis, whereas putative Yntp and Yntent complexes share 99 and 91.6% of residues, respectively, with Ynt proteins from Y. pseudotuberculosis. Phylogenetic analysis of the putative periplasmic nickel-binding protein encoded by the yntA gene from these three pathogenic Yersinia species showed that YntA, YntAp, and YntAent cluster with the serovar Typhimurium nickel-binding-protein-related Oxd-6a polypeptide and also with the orf1 gene product from urease-producing E. coli strains. Hence, besides the Nik transport system group, the bacterial nickel-ABC transporter family includes another subclass, with Ynt as a prototype carrier and two other members produced by Salmonella and some E. coli isolates.
For several bacterial species in which nickel-transport systems were characterized, genes specifying these carriers were found within or in proximity to genetic loci encoding the nickel-requiring enzyme urease (1
) or hydrogenase (13
). In addition, nickel permeases of the ABC family are encoded by gene clusters which are harbored either on a plasmid (10
), on a pathogenicity island (26
), or adjacent to an insertion sequence (2
). In Y. pseudotuberculosis
flanks the yut
), located just downstream of the urease accessory gene ureD
, whereas the yntABCDE
polycistronic unit is upstream of the ureA
structural urease gene. The fact that the intergenic space (1,198 bp) between yntE
has a low G+C content (34 versus 47% for the whole Y. pseudotuberculosis
genome) and includes many repeated sequences is noteworthy. Furthermore, a copy of insertion sequence IS285
is present 1,848 bp upstream of the Y. pestis yntA
gene. Taken together, these genetic features suggest that the chromosomal region containing the ynt
operon has been the site of DNA recombination events and that the nickel ABC transport system could have been acquired by yersiniae through horizontal gene transfer.
Nickel uptake assays with Y. pseudotuberculosis ureH
mutants revealed that cellular entry of this divalent cation occurs principally via the Ynt ABC transporter. At the nickel concentration used for the assay, initial rates of nickel uptake by ynt
mutants reached approximately 15 and 60%, respectively, of the wild-type value (Fig. ), although the KT
values of UreH and Ynt are similar. This discrepancy could be due to better production of Ynt under our in vitro bacterial growth conditions. Surprisingly, urease activity was not found to correlate with nickel accumulation inside the cells, since it was shown to be strongly reduced (99%) after ynt
inactivation but did not significantly differ from that of wild-type Y. pseudotuberculosis
knockout, regardless of nickel or magnesium concentrations in the growth medium. This was not due to a polar effect of the ynt
mutation on the downstream ure
locus, since urease activity of the ynt
mutant was fully restored after trans
-complementation with the ynt
operon. These discrepancies between the mutants' ureolytic and nickel uptake capacities could be due to regulation by nickel concentration of the Y. pseudotuberculosis
urease gene cluster expression, as has been recently demonstrated for H. pylori
urease, which is induced by Ni2+
at the transcriptional level (35
). Mutation of ynt
would thus reduce the intracellular nickel concentration below the threshold necessary for induction.
Although weakly homologous (between 25 and 32% identity) to E. coli Nik permease, the Y. pseudotuberculosis Ynt complex is functionally interchangeable with this ABC transporter. However, E. coli cells incorporated much more nickel when expressing ynt instead of the nik gene cluster (Fig. ). In the same heterogenous genetic background, UreH is also functional and is as efficient as the endogenous Nik transport system (Fig. ). The differences in these systems' nickel transport capacities may reside in their expression in E. coli and could also be linked to their conformation in the cell membrane.
The production of redundant nickel-specific permeases by yersiniae emphasizes the importance of the penetration of this divalent cation into the cell in relation to the biosynthesis of urease—and possibly that of other nickel-dependent enzymes. It also poses the question of their physiological role and raises the possibility that the two systems are expressed under different growth conditions at various stages of the life cycle.