An Intact Urease Assembly Pathway Is Required To Compete with NikR for Nickel Ions in Helicobacter pylori

Abstract

We examined the effects of urease and hydrogenase assembly gene deletions on NikR activation in H. pylori strains 26695 and G27. The loss of any component of urease assembly increased NikR activity under Ni 2؉ -limiting conditions, as measured by reduced transcript levels and 63 Ni accumulation. Additionally, SlyD functioned in urease assembly in strain 26695.A diverse complement of proteins is dedicated to the acquisition, trafficking, and regulation of intracellular transition metal ions. The mechanisms by which these activities are integrated to allocate the appropriate proportion of metal to different metal-binding proteins are not yet understood. Additionally, studies of the equilibrium metal-binding properties of transcriptional regulatory proteins important for metal homeostasis have revealed that they avidly bind their cognate metal ions (10 8,9,22,33,42]). These observations suggest that competition may exist between metalloenzyme assembly and metalloregulation. Detailed investigations of this hypothesis are encumbered by the presence of numerous essential metalloenzymes for metals such as zinc and iron. Microbial nickel physiology provides an ideal system for studying intracellular metal competition due to the small number of enzymes that require nickel ions (30) and their nonessentiality under laboratory growth conditions.We have studied the effect of disrupting Ni 2ϩ -dependent enzyme assembly pathways on nickel-dependent gene regulation in the gram-negative gastric pathogen Helicobacter pylori (3). The two Ni 2ϩ -dependent enzymes of H. pylori, urease and hydrogenase, are required for efficient colonization of animal models of infection (15,16,31). Both enzymes require conserved, GTP-dependent pathways for metal cofactor assembly that include an absolute requirement for nickel insertion chaperones under metal-limiting conditions (30). Hausinger and coworkers identified UreE as the Ni 2ϩ -binding protein required for urease assembly in Klebsiella aerogenes (10,38 before NikR, and the subsequent repression of nickel uptake genes. Such competition, if present, would be manifested as a change in NikR activity independently of a change in total nickel levels. In the absence of competition, NikR activity would correlate with a fixed total nickel concentration, independent of Ni 2ϩ -dependent enzyme expression or biosynthesis. Competition between metalloenzymes and metalloregulatory proteins has not been tested. Demonstration of the nature of such competition would facilitate subsequent studies to understand the molecular basis of metal ion partitioning within cells.We examined the effects of Ni 2ϩ -dependent enzyme assembly pathway gene deletions on NikR activity using several assays. In each case, cells were grown under identical conditions and manipulated in the same way for the same length of time. Cells were grown for 20 h (26695) or 24 h (G27) to an optical density at 600 nm of 1.0 in brucella broth (BD Difco) with 5% fetal bovine serum (Sigma) and then exposed to either 100 M dimethylglyoxime (DMG),...

Genetic Identification of a High-Affinity Ni Transporter and the Transcriptional Response to Ni Deprivation in Synechococcus sp. Strain WH8102

“…The reduction in Ni uptake rates for the sodN::sodB lines relative to those for the wild type supports this model; by removing Ni-SOD, only urease is removing Ni from SodT, thus resulting in lower Ni uptake rates. The reduction of competition between Ni metalloenzymes has been shown to influence the induction of Ni uptake in Helicobacter pylori (5). Similar allosteric regulation of transporter activity has been observed for transporters of methionine (23) and molybdate (21), though not for Ni.…”

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

“…6c). Although mutation of slyD in H. pylori does not result in decreased nickel uptake (Benanti & Chivers, 2009), the protein has recently been shown to bind nickel and to recognize the TAT signal peptide of HydA (Cheng et al, 2012). The physiological role of SlyD and the pathway of nickel into the hydrogenase in C. jejuni await further investigation.…”

Nickel Binding and [NiFe]-Hydrogenase Maturation by the Metallochaperone SlyD with a Single Metal-Binding Site in Escherichia coli

“…11 Whether this also holds true for SlyD from Helicobacter pylori remains to be verified as SlyD is considered to contribute to both [NiFe]-hydrogenase and urease maturation in this organism. 24,30,31 It becomes clear that SlyD has a multifunctional role in the hydrogenase biosynthetic pathway of E. coli. 9,32 Some organisms produce hydrogenase enzymes in the absence of slyD, suggesting that either SlyD is not required under all growth conditions or there are alternative means to achieve the activities of this protein, perhaps through variants of HypB that can bind multiple nickel ions.…”