Characterization of NikR-responsive promoters of urease and metal transport genes of Helicobacter mustelae

Abstract

The NikR protein is a nickel-responsive regulator, which in the gastric pathogen Helicobacter pylori controls expression of nickel-transporters and the nickel-cofactored urease acid resistance determinant. Although NikR-DNA interaction has been well studied, the Helicobacter NikR operator site remains poorly defined. In this study we have identified the NikR operators in the promoters of two inversely nickel-regulated urease operons (ureAB and ureA2B2) in the ferret pathogen Helicobacter mustelae, and have used bioinformatic approaches for the prediction of putative NikR operators in the genomes of four urease-positive Helicobacter species. Helicobacter mustelae NikR bound to the ureA2 promoter to a sequence overlapping with the -35 promoter region, leading to repression. In contrast, NikR binding to a site far upstream of the canonical sigma(80) promoter in the H. mustelae ureA promoter resulted in transcriptional induction, similar to the situation in H. pylori. Using H. pylori NikR operators and the newly identified H. mustelae NikR operators a new consensus sequence was generated (TRWYA-N(15)-TRWYA), which was used to screen the genomes of four urease-positive Helicobacter species (H. mustelae, H. pylori, H. acinonychis and H. hepaticus) for putative NikR-regulated promoters. One of these novel putative NikR-regulated promoters in H. mustelae is located upstream of a putative TonB-dependent outer membrane protein designated NikH, which displayed nickel-responsive expression. Insertional inactivation of the nikH gene in H. mustelae resulted in a significant decrease in urease activity, and this phenotype was complemented by nickel-supplementation of the growth medium, suggesting a function for NikH in nickel transport across the outer membrane. In conclusion, the H. mustelae NikR regulator directly controls nickel-responsive regulation of ureases and metal transporters. The improved consensus NikR operator sequence allows the prediction of additional NikR targets in Helicobacter genomes, as demonstrated by the identification of a new nickel-repressed outer membrane protein in H. mustelae.

An ABC Transporter and a TonB Ortholog Contribute to Helicobacter mustelae Nickel and Cobalt Acquisition

“…As for ferrous iron, the transport of nickel was until recently thought not to require an outer membrane component but only an ABC transporter or single-component NiCoT transporter (18,19). However, it has become apparent that there is much more variation in these themes and that TonB-dependent outer membrane proteins, annotated as putative iron acquisition systems, may be involved in the transport of a range of other metals or compounds (10,14,28,29,34). Examples of these new insights stem from recent work with H. pylori, where two out of three FecA and FrpB orthologs were shown to be regulated by iron and Fur, whereas the third FecA and FrpB ortholog is NikR and nickel repressed (9,14,39) and has been proposed to function in nickel acquisition (10,28).…”

“…Examples of these new insights stem from recent work with H. pylori, where two out of three FecA and FrpB orthologs were shown to be regulated by iron and Fur, whereas the third FecA and FrpB ortholog is NikR and nickel repressed (9,14,39) and has been proposed to function in nickel acquisition (10,28). Similarly, the H. mustelae putative TonB-dependent outer membrane protein NikH contributes to urease activity, probably by mediating nickel acquisition (34). Caution also needs to be taken when annotating ABC transporters, as the proposed ferric citrate permease and ATPase FecD and FecE, respectively, were shown not to contribute to iron acquisition in H. pylori (41), suggesting a role for these H. pylori genes in the acquisition of other metals or compounds.…”

“…Construction of isogenic H. mustelae mutants. The H. mustelae nikR and fur mutants were described previously (33,34). For the insertional mutagenesis of the other genes studied here (see Tables S1 and S2 in the supplemental material), a ϳ1-kb fragment of the gene of interest was PCR amplified from DNA from H. mustelae strain NCTC 12198 by using knockout (KO) primers (Table S2).…”

“…Total RNA was isolated by using Trizol (Gibco) according to the manufacturer's instructions. RNA was separated on 2% formaldehyde-1.5% agarose gels in 20 mM sodium phosphate buffer (pH 7), transferred onto positively charged nylon membranes (Roche Diagnostics), and covalently bound to the membrane by UV cross-linking (33,34). RNA was visualized by methylene blue staining, and RNA concentrations were normalized based on 16S and 23S rRNA band intensities.…”

“…The PCR fragments contained a T7 promoter sequence and were used for the production of an antisense RNA probe labeled with digoxigenin by transcription with T7 RNA polymerase (Roche Diagnostics). Northern hybridization and stringency washes were performed at 68°C, and bound probe was visualized with the digoxigenin (DIG) detection kit (Roche Diagnostics) and the chemiluminescent substrate CPD-Star (Amersham Pharmacia) as described previously (33,34).…”