Helicobacter hepaticus NikR controls urease and hydrogenase activities via the NikABDE and HH0418 putative nickel import proteins

Abstract

Helicobacter hepaticus open reading frame HH0352 was identified as a nickel-responsive regulator NikR. The gene was disrupted by insertion of an erythromycin resistance cassette. The H. hepaticus nikR mutant had five- to sixfold higher urease activity and at least twofold greater hydrogenase activity than the wild-type strain. However, the urease apo-protein levels were similar in both the wild-type and the mutant, suggesting the increase in urease activity in the mutant was due to enhanced Ni-maturation of the urease. Compared with the wild-type strain, the nikR strain had increased cytoplasmic nickel levels. Transcription of nikABDE (putative inner membrane Ni transport system) and hh0418 (putative outer membrane Ni transporter) was nickel- and NikR-repressed. Electrophoretic mobility shift assays (EMSAs) revealed that purified HhNikR could bind to the nikABDE promoter (P(nikA)), but not to the urease or the hydrogenase promoter; NikR-P(nikA) binding was enhanced in the presence of nickel. Also, qRT-PCR and EMSAs indicated that neither nikR nor the exbB-exbD-tonB were under the control of the NikR regulator, in contrast with their Helicobacter pylori homologues. Taken together, our results suggest that HhNikR modulates urease and hydrogenase activities by repressing the nickel transport/nickel internalization systems in H. hepaticus, without direct regulation of the Ni-enzyme genes (the latter is the case for H. pylori). Finally, the nikR strain had a two- to threefold lower growth yield than the parent, suggesting that the regulatory protein might play additional roles in the mouse liver pathogen.

Fig. 1.

Nucleotide sequence of the nikA–hh0418 and nikR–exbB intergenic regions. Putative open reading frames are shown in bold. hh numbers refer to annotated genes from strain H. hepaticus 51449 (Suerbaum et al., 2003). PCR products used for EMSAs are underlined. Putative NikR binding boxes for both nikA and hh0418, as defined by Stoof et al. (2010b), are highlighted in grey. Putative alternative NikR binding motifs are indicated by boxes.

Fig. 2.

NikR protein sequences of H. hepaticus, H. pylori, H. mustelae and E. coli NikR. The residues shaded in grey and located toward the N-terminal region are conserved residues previously identified to be involved in DNA binding in H. pylori or E. coli. The H and C residues shaded in grey and located towards the C terminus have been shown to be involved in nickel binding in E. coli (Schreiter et al., 2003).

Fig. 3.

Amount of nickel detected in cells by ICP-MS. H. hepaticus wild-type (white bars) and nikR mutant (black bars) cells were grown with the indicated nickel supplement (0–50 µM). Total protein and nickel content were determined by BCA assay and ICP-MS, respectively. Bars show mean±sd and are from one experiment and six measurements. The experiment was performed three other times and the same trend was observed. The mutant values were greater than the parent for all nickel supplementation levels: P<0.005, as determined by Student’s t-test.

Fig. 4.

Transcript levels of nikA, hh0418, exbB and nikR as a function of nickel and NikR. The level of transcripts after culture on BA without added NiCl₂ or with supplement of 1, 10 or 100 µM NiCl₂ was determined relative to that of the level of the housekeeping gene gyrA (hh1633) under the same conditions. For each gene, fold changes are relative to transcript levels of the wild-type grown on BA without added Ni, using the $2^{-\Delta \Delta C_{\text{t}}}$. The average fold changes are from two independent experiments, each with three to five measurements.

Fig. 5.

EMSAs. Increasing concentrations of purified T7-HhNikR protein (in μM, indicated above the lanes) were incubated with (a) the P_ureA promoter (220 pM) or the P_hyaA promoter (240 pM) in presence of 0.8 mM NiSO₄, or the P_nikA promoter (220 pM) in presence of 1 mM EDTA (b) or 0.8 mM NiSO₄ (c). Free or bound ³²P-labelled DNA probes are indicated on the side of each panel.