Iron-responsive repression of urease expression in Helicobacter hepaticus is mediated by the transcriptional regulator Fur

Abstract

Persistent colonization of mucosal surfaces by bacteria in the mammalian host requires concerted expression of colonization factors, depending on the environmental conditions. Helicobacter hepaticus is a urease-positive pathogen that colonizes the intestinal and hepatobiliary tracts of rodents. Here it is reported that urease expression of H. hepaticus is iron repressed by the transcriptional regulator Fur. Iron restriction of growth medium resulted in a doubling of urease activity in wild-type H. hepaticus strain ATCC 51449 and was accompanied by increased levels of urease subunit proteins and ureA mRNA. Insertional inactivation of the fur gene abolished iron-responsive repression of urease activity, whereas inactivation of the perR gene did not affect iron-responsive regulation of urease activity. The iron-responsive promoter element was identified directly upstream of the H. hepaticus ureA gene. Recombinant H. hepaticus Fur protein bound to this ureA promoter region in a metal-dependent matter, and binding resulted in the protection of a 41-bp, Fur box-containing operator sequence located at positions -35 to -75 upstream of the transcription start site. In conclusion, H. hepaticus Fur controls urease expression at the transcriptional level in response to iron availability. This represents a novel type of urease regulation in ureolytic bacteria and extends the already diverse regulatory repertoire of the Fur protein.

FIG. 1.

Induction of H. hepaticus urease transcription, expression, and activity under iron-restricted conditions. (A) H. hepaticus ATCC5 1449 was grown in standard BBC (gray squares), iron-restricted medium (white diamonds), and iron-replete medium (black triangles), and the OD₆₀₀ was monitored over a time period of 36 h. The arrow indicates the 24-h time point, representing late log phase, which was chosen for comparison of iron-restricted and iron-replete conditions. (B) Urease activity in H. hepaticus is iron repressed, as measured after 24 h of growth in iron-restricted medium (white bar) or iron-replete medium (black bar). Each bar represents data from seven independent experiments; error bars denote standard deviations. An asterisk indicate a significant increase in urease activity after growth under iron-restricted conditions compared to growth under iron-replete conditions (P < 0.05 [Mann-Whitney U test]). (C) The increase in H. hepaticus urease activity under iron-restricted conditions is associated with increased expression of the UreA and UreB enzyme subunits, as shown by SDS-PAGE (left panel) and immunoblotting (right panel) with antibodies to H. felis urease. The left panel displays the relevant section of the protein profile of the lysates used for immunoblots, stained with Coomassie brilliant blue. Relevant marker sizes are indicated on the left. (D) Identification of the H. hepaticus ureA transcription start site by primer extension analysis using RNA purified from H. hepaticus ATCC 51449 grown under iron-restricted (−Fe) and iron-replete (+Fe) conditions. The sequence of the corresponding promoter region is displayed on the right, with the +1 residue and the −10 promoter sequence indicated. Note that the primer extension product displays iron-responsive repression of transcription.

FIG. 2.

Fur, but not PerR, mediates iron-responsive regulation of urease expression in H. hepaticus. Urease activity of H. hepaticus strain ATCC 51449 and the fur and perR mutants was assessed after 24 h of growth in iron-restricted medium (white bars) or iron-replete medium (black bars). Each bar represents data from three independent experiments for each strain; error bars denote standard deviations. An asterisk indicate a significant increase in urease activity after growth under iron-resticted conditions compared to growth under iron-replete conditions (P ≤ 0.05 [Mann-Whitney U test]); NS, not significant.

FIG. 3.

H. hepaticus Fur displays metal-dependent binding to the ureA promoter. Shown is an electrophoretic mobility shift assay using recombinant H. hepaticus Fur protein and DIG-labeled ureA promoter DNA (P_ureA). In the absence of the iron substitution manganese (+EDTA, middle panel), Fur is unable to complex with the ureA promoter region and a shift is not observed. Only in the presence of manganese (+MnCl₂, top panel) is Fur able to bind to the ureA promoter region and cause a mobility shift (indicated as P_ureA + Fur). No shift is observed when the Fur protein is incubated with the promoter region of the ksgA gene (P_ksgA) in the presence of manganese (+MnCl₂, bottom panel). The concentration of Fur is indicated above the lanes; the concentration of DNA was 0.5 nM ureA or ksgA promoter DNA.

FIG. 4.

Identification of the Fur operator sequence in the H. hepaticus ATCC 51449 ureA promoter. (A) DNase I footprinting assay performed in the presence and absence of the iron substitute MnCl₂, using 50 nM DIG-labeled H. hepaticus ATCC 51449 ureA promoter DNA and 500 nM Fur. On the left side are indicated the positions relative to the ureA transcription start site. On the right-hand side is indicated the position of the protected region (located from −35 to −75 relative to the transcription start site). Note that a protected region is observed only in the presence of both Fur and MnCl₂. (B) Binding of ureA promoter DNA is dependent on the Fur concentration. Increasing concentrations of Fur were mixed with 20 nM DIG-labeled H. pylori ATCC 51449 ureA promoter in the presence of 200 μM MnCl₂. The concentration of Fur protein used is indicated above the lanes. Positions relative to the ureA transcription start site are indicated on the left; the protected region is indicated on the right.

FIG. 5.

(A) Schematic representation of the H. hepaticus ureA promoter region with the location and sequence of the Fur-binding site indicated. The proposed ribosome-binding site (RBS), −10 promoter sequence, and transcription start site (+1) are indicated. (B) Annotation of the proposed Fur box present in the Fur-bound sequence in the H. hepaticus ureA promoter region, according to the F-F-x-R hexamer consensus sequence. Residues indicated in bold type match the 5′-NAT(A/T)AT consensus hexamer sequence.