The fdxA ferredoxin gene can down-regulate frxA nitroreductase gene expression and is essential in many strains of Helicobacter pylori

Abstract

Very few examples of metabolic regulation are known in the gastric pathogen Helicobacter pylori. An unanticipated case was suggested, however, upon finding two types of metronidazole (Mtz)-susceptible strains: type I, in which frxA (which encodes a nitroreductase that contributes to Mtz susceptibility) is quiescent, and type II, in which frxA is well expressed. Here we report that inactivation of the fdxA ferredoxin gene (hp277) in type I strains resulted in high-level frxA expression (in effect, making them type II). However, fdxA null derivatives were obtained from only 6 of 32 type I strains tested that were readily transformed with an frxA::aphA marker. This suggested that fdxA is often essential. This essentiality was overcome in 4 of 20 strains by inactivating frxA, which suggested both that frxA overexpression is potentially deleterious and also that fdxA has additional, often vital roles. With type II strains, in contrast, fdxA null derivatives were obtained in 20 of 23 cases tested. Thus, fdxA is dispensable in most strains that normally exhibit (and tolerate) strong frxA expression. We propose that restraint of frxA expression helps maintain balanced metabolic networks in most type I strains, that other homeostatic mechanisms predominate in type II strains, and that these complex results constitute a phenotypic manifestation of H. pylori's great genetic diversity.

FIG. 1.

RAPD analysis of relatedness. RAPD tests were carried out on the related strain pairs 88-3887 and 26695 and also on X47 and 98QM3, as discussed in the text, by using primers 1247 (left eight lanes), 1254 (top right eight lanes), 1281 (bottom left), and 1283 (bottom right) (3). The first and second lanes in each set contain products of duplicate RAPD tests, carried out with 5 and 20 ng of template DNA, to ensure that any differences seen are reproducible (or to learn when they are not). Lanes labeled m contain 1-kb marker size standards from Gibco-BRL.

FIG. 2.

RT-PCR analysis of mRNA levels. H. pylori cells were grown, RNA was extracted, and RT-PCR was carried out as detailed in Materials and Methods. Threshold deleterious levels of Mtz were included in BHI agar where indicated (0.2 μg/ml for strain SS1; 1.5 μg/ml for strain 26695). WT, wild type.

FIG. 3.

Profiles of susceptibility to Mtz of strain 26695 wild type (WT) and isogenic mutant derivatives of it. Each test was carried out at least three times.

FIG. 4.

Kinetics of death in stationary phase. Young exponentially growing cells were spread on BHI agar and were incubated. Aliquots were withdrawn daily, and efficiencies of colony formation, relative to culture optical density, were determined. WT, wild type.