Stable Accumulation of σ 54 in Helicobacter pylori Requires the Novel Protein HP0958

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Abstract
Several flagellar genes in Helicobacter pylori are dependent on σ 54 (RpoN) for their expression. These genes encode components of the basal body, the hook protein, and a minor flagellin, FlaB. A protein-protein interaction map for H. pylori constructed from a high-throughput screen of a yeast two-hybrid assay ( http://pim.hybrigenics.com/pimrider ext/common/) revealed interactions between σ 54 and the conserved hypothetical protein HP0958. To see if HP0958 influences σ 54 function, the corresponding gene was disrupted with a kanamycin resistance gene ( aphA3 ) in H. pylori ATCC 43504 and the resulting mutant was analyzed. The hp0958:aphA3 mutant was nonmotile and failed to produce flagella. Introduction of a functional copy of hp0958 into the genome of the hp0958:aphA3 mutant restored flagellar biogenesis and motility. The hp0958:aphA3 mutant was deficient in expressing two σ 54 -dependent reporter genes, flaB ′ - ′ xylE and hp1120 ′ - ′ xylE . Levels of σ 54 in the hp0958 mutant were substantially lower than those in the parental strain, suggesting that the failure of the mutant to express the genes in the RpoN regulon and produce flagella was due to reduced σ 54 levels. Expressing σ 54 at high levels by putting rpoN under the control of the ureA promoter restored flagellar biogenesis and motility in the hp0958:aphA3 mutant. Turnover of σ 54 was more rapid in the hp0958:aphA3 mutant than it was in the wild-type strain, suggesting that HP0958 supports wild-type σ 54 levels in H. pylori by protecting it from proteolysis.