Allelic exchange mutagenesis of rpoN encoding RNA-polymerase sigma54 subunit in Helicobacter pylori

The rpoN gene, encoding the alternative sigma factor (sigma54) of Helicobacter pylori, was amplified from genomic DNA. H. pylori rpoN has an overall similarity to the rpoN of other bacteria, but lacks a glutamine (Q)-rich region in region I and an acidic region in region II. When the rpoN gene was disrupted, the mutant was found to be completely nonmotile. Because the flaB gene has an rpoN consensus sequence in its promoter region, we assessed the transcriptional activity of the flaB gene, using xylE transcriptional fusion. In the isogenic mutant of rpoN, transcription of the flaB gene was severely affected, but transcription of the ureA gene (control) was intact. In late stationary phase, the rpoN mutant showed marked decreases in viability: i.e., the number of colony-forming units (CFU) at 100 h was 4 log lower in the rpoN mutant than in the wild-type strain. By morphological examination with acridine orange staining, the rpoN mutant showed green and faintly orange-stained irregularly shaped cells with a few orange-stained rod/spiral cells. In contrast, the wild-type strain and the non-flagella flgE mutant (control) contained many orange-stained rod/spiral and coccoid cells. These results indicated that in H. pylori, RpoN is involved not only in motility but also in viability, through the morphological changes in the stationary phase.