Isolation of the Helicobacter pylori recA gene and involvement of the recA region in resistance to low pH

Abstract

To understand the potential roles of the important DNA repair protein RecA in Helicobacter pylori pathogenesis, we cloned the recA gene from H. pylori 84-183. Degenerate PCR primers based on conserved RecA protein regions were used to amplify a portion of H. pylori recA, which was used as a probe to isolate the full-length recA gene from H. pylori genomic libraries. The H. pylori recA gene encoded a protein of 347 amino acids with a molecular mass of 37.6 kDa. As expected, H. pylori RecA was highly similar to other RecA proteins and most closely resembled that of Campylobacter jejuni (75% identity). Immediately downstream of recA was an open reading frame whose predicted product showed 58% identity to the Bacillus subtilis enolase protein. recA and eno were disrupted in H. pylori 84-183 by insertion of antibiotic resistance genes. Reverse transcription-PCR demonstrated that recA and eno were cotranscribed and that insertion of the kanamycin resistance gene into recA had polar effects on expression of the downstream eno. The H. pylori recA mutants were severely impaired in their ability to survive treatment with UV light and methyl methanesulfonate and with the antimicrobial agents ciprofloxacin and metronidazole. The eno mutant had sensitivities to UV light and metronidazole intermediate to those of wild-type and recA strains, suggesting that truncation of the recA-eno transcript resulted in lowered recA expression. For survival at low pH, a recA mutant was approximately 10-fold more sensitive than strain 84-183, while the eno mutant demonstrated intermediate susceptibility. This difference occurred in the presence or absence of urea, implying the involvement of a gene in the recA region in an acid resistance mechanism distinct from that mediated by urease.