Identification of genes associated with natural competence in Helicobacterpylori by transposon shuttle random mutagenesis

To identify genes involved in DNA transformation, we generated 1500 inserti on mutants of a Helicobacter pylori strain by transposon shuttle mutagenesi s. All mutant strains were screened for their frequency of natural transfor mation. A total of 20 mutant strains were found to exhibit a significantly decreased transformation frequency. DNA sequencing revealed seven genetic l oci, including the reported comB locus, HP0017 (a putative virB4 homologue) and five loci without database match (HP0015, HP1089, HP1326, HP1424, and HP1473) from the 20 mutants. Reknock-out of HP1326 revealed no impairment i n natural transformation, while the other 5 mutants showed the same defecti ve in natural transformation. Mutation of HP0017 severely impaired natural transformation both chromosome and plasmid DNA. Slot blot analysis revealed that some noncompetent strains had decreased virB4 RNA expression levels c ompared with competent strains. Nineteen ORFs had decreased expression leve ls in virB4 knockout mutant by microarray. Therefore, our data indicate tha t HP0017 is a virB4 homologue and is essential in the natural competence of H. pylori. HP0015, HP1089, HP1424, and HP1473 genes could be also involved in natural transformation. (C) 2001 Academic Press.