Identification of nonessential Helicobacter pylori genes using random mutagenesis and loop amplification

Analysis of the published genome sequences of Helicobacter pylori revealed that approximately 40% of the predicted open reading frames (ORFs) were of unknown function. We have developed the random mutagenesis and loop amplifi cation (RMLA) strategy, and used this approach both to characterize individ ual virulence factors and to collectively screen comparatively large number s of H. pylori mutants to identify genes that are not essential for viabili ty in vitro. The mini-Tn3-Km transposon was used to generate a random mutan t library in H. pylori strain G27. By screening the library of mutants we w ere able to demonstrate that the transposon integrated randomly into the ch romosome of H. pylori and that RMLA was able to identify mutants in known v irulence genes (urease and catalase). To test whether this strategy could b e used as a high-throughput approach for the simultaneous identification of a series of nonessential genes of H. pylori, the transposon-chromosomal ju nctions of a pool of mutants were amplified by inverse PCR using circular f ragments of genomic DNA obtained after chromosomal DNA extracted from the p ool of mutants had been digested with HindIII and self-ligated. The amplifi cation products were radioactively labelled and hybridized to a high densit y macroarray membrane containing a duplicated target sequence for every gen e of H. pylori strain 26695. For the positive ORFs the precise site of tran sposon insertion was confirmed by PCR mapping. In total 78 H. pylori genes were unambiguously identified as nonessential for viability in vitro, inclu ding 20 with orthologues of unknown function in other species and 21 which were H. pylori-specific. (C) 2001 Editions scientifiques et medicales Elsev ier SAS.