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 amplification (RMLA) strategy, and used this approach both to characterize individual virulence factors and to collectively screen comparatively large numbers of H. pylori mutants to identify genes that are not essential for viability in vitro. The mini-Tn3-Km transposon was used to generate a random mutant library in H. pylori strain G27. By screening the library of mutants we were able to demonstrate that the transposon integrated randomly into the chromosome of H. pylori and that RMLA was able to identify mutants in known virulence genes (urease and catalase). To test whether this strategy could be used as a high-throughput approach for the simultaneous identification of a series of nonessential genes of H. pylori, the transposon-chromosomal junctions of a pool of mutants were amplified by inverse PCR using circular fragments of genomic DNA obtained after chromosomal DNA extracted from the pool of mutants had been digested with HindIII and self-ligated. The amplification products were radioactively labelled and hybridized to a high density macroarray membrane containing a duplicated target sequence for every gene of H. pylori strain 26695. For the positive ORFs the precise site of transposon insertion was confirmed by PCR mapping. In total 78 H. pylori genes were unambiguously identified as nonessential for viability in vitro, including 20 with orthologues of unknown function in other species and 21 which were H. pylori-specific.