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.
