Global transposon mutagenesis and essential gene analysis of Helicobacter pylori

Abstract

We have constructed a genome-saturating mutant library of the human gastric pathogen Helicobacter pylori. Microarray tracking of transposon mutants (MATT) allowed us to map the position of 5,363 transposon mutants in our library. While we generally found insertions well distributed throughout the genome, 344 genes had no detectable transposon insertions, and this list is predicted to be highly enriched for essential genes. Comparison to the essential gene set of other bacteria revealed a surprisingly limited overlap with all organisms tested (11%), while 55% were essential in some organisms but not others. We independently verified the essentiality of several gene products, including an HtrA family serine protease, a hypothetical protein with putative phospholipase D activity, and a riboflavin specific deaminase. A limited screen for motility mutants allowed us to estimate that 4.5% of the genome is dedicated to this virulence-associated phenotype.

FIG. 1.

MATT method for labeling DNA adjacent to transposon insertions. The first PCR is run with genomic DNA template, primer S reading out from the left arm of the transposon and primer CEKG2C, which contains an anchored degenerate sequence. The second PCR uses the product of the first as template with nested transposon primer S2 and primer CEKG4, complementary to the conserved portion of primer CEKG2C. Amino-allyl dUTP is incorporated in the second PCR to allow conjugation of the resulting product to Cy3 or Cy5 fluorescent dyes for hybridization to the microarray.

FIG. 2.

Number of transposon (Tn) insertions per gene weakly correlates with gene size in base pairs. A linear regression curve is shown according to y = 0.0013x + 2.3144, where R² = 0.0696.

FIG. 3.

Genes with no transposon (Tn) insertions are enriched in some functional classes but not in others. The percentage of genes with no transposon insertions in each functional class is indicated. Abbreviations: Biosyn/Metab, biosynthesis and metabolism; Cell Div, cell division; Cell Env, cell envelope; Chap, chaperone; Comp, DNA competence; Detox, detoxification; DNA/RNA, DNA and RNA metabolism; Mot, motility; Out Mem, outer membrane; PAI, pathogenicity island; Prot Syn, protein synthesis; Reg, regulation; Res Mod, restriction modification; Trans, transporters; Unknown Hyp, unknown hypothetical.

FIG. 4.

HP0190 is an essential gene. (A) Genomic region surrounding HP0190. Gene annotation is according to Tomb et al. (47). Numbers above each open reading frame indicate the number of transposon insertions detected by MATT. (B) A knockout cassette can integrate at either locus containing HP0190 in a strain containing a second copy at the rdxA locus. The number above each lane indicates independent chloramphenicol-resistant clones. The letters represent the primer pair used for amplification. A lanes (e.g. 1A, 2A, etc.) depict PCR results with primers rdx1 and cat2, which will only give a product if integration occurred at the rdxA locus. B lanes indicate PCR with primers HP0190-1 and cat2, which give a product for integration at the native locus and the rdxA locus. Reactions were fractionated on a 0.8% agarose gel, and DNA was visualized by ethidium bromide staining. The far left lane contains a 1-kb ladder DNA standard (Gibco-BRL). Clones 1, 2, and 5 have integrated the knockout cassette into the gene copy at the rdxA locus. Clones 3 and 4 have integrated the knockout cassette into the gene copy at the native HP0190 locus.