Strain-specific genes of Helicobacter pylori: genome evolution driven by a novel type IV secretion system and genomic island transfer

Abstract

The availability of multiple bacterial genome sequences has revealed a surprising extent of variability among strains of the same species. The human gastric pathogen Helicobacter pylori is known as one of the most genetically diverse species. We have compared the genome sequence of the duodenal ulcer strain P12 and six other H. pylori genomes to elucidate the genetic repertoire and genome evolution mechanisms of this species. In agreement with previous findings, we estimate that the core genome comprises about 1200 genes and that H. pylori possesses an open pan-genome. Strain-specific genes are preferentially located at potential genome rearrangement sites or in distinct plasticity zones, suggesting two different mechanisms of genome evolution. The P12 genome contains three plasticity zones, two of which encode type IV secretion systems and have typical features of genomic islands. We demonstrate for the first time that one of these islands is capable of self-excision and horizontal transfer by a conjugative process. We also show that excision is mediated by a protein of the XerD family of tyrosine recombinases. Thus, in addition to its natural transformation competence, conjugative transfer of genomic islands has to be considered as an important source of genetic diversity in H. pylori.

Figure 1.

Strain-specific genes in the H. pylori P12 genome. (A) Circular representation of the genome. Genes predicted on the plus and minus strands are shown as bars on the outer circles. Third and fourth circles: Positions of strain-specific genes (being absent from at least three out of six complete genome sequences). Fifth and sixth circles: GC content and GC skew calculated with a window size of 10 000 and steps of 100 bp. Positions of the putative origin and terminus of replication, the cag pathogenicity island (cagPAI), the comB genes and the plasticity zones (PZ1-3) with their corresponding type IV secretion systems (tfs) are indicated. (B) Venn diagrams showing numbers of common and strain-specific genes for the complete genome sequences indicated. (C) Power law regression fit for the identification of new genes in H. pylori genomes [according to (1)]. The x-axis indicates the number N of complete genome sequences examined, and the y-axis the number n of newly identified genes in each genome. The straight green line, representing a regression fit of the mean numbers of n, indicates a power law progression (n ∼ N^−α), and a power law coefficient (α = 0.339) <1 (dashed line) indicates that the rate of newly identified genes is decreasing very slowly and that H. pylori has thus an open pan-genome.

Figure 2.

Type IV secretion systems in plasticity zones. (A) Gene arrangement in PZ1 and comparison with corresponding regions in other genome sequences. PZ1 of strain P12 is inserted into a restriction–modification system pseudogene (similar to gene hp464 in strain 26695). Genes encoding type IV secretion system components are indicated as black arrows, and frameshift mutations are indicated by asterisks. Genes encoding proteins with 90–95% sequence similarity to the P12 proteins are shown in full colour, genes encoding proteins with 50–75% sequence similarity are hatched. Note that tfs4 has been termed tfs3a (for strains Shi470 and G27) or tfs3b (for strain P12) previously (32). PeCan18B plasticity zone, GenBank accession AF487344.3. (B) Neighbor-joining tree showing relationships between PZ type IV secretion systems and other Helicobacter and Campylobacter type IV secretion systems, based on average distances of the corresponding VirB4, VirB9 and VirB10 homologs. The TFS4 systems of strains P12 (tfs4-HPP12), G27 (tfs4-G27) and Shi470 (tfs4-HPSH) are depicted individually to show their mutual relationships. pTet, C. jejuni 81–176 pTet plasmid; pVir, C. jejuni 81–176 pVir plasmid.

Figure 3.

Gene content analysis of plasticity zones in H. pylori isolates by microarray hybridization. Fragmented and biotin-labeled genomic DNA preparations of the indicated strains were used as probes for array hybridization. Plasticity zone genes are indicated by their hpp12 gene numbers on the left, and putative gene functions are indicated on the right. The presence of individual genes is indicated in white, and their absence in black color. The tfs3 and tfs4 gene clusters are boxed.

Figure 4.

Horizontal gene transfer mediated by the plasticity zone 1 T4SS. (A) DNA transfer experiments were performed in the presence of DNase with a virB4/topA_TFS4-reconstituted P12 donor strain containing a chloramphenicol resistance cassette inserted into an intergenic region of PZ1, and a recA deletion to render the donor strain non-transformable. As recipient strains, we used a P12 wild-type strain with a ΔmoeB(hpp12_765)::aphA-3 insertion conferring kanamycin resistance, or a tfs4 deletion variant with the same aphA-3 insertion. Growth of chloramphenicol/kanamycin double-resistant clones indicated a unidirectional transfer of (parts of) PZ1 to the recipient strains. (B) DNA transfer rates from co-cultivation experiments using the donor and recipient strains indicated. Data shown are mean values of at least four independent experiments including standard deviations (C) Chromosomal DNA of transconjugant clones was examined by PCR and sequencing for co-transfer of the intact (non-frameshifted) virB4 allele, indicating transfer of the whole PZ or the whole tfs4 system. Proportions of transconjugants containing intact virB4 alleles are expressed as percentages of at least 12 independent clones sequenced. (D) Detection of a circular PZ1 intermediate. PCR products spanning the junction sites of circular intermediates were generated using primers WS362 and WS363 from the donor strains indicated. (E) Empty-site PCR with primers WS429 and WS432 was used to obtain DNA fragments from chromosomal DNA prepared from a co-cultivation mixture. Sequences of empty-site PCR fragments and circular intermediates are shown.