Evolutionary history of hrgA, which replaces the restriction gene hpyIIIR in the hpyIII locus of Helicobacter pylori

Abstract

A recently identified Helicobacter pylori gene, hrgA, was previously reported to be present in 70 (33%) of 208 strains examined (T. Ando, T. M. Wassenaar, R. M. Peek, R. A. Aras, A. I. Tschumi, L.-J. Van Doorn, K. Kusugami, and M. J. Blaser, Cancer Res. 62:2385-2389, 2002). Sequence analysis of nine such strains indicated that in each strain hrgA replaced hpyIIIR, which encodes a restriction endonuclease and which, together with the gene for its cognate methyltransferase, constitutes the hpyIII locus. As a consequence of either the hrgA insertion or independent mutations, hpyIIIM function was lost in 11 (5%) of the 208 strains examined, rendering chromosomal DNA sensitive to MboI digestion. The evolutionary history of the locus containing either hpyIII or hrgA was reconstructed. By homologous recombination involving flanking sequences, hrgA and hpyIIIR can replace one another in the hpyIII locus, and there is simultaneous replacement of several flanking genes. These findings, combined with the hpyIM/iceA2 locus discovered previously, suggest that the two most strongly conserved methylase genes of H. pylori, hpyIIIM and hpyIM, are both preceded by alternative genes that compete for presence at their loci.

FIG. 1.

Alignment of deduced amino acid sequences encoded by hrgA from seven H. pylori strains. The JP26 sequence was used as the reference. Conserved amino acids are indicated by dots. Gaps introduced for optimal alignment are indicated by hyphens. The grey bars indicate three regions with frequent indels and substitutions. The stop codons are indicated by asterisks. The numbers at the top include introduced gaps.

FIG. 2.

Plot of Ka and Ks in hrgA sequences derived from seven strains, obtained by using sliding windows of 30 amino acids. The grey bars indicate the regions with frequent indels and substitutions identified in Fig. 1.

FIG. 3.

Phylogeny of hrgA based on nucleotide sequences. Bar = 10 nucleotide changes. The numbers at the nodes are bootstrap values per 100 runs.

FIG.4.

(A) Reconstruction of the evolution of the hrgA/hpyIIIR locus in H. pylori strains, as deduced from the sequences of 13 strains. (B) Sequences of the regions upstream of hpyIIIR (strains 26695, 99517, 88-29, and J99) or hrgA (strains 99515, JP2, JP28, JP26, B128, 4602, 60190, 9627, and J54). Nucleotides identical to nucleotides in the 26695 sequence are indicated by dots, while gaps introduced for the alignment are indicated by hyphens. The positions where hrgA was introduced, replacing the upstream region of hpyIIIR, are indicated by arrows. Nucleotides identical to nucleotides in the JP2 sequence are indicated by colons. Translational start sites of both genes are underlined. (C) Sequences of the boundaries between hpyIIIR or hrgA and hpyIIIM of the strains shown in panel A. The start codon of hpyIIIM is underlined for each strain. An alternative start codon of hpyIIIM is shown for hrgA-containing strains. The 3′ ends of hpyIIIR and hrgA are enclosed in boxes, and each stop codon is indicated by italics. Nucleotides identical to nucleotides in the 26695 sequence are indicated by dots, and nucleotides identical to nucleotides in the JP2 sequence are indicated by colons. Gaps introduced into the alignment are indicated by hyphens. The 3′ end of the insertion containing hrgA for nine genes is indicated by an arrow. The insertion end could not be determined for strain 99515.

FIG. 5.

Schematic diagram of transformation experiments exchanging hrgA and hpyIIIR. The donor DNA was derived from a mutant in which a selectable cat cassette was introduced into hrgA (in strain JP26) (top) or into hpyIIIR (in strain 26695) (bottom). The positions of the crossovers were determined by sequence analysis for each transformant, based on the polymorphisms between JP26 and 26695. The size of the transformed DNA is indicated. Sequences and lengths of the DNA segment with 100% identity around the crossover points are indicated by grey (for 26695 sequences) and black (for JP26 sequences).