Evolutionary History of hrgA, Which Replaces the Restriction Gene hpyIIIR in the hpyIII Locus of Helicobacter pylori

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. Type II restriction-modification (R-M) systems are comprised of paired enzymes, a restriction endonuclease that cleaves DNA within a specific 4- to 8-bp sequence and a methyltransferase that specifically methylates the DNA within the same sequence, protecting the sequence from cleavage (4, 5, 14). Helicobacter pylori, a gram-negative bacterium that colonizes the human stomach, affects the risk of getting upper gastrointestinal tract diseases, including gastric cancer (13). H. pylori strains are highly heterogeneous in terms of the number and nature of the R-M systems that they carry (2, 10, 11, 18, 19, 21, 23). During characterization of the hpyIIIR-hpyIIIM locus in Asian and Western strains, we found numerous strains with a novel gene that we designated hrgA in place of hpyIIIR (encoding an isoschizomer of Moraxella bovis MboI). The presence of hrgA appears to have predictive value for virulence in cagA-positive strains from Asia (3). Neither gene is essential, but since no strain that lacks or contains both genes has been identified thus far, it is hypothesized that there is selection for the presence of either gene. The work described here addressed the following questions. How conserved is hrgA? How did the hrgA-hpyIII locus evolve? And are hrgA and hpyIIIR functional, and can the two genes be exchanged by natural transformation? Mutants bearing antibiotic resistance cassettes were constructed to investigate exchange of the genes between strains by natural transformation. The results indicate that exchange of these genes is possible and may involve transfer of a DNA fragment containing substantial flanking sequences, thus increasing the potential for genomic plasticity in H. pylori. MATERIALS AND METHODS