PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Abstract

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

Figure 1

Bacterial genome subtractive DNA method. Solid lines represent AluI-digested tester and driver DNA fragments. Boxes represent outer part of PCR adaptors, which lack phosphate groups at their 5′ ends. Adaptors 1 and 2 are identical near their 5′ ends but differ near their 3′ ends. Note that after recessed 3′ ends are filled, types a, b, and c molecules having adaptor 2 are also present, but are not shown. This method is adapted from the subtraction subtractive hybridization method for studies of mRNAs in eukaryotes (18, 19).

Figure 2

Map locations of 14 subtracted clones in the cag PAI. Coordinates refer to distance in kb from left end of the 37-kb cag PAI, as in ref. , which corresponds to position 537 kb in the entire H. pylori 26695 genome sequence (30).

Figure 3

Representative dot-blot hybridization of products of subtractive hybridization by using genomic DNAs of H. pylori strains J166 as tester and 26695 as driver. Cloned subtractive hybridization products were PCR-amplified using primers NP1 and NP2 and spotted on Hybond N+ filters (Materials and Methods) and probed with labeled genomic DNAs, as indicated. The clones shown by DNA sequencing to have J166 DNAs either not present in the 26695 genome or substantially different from sequences in 26695 (as summarized in Table 3) are circled.