Sequence organization and insertion specificity of the novel chimeric ISHp609 transposable element of Helicobacter pylori

Abstract

Here we describe ISHp609 of Helicobacter pylori, a new member of the IS605 mobile element family that is novel and contains two genes whose functions are unknown, jhp960 and jhp961, in addition to homologs of two other H. pylori insertion sequence (IS) element genes, orfA, which encodes a putative serine recombinase-transposase, and orfB, whose homologs in other species are also often annotated as genes that encode transposases. The complete four-gene element was found in 10 to 40% of strains obtained from Africa, India, Europe, and the Americas but in only 1% of East Asian strains. Sequence comparison of 10 representative ISHp609 elements revealed higher levels of DNA sequence matches (99%) than those seen in normal chromosomal genes (88 to 98%) or in other IS elements (95 to 97% for IS605, IS606, and IS607) from the same H. pylori populations. Sequence analysis suggested that ISHp609 can insert at many genomic sites with its left end preferentially next to TAT, with no target specificity for its right end, and without duplicating or deleting target sequences. A deleted form of ISHp609, containing just jhp960 and jhp961 and 37 bp of orfA, found in reference strain J99, was at the same chromosomal site in 15 to 40% of the strains from many geographic regions but again in only 1% of the East Asian strains. The abundance and sequence homogeneity of ISHp609 and of this nonmobile remnant suggested a recent bottleneck and then rapid spread in H. pylori populations, possibly selected by the contributions of the elements to bacterial fitness.

FIG. 1.

Structures of ISHp609 and related elements. (A) Full-length predominant ISHp609 type with four characteristic open reading frames and 99% DNA identity, independent of geographic origin. Sequence analysis of 10 full-length elements from Spanish strains (HUP-B43, HUP-B79, and HUP-B80; accession numbers AY487825, AY639112, and AY639113), Lithuanian strains (Lit-7 and Lit38; accession numbers AY639110 and AY639111), Alaskan strains (Al64 and Al97; accession numbers AY639115 and AY639116), a Peruvian strain (SJM27; accession number AY639118), an Indian strain (I-86; accession number AY639117), and an African strain (R48; accession number AY639114) showed limited internal divergence. In strain Lit7 orfA contained a frameshift due to a 14-bp duplication; in strain HUP-B79 orfA contained an in-frame stop codon due to a G-to-T substitution; and in strain I-86 orf1 contained an in-frame stop codon due to a C-to-T substitution. (B) ISHp609var. This rare variant element was found in one Indian strain (Chennai4; accession number AY639119) with 81% DNA identity to the predominant type. ISHp609var has full-length orfA and orfB genes, although orfB is probably inactive due to a frameshift. It lacks orf2 and most of orf1, and it has the first 79 bp of orf1, but there is no start codon at its left end. (C) orf1-2 remnant. This DNA segment consists of orf1 (jhp960 in strain J99), orf2 (jhp961 in strain J99), and the first 37 bp of orfA and is located between homologs of jhp959 and jhp962 in most or all strains (as determined by PCR with primers jhp959 and jhp962, primers jhp959 and 609.R6, and primers FlankL and jhp962).

FIG. 2.

Phylogenetic relationships among H. pylori IS elements. (A) OrfA and homologs. Two subfamilies were identified, one represented by OrfAs of IS605 (accession number NP_208326), IS606 (accession number AAD11513), and ISHp608 (accession number AAL06576), which are not considered to encode serine recombinases based on amino acid homologies, and the other represented by IS607 (accession number AAF05600), ISHp609 (accession number AAR83266.1), ISHp609var (accession number AY639119), and the closest homolog in T. tengcongensis (T. teng.) (tte0714; accession number AAM23976), which are thought to encode serine recombinases. Branches with significant bootstrap support (≥50) are indicated. Bar = 1 amino acid substitution per site. (B) OrfB and homologs. OrfBs of H. pylori IS605 (accession number NP_208324), IS606 (accession number AAD11514), IS607 (accession number AAF05601), ISHp608 (accession number AAL06577), and Salmonella's GipA protein (accession number NP_752781) form an OrfB subfamily different from that of ISHp609 (accession number AAR83267.1), ISHp609var (accession number AY639119), and the closest homolog in T. tengcongensis (tte0715; accession number AAM23977). (C) Partial C-terminal sequence alignment of H. pylori IS element OrfBs, GipA (22), and the corresponding sequence in T. tengcongensis. A single C-terminal Zn(II) binding tetracysteine motif, CX₍₂₎CX₍₁₅₎CX₍₂₎C (C4-type zinc finger), is well conserved among IS605, IS606, IS607, and ISHp608 OrfBs and GipA and might potentially facilitate DNA or RNA binding or protein-protein interaction. Notably, this motif is not present in ISHp609 OrfB (both predominant and variant).

FIG. 3.

Terminal sequences of ISHp609, ISHp609var, and the orf1-2 remnant and their sites of insertion in H. pylori. ISHp609, ISHp609var, and orf1-2 remnant termini are in uppercase type. Flanking DNA and empty sites in reference strains 26695 and J99 (gene designations beginning with hp and jhp, respectively) are in lowercase type. (A) ISHp609 predominant type. (B) ISHp609var type. Terminal sequences were extrapolated from a comparison with sequences of the predominant ISHp609 type, because its flanking sequences did not have homology with known H. pylori sequences and therefore the site of insertion (empty site) was not known. (C) Predicted left end of the orf1-2 remnant and its flanking sequences. Sites of insertion could not be determined precisely due to local sequence heterogeneity in strains lacking this element (intergenic region between jhp959 and jhp962) (see Fig. S2 in the supplemental material). (D) Predicted right end of the orf1-2 remnant compared to the corresponding region of orfA in the ISHp609 sequence.