Transposable element ISHp608 of Helicobacter pylori: nonrandom geographic distribution, functional organization, and insertion specificity

Abstract

A new member of the IS605 transposable element family, designated ISHp608, was found by subtractive hybridization in Helicobacter pylori. Like the three other insertion sequences (ISs) known in this gastric pathogen, it contains two open reading frames (orfA and orfB), each related to putative transposase genes of simpler (one-gene) elements in other prokaryotes; orfB is also related to the Salmonella virulence gene gipA. PCR and hybridization tests showed that ISHp608 is nonrandomly distributed geographically: it was found in 21% of 194 European and African strains, 14% of 175 Bengali strains, 43% of 131 strains from native Peruvians and Alaska natives, but just 1% of 223 East Asian strains. ISHp608 also seemed more abundant in Peruvian gastric cancer strains than gastritis strains (9 of 14 versus 15 of 45, respectively; P = 0.04). Two ISHp608 types differing by approximately 11% in DNA sequence were identified: one was widely distributed geographically, and the other was found only in Peruvian and Alaskan strains. Isolates of a given type differed by < or = 2% in DNA sequence, but several recombinant elements were also found. ISHp608 marked with a resistance gene was found to (i) transpose in Escherichia coli; (ii) generate simple insertions during transposition, not cointegrates; (iii) insert downstream of the motif 5"-TTAC without duplicating target sequences; and (iv) require orfA but not orfB for its transposition. ISHp608 represents a widespread family of novel chimeric mobile DNA elements whose further analysis should provide new insights into transposition mechanisms and into microbial population genetic structure and genome evolution.

FIG. 1.

IS element maps. (A) Structures of ISs of H. pylori. ISHp608 is 1,833 bp long, making it slightly smaller than its three other known H. pylori relatives (size range, 1,888 to 2,028 bp [18, 19]). Boxes represent ORFs: those with the same shading pattern are related at the protein level (range of 28 to 36% amino acid sequence identities). orfA sizes are 156, 148, 139, and 218 codons and orfB sizes are 384, 427, 446, and 420 codons in ISHp608, IS605, IS606, and IS607, respectively. orfA and orfB overlap by 10 codons in ISHp608, which is reminiscent of the arrangement in IS607 (nine-codon overlap). Distances from left and right ends to the 5" end of orfA and 3" end of orfB in a representative ISHp608 are 171 bp and 75 bp, respectively (accession no. AF357224). GenBank accession nos. for IS605, IS606, and IS607 are AC000108, U95957, and AF189015, respectively. (B) Positions of oligonucleotide primers, the subtractive clone that led to the discovery of ISHp608, and mutations generated in ISHp608. The locations and orientations of PCR and sequencing primers are denoted by arrowheads; these primers are described in detail in Table 1. Primers 1 and 20 are specific to sequences in jhp0924 (an ORF in strain J99 [4]) located to the left and right of ISHp608, respectively, in strain PeCan2A. ∗ and ∗∗ identify primers specific for ISHp608 types 2 and 3, respectively (defined in Results). Subtractive clone D7 is a 242-bp AluI fragment from strain PeCan18B that begins approximately 30 to 34 bp from the left end of ISHp608 copy 2. cam#2, cam#3, cam#4, and cam#5 represent mutant derivatives of ISHp608 from strain PeCan2A that contain the cam (resistance gene) cassette inserted into (cam#4 and cam#5) or replacing (cam#2 and cam#3) particular ISHp608 sequences. In cam#4 (A⁺B⁺), cam was inserted just downstream of orfB and 68 bp from the rightmost end of ISHp608 (generated with primers 8 and 18); in cam#2 (A⁻B⁺), 154 bp of orfA sequence was replaced by the cam gene (generated with primers 2 and 12); in cam#3 (A⁺B⁻), 948 bp of orfB sequence was replaced by cam (generated with primers 4 and 16); in cam#5 (A⁺B⁺), cam was inserted just 22 bp from the ISHp608 right end, within a 22-bp direct repeat (putative transposase binding site) (generated with primers 9 and 19), as detailed in Results.

FIG. 2.

ISHp608 terminal sequences in several H. pylori strains. Left and right termini of ISHp608s determined by sequence comparison are shown in uppercase, and flanking DNA and empty sites in reference strains 26695 and J99 (HP and jhp gene designations, respectively [4, 35]) are shown in lowercase letters. The left end of ISHp608 copy 1 in strain PeCan18B and flanking sequence were determined by sequencing adapter PCR products. This left end was located in a gene with low homology to HP0456 of strain 26695 and was absent from strain J99. The sequence of the right end was obtained directly from genomic DNA, but flanking sequence was not determined because this strain contained two ISHp608 copies. The right end of ISHp608 copy 2 from strain PeCan18B and flanking sequence were determined by adapter PCR and sequencing. Attempts to obtain the leftmost ∼30 bp of ISHp608 copy 2 by PCR with jhp0942-specific primers and by sequencing directly on genomic DNA were not successful. The left and right ends of ISHp608 in strain PeCan2A and flanking sequences were determined by adapter PCR and by direct chromosomal sequencing using a primer matching the inferred flanking sequence, respectively. The left end of ISHp608 in strain PeCan16A and the flanking sequence were determined by adapter PCR; this left end was located in an IS605 orfA sequence. The right end was identified by genomic DNA sequencing, but the flanking sequence was not determined because this strain contained two ISHp608 copies and because no product was obtained after PCR using primers specific for ISHp608 and for the anticipated flanking IS605 sequence. Both ends and flanking sequences of the single copy of ISHp608 in strain India49 were determined directly from genomic DNA. The element was found inserted into the gene corresponding to jhp0974 of reference strain J99 (4). Both ends and flanking sequences of the single copy of ISHp608 in strain India77 were determined directly from genomic DNA and were found inserted into an intergenic (noncoding) region (between genes jhp1113 and jhp1114 in J99 and HP1188 and HP1189 in 26695).

FIG.3.

DNA sequence diversity of ISHp608 elements. (A) Maximum-likelihood phylogenetic tree generated using DNA sequences of type 1 and type 2 elements. DNA sequence divergence between types was about 11%; divergence within a given type was about 1 to 2%. The four ISHp608s that seemed to have resulted from recombination between elements of different types were not included because this tree analysis assumes divergence by mutation alone, without recombination between divergent lineages. (B) Recombinant ISHp608 elements identified by DNA sequencing. Areas of type 1, type 2, and type 3 sequence are depicted as white, grey, and black boxes, respectively. Strain PeCan4A contains just one copy of ISHp608, which was inferred to be recombinant, based on the following DNA sequence matches: bp 1 to 114, 93% identity with type 2 but 80% with type 1; bp 123 to 715, 99% identity with type 1 but 93% with type 2; bp 732 to 1832, 98% identity with type 2 but 87% with type 1. Strain PeCan18B contained two copies of ISHp608, designated PeCan18B-1 and PeCan18B-2 (for which we do not have the leftmost approximately 34 bp of sequence, as detailed in the Fig. 2 legend). The only difference detected between the two copies was in the leftmost 160 bp, in which copy 1 matched type 1, and the available 130 bp of copy 2 matched type 2 (97% identity with type 2 but 78% with type 1). In other regions the two copies seemed to be identical, although a mosaic of type 1 and type 2 sequences. DNA sequence matches for PeCan18B-2 were as follows: bp 1 to 160, 97% type 1 and 84% type 2; bp 160 to 220, 97% type 2 and 89% type 1; bp 220 to 680, 99% type 1 and 94% type 2; bp 680 to 1270, 97% type 2 and 89% type 1; bp 1270 to 1310, 100% type 1 and 88% type 2; bp 1310 to 1630, 97% type 2 and 87% type 1; bp 1630 to 1750, 97% type 1 and 82% type 2; bp 1750 to 1833, 98% type 2 and 87% type 1. Most of the sequencing of these two copies was done by primer walking on chromosomal DNA, but with the right end of copy 1 also determined by the adapter PCR method (which showed that this copy was inserted into gene jhp0942). The nearly complete copy 2 left-end sequence was obtained from subtractive clone D7 (lacks 30 to 34 bp to the very end); the left end of copy 1 was from sequencing an adapter PCR product. The genomic DNA sequence profiles were carefully searched for mixed traces that would indicate sequence differences between the copies, and none was found except at the left end, as indicated in the figure. Strain India77 contained just one copy of ISHp608, which was inferred to be recombinant based on the following DNA sequence matches: bp 1 to 1021 and also bp 1445 to 1833, 97% identity with type 1 in each segment; bp 1029 to 1419, 75% identity with type 1 and 72% with type 2. This divergent 400 bp was interpreted to indicate the existence of a third type (type 3) of ISHp608 element.

FIG. 4.

Multiple alignments of amino acid sequences in region of putative DDE(K) motifs in OrfB and homologs. Asterisks identify residues of the DDE(K) motif that, by extrapolation from other systems, could be critical features of an active site (namely, coordination of divalent cations and nucleophilic attack leading to DNA cleavage early in transposition [14, 21]). The consensus sequence (Cons) was developed by multiple alignment in ClustalW in the NTI vector program. Underlining identifies residues also conserved in the consensus based on 26 sequences of OrfB homologs found in GenBank as of September 2001. GenBank accession nos. used in developing this alignment are as follows: AC000108, IS605; U95957, IS606; AF189015, IS607; AF357223, ISHp608 type 1; AF411944, ISHp608 type 2; C64895, E. coli B1432; AAF98319, GipA. The other OrfB homolog accession nos. used here are AC000108, E64644, U95957, A33489, AAC97568, CAC03683, G75376, CAB41498, BAA15060, C64895, CAA60219, F70884, E70811, AAB12365, AAF05601, AF357223, JC4292, AAK40078, AAA26505, AAF98319, T36649, AAA83564, S74909, F75401, C72305, and A82794.

FIG. 5.

Repeat sequences in the termini of ISHp608 elements. (A) Size and spacing of direct repeats of type 1 and type 2 element termini. Boxed sequences represent a well-conserved 22 (or 23)-bp direct repeat present in both ISHp608 ends. Horizontal lines identify shorter direct repeats: octanucleotide (5"AACGCCTT) and related penta- or hexanucleotides (5"AACGC and 5"AACGCC) in the left ends of type 1 and type 2 elements, respectively; and decamer (5"-GCTTTAGCTA) or dodecamer (5"-TAGCTTTAGCTA) in the right ends of type 1 and type 2 elements, respectively. TAG is the stop codon at the 3" end of orfB. ∗ identifies the site of insertion of the cam cassette in ISHp608 right end (insertions 4 and 5; see Fig. 1B). Lowercase letters represent flanking sequences. (B) Subterminal imperfect inverted repeats of ISHp608. Left and right ends of ISHp608 (same strand) are aligned to highlight inverted repeat sequence (capital letters).

FIG. 6.

ISHp608 insertions in F factor in E. coli. Termini and sites of ISHp608cam insertions in F factor DNA (GenBank accession no. NC002483). Left and right termini of ISHp608s are shown in uppercase and flanking DNAs are shown in lowercase letters. The products of transposition from donor plasmid 3 (A⁺B⁻) (eight sites of insertion) were as follows: 2-1, position 60704/5; 5-2, position 62725/6; 6-1, position 64675/6; 2-2, position 65422/3; 1-1, position 80949/50; 5-1, position 85934/5; 6-2, position 86796/7; and 1-2, position 99/100. The products of transposition from donor plasmid 4 (A⁺B⁺) (five sites of insertion) were: 18-1, position 65422/3; 18-2 and 23-1 each at (although independent), position 95172/3; 23-2, position 59288/9; and 24-1, position 70860/1.