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Conserved extragenic DNA elements in Haemophilus influenzae

We have identified a DNA sequence (58-GTAGGGTGGGCTTYAGCCCACCA-38) that is present in multiple copies at extragenic locations in the chromosome of Haemophilus influenzae and associated with DNA duplications. The sequence was described as part of a repetitive extragenic palindromic (REP)-like unit in the pilus gene cluster of H. influenzae type b by van Ham et al. (1994, Mol Microbiol 13: 673–684). We detected the 23 bp element while sequencing the duplicated pilus gene complexes of Brazilian purpuric fever (BPF)-associated H. influenzae (Read et al., 1996, J Bacteriol 178: 6564– 6570), and noticed it at different locations in the otherwise highly similar clusters. A full and systematic BLAST search of the combined EMBL and GenBank databases (parameters: EXPECT = 10 and CUTOFF = 60) produced 20 matches to extragenic regions of the recently released H. influenzae Rd chromosome sequence (Fleischmann et al., 1995, Science 269: 496–512), several matches with H. influenzae pilus-gene clusters (which are absent from the Rd genome) and, intriguingly, similarities to extragenic portions of the frpC gene of Neisseria meningitidis (Thompson et al., 1993, Mol Microbiol 9: 85–96). Subsequent analysis of the regions around many of the elements identified by the BLAST search revealed additional sequences similar to the consensus. The elements are often found in pairs as inverted repeats (IRs) separated by AT-rich spacer regions of 12–26 bases (Table 1). In general, the intervening regions are not conserved, although clusters of inverted repeats have identical or near-identical spacers (i.e. the IRs at H. influenzae Rd genome co-ordinates 614950, 677373, 677527, 706237 and 706453; and the IRs at 234084 and 234711). We found two other individual conserved elements located extragenically at co-ordinates 156992 and 1306795 on the 1.8 Mb H. influenzae Rd genome and also a single element in an extragenic portion of the carA gene of Neisseria gonorrhoeae (Lawson et al., 1995, Microbiol 141: 1183–1191). The first three IRs in the H. influenzae Rd genome shown in Table 1 are interspersed among a set of three open reading frames (ORFs), two of which encode putative HsdS and HsdM subunits of a type I restriction system. Six sets of IRs are spread over 100 000 bp containing approx. 70 ORFs. There is a duplicated DNA sequence associated with some of these IRs. Portions of a 62 bp sequence between co-ordinates 706520–706581 are repeated almost perfectly at locations 615016– 615077, 677463–677491 and 706323–706368. Additionally, the same DNA region is duplicated in inverted orientation between 1632817 and 1632756. These DNA configurations are reminiscent of Haemophilus pilus-gene clusters, where both ourselves and van Ham et al. (1994, ibid.) have observed that duplications of DNA are also linked with single and tandem copies of the 23 bp element. For instance, the 6.5 kb haf1 and haf2 clusters of BPF-associated H. influenzae are flanked by direct repeats of sequences corresponding to H. influenzae Rd co-ordinates 1220363–94 and 1682304–62, respectively (Read et al., 1996, ibid.). Thus, we speculate that the pilus-gene clusters were inserted as a genetic unit into the chromosome of H. influenzae by a mechanism involving the 23 bp elements that generated 32–60 bp flanking duplications. The potential role of repetitive extragenic palindromic sequences in mRNA stabilization, differential gene expression and gene conversion have been reviewed elsewhere (Higgins et al., 1988, Gene 72: 3–14). Finally, it is notable that the only bacterial genus other than Haemophilus identified as containing these elements is Neisseria. With the recent discovery of IS1016 in both genera (Thompson et al., 1993, ibid.), the shared possession of these 23 bp sequences may represent horizontal gene exchange between these distantly related bacteria that share the ability for natural transformation and a common environment on human mucosal surfaces.