Potential role of two Helicobacter pylori relaxases in DNA transfer?

Sir, The recent determination of the complete Helicobacter pylori genome sequence (Tomb et al., 1997, Nature388: 539–547) and functional studies on the CAG pathogenicity island (Censini et al., 1997, Proc Natl Acad Sci USA93: 14648–14653; Covacci et al., 1997, Trends Microbiol5: 205–208) are major contributions to our understanding of this important pathogen. Current investigations focus on the CAG genes coding for a cytotoxin-associated antigen (CagA, ORF 547) and potential virulence factors such as homologues of Agrobacterium VirB4 (ORF 544), VirB7 (lipoprotein CagT, ORF 532), VirB9 (ORF 528), VirB10 (ORF 527), VirB11 (ORF 525) and VirD4 (ORF 524) proteins. Three additional virb4 gene copies (ORFs 017, 441 and 459) as well as a gene encoding a vacuolating toxin (VacA, ORF 887) are located outside the CAG region. CagA, VacA and a few other proteins were found to be secreted and to enhance the inflammatory response in the gastric mucosa. The presence of vir genes and several GC-rich islands further suggests the existence of an adapted DNA-transfer apparatus for delivering virulence genes across bacterial boundaries. Evidence for conjugation-like DNA-transfer mechanisms between Helicobacter pylori strains has already been demonstrated in vitro (Kuipers et al., 1998, J Bacteriol180: 2901–2905), but genetic determinants remain unknown. An intriguing aspect is the sequence homology of the CAG-encoded proteins to the membrane pore-forming VirB proteins of the Agrobacterium tumour-inducing (Ti) plasmid for interkingdom export of transfer (T)-DNA from bacteria to plant cells. On the basis of this homology, the CAG island was recently suggested to code for an ancient secretion apparatus that is capable of exporting a variety of proteinaceous material, and possibly also nucleoprotein particles, from Helicobacter pylori (Christie, 1997, Trends Microbiol5: 264–265). Each of the six proposed proteins in this system is also related to components forming the export machinery for the Bordetella pertussis toxin and broad-host-range DNA plasmids (Pansegrau and Lanka, 1996, Prog Nucleic Acid Res Mol Biol54: 197–251). Interestingly, members of the VirD4 protein family that have been suggested to link the T-DNA complex directly to the exporting membrane channel have to date been detected only in agrobacterial Ti-plasmid and conjugative plasmid DNA-transfer systems but not in protein transporters. Besides the VirD4 encoded in the CAG island, there is another putative VirD4 homologue (TraG, ORF 1006) present in the Helicobacter pylori sequence. Other VirD proteins representing essential components of a hypothetical DNA-transfer apparatus of Helicobacter pylori have not been identified so far. Likewise, a DNA-processing machinery including relaxase VirD2 as a second major component would be essential for the specific cleavage and binding of the T-DNA. By searching the Helicobacter pylori genomic sequence in the TIGR database for potential homologues of VirD2, we found two ORFs in a third GC-rich island downstream of the CAG region with significant homology to relaxases. Figure 1 shows the three conserved functional domains between Helicobacter ORFs 996 and 1004, relaxases from Agrobacterium Ti-plasmids and relaxases from mobilizable plasmids of Gram-negative and Gram-positive bacteria. These motifs are usually located at the N-terminus of the proteins and are arranged in a specific order (Pansegrau and Lanka, 1996, Prog Nucleic Acid Res Mol Biol54: 197–251). Two regions designated as motifs I and III are involved in catalysing the nicking–closing reaction at specific DNA target sequences. Motif I carries the tyrosine residues that covalently attach the relaxase in a transesterification reaction to the 5′ terminus of the cleaved single-stranded T-DNA. Motif II is thought to function as a DNA recognition domain. Motif III contains an HUH sequence pattern essential for relaxase activity and is therefore believed to activate the aromatic hydroxyl group of tyrosine in motif 2 by proton abstraction. . Alignment of three conserved functional motifs (I–III) in DNA relaxases encoded in mobilizable plasmids of Gram-negative (RP4; accession no. Q00191) and Gram-positive (pC221; accession no. A04494) bacteria, in Agrobacterium Ti-plasmids (pTiC58; accession no. M33673) and ORFs 996 (accession no. D64644) and 1004 (accession no. D64645) from Helicobacter pylori. Identical amino acids are shaded with dark grey and at least three identical or similar functional residues with grey. Motif boundaries are given by the corresponding number of amino acid positions. The detection of two ORFs with significant similarity to conjugative and T-DNA transfer systems strongly supports the notion of a DNA-transfer apparatus in Helicobacter pylori. The relaxases might act in concert with the VirD4 and VirB proteins of the proposed membrane pore described above. Whether the relaxases participate in normal bacterial conjugation processes and/or the transfer of DNA to infected target cells, e.g. gastric epithelial cells, remains to be elucidated in future studies.