Abstract
Four different type IV secretion systems are variously represented in the genomes of different Helicobacter pylori strains. Two of these, encoded by tfs3 and tfs4 gene clusters are contained within self-transmissible genomic islands. Although chromosomal excision of tfs4 circular intermediates is reported to be dependent upon the function of a tfs4-encoded XerD tyrosine-like recombinase, other factors required for transfer to a recipient cell have not been demonstrated. Here, we characterize the functional activity of a putative tfs4-encoded VirD2-like relaxase protein. Tfs4 VirD2 was purified as a fusion to maltose-binding protein and demonstrated to bind and nick both supercoiled duplex DNA and oligonucleotides in vitro in a manner dependent upon the presence of Mg(2+) but independently of any auxiliary proteins. Unusually, concentration-dependent nicking of duplex DNA appeared to require only transient protein-DNA interaction. Although phylogenetically distinct from established relaxase families, site-specific cleavage of oligonucleotides by Tfs4 VirD2 required the nick region sequence 5'-ATCCTG-3' common to transfer origins (oriT) recognized by MOBP conjugative relaxases. Cleavage resulted in covalent attachment of MBP-VirD2 to the 5'-cleaved end, consistent with conventional relaxase activity. Identification of an oriT-like sequence upstream of tfs4 virD2 and demonstration of VirD2 protein-protein interaction with a putative VirC1 relaxosome component indicate that transfer initiation of the tfs4 genomic island is analogous to mechanisms underlying mobilization of other integrated mobile elements, such as integrating conjugative elements, requiring site-specific targeting of relaxase activity to a cognate oriT sequence.
Highlights
The complement of factors involved in mobilization of the Helicobacter pylori disease-associated tfs4 genomic island are presently unknown
Identification of an oriT-like sequence upstream of tfs4 virD2 and demonstration of VirD2 proteinprotein interaction with a putative VirC1 relaxosome component indicate that transfer initiation of the tfs4 genomic island is analogous to mechanisms underlying mobilization of other integrated mobile elements, such as integrating conjugative elements, requiring site-specific targeting of relaxase activity to a cognate oriT sequence
Sequence Analysis of tfs4 oriT nick sequence (Tfs4) VirD2—Two VirD2-like proteins can be identified within the genomes of some sequenced H. pylori strains based on sequence similarity in the N-terminal region of the proteins to the conserved VirD2 relaxase domain, COG3843 in the conserved domain database (E value ϭ 1.19eϪ65)
Summary
The complement of factors involved in mobilization of the Helicobacter pylori disease-associated tfs genomic island are presently unknown. In addition to XerD and the T4SS structural vir gene complement, the tfs element encodes a putative VirD2-like relaxase, which we considered might function to initiate transfer of XerD-excised tfs intermediates. To address this possibility, we studied the biochemical properties of Tfs VirD2, demonstrating it to have a distinctive in vitro site- and strandspecific nicking activity consistent with conjugative relaxase function. We identified a putative tfs oriT region within tfs and demonstrate interaction of Tfs VirD2 with a putative VirC1-like relaxosome protein These studies suggest that the tfs PZ cluster encodes a complete complement of proteins enabling self-transmission via a conjugative mechanism analogous to other self-transmissible mobile genetic elements
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