Journal of Biological Chemistry
Volume 288, Issue 37, 13 September 2013, Pages 26385-26396
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Microbiology
Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylori*

https://doi.org/10.1074/jbc.M113.496430Get rights and content
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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 Mg2+ 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.

Background: The complement of factors involved in mobilization of the Helicobacter pylori disease-associated tfs4 genomic island are presently unknown.

Results: tfs4 encodes a VirD2-like relaxase with distinctive DNA binding and nicking activity.

Conclusion: Tfs4 VirD2 probably initiates mobilization of tfs4 by specific interaction at a chromosomal transfer origin sequence.

Significance: Tfs4 VirD2-mediated mobilization of tfs4 may increase pathogenic potential of H. pylori strains.

Bacterial Conjugation
Bacterial Pathogenesis
DNA Enzymes
Helicobacter pylori
Protein-DNA Interaction
Protein Domains
Integrating Conjugative Element
Relaxase
Tfs4
VirD2

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The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) KF438085, KF438086, KF438087, and KF438088.

*

This work was performed in facilities supported by the Nottingham Digestive Diseases Centre National Institute for Health Research Biomedical Research Unit and supported by Medical Research Council Grant G0901104 (to R. M. D.).

This article contains supplemental Table 1.