Antirestriction protein ard (Type C) encoded by IncW plasmid pSa has a high similarity to the "protein transport" domain of TraC1 primase of promiscuous plasmid RP4

The IncW plasmid pSa contains the gene ard encoding an antirestriction func tion that is specific for type I restriction and modification systems. The nucleotide sequence of ard was determined and an appropriate polypeptide of about 33 kDa was identified in Escherichia coli T7 expression system. Anal ysis of deduced amino acid sequence of Ard encoded by pSa revealed that thi s protein has no significant similarities with the known Ard proteins (ArdA and ArdB types) except the "antirestriction" motif (14 amino acid residues in length) conserved for all known Ard proteins. This finding suggests tha t pSa Ard may be classified as a new type of Ard proteins which we designat ed ArdC. The remarkable feature of ArdC is that it has a high degree of sim ilarity (about 38 % identity) to the N-terminal region of RP4 TraC1 primase which includes about 300 amino acid residues and seems to be essential for binding to the single-stranded DNA and TraC1 protein transport to the reci pient cells during the conjugal transfer of plasmid DNA. ArdC also binds to single-stranded DNA. Zn addition, this protein is able in vitro to protect the single-stranded but not double-stranded plasmid DNA against the activi ty of type II restriction endonuclease Hha1 that cleaves both single and do uble-stranded DNA. We suggest that like TraC1, ArdC would be transported as a result of their interaction with the single-stranded DNA of transferred plasmid strand during conjugative passage through the cell envelope to the recipient bacterium. Such properties of ArdC protein might be useful to pro tect immediately the incoming single-stranded DNA from the host endonucleas es. (C) 2000 Academic Press.