PLASMID PKM101 ENCODES 2 NONHOMOLOGOUS ANTIRESTRICTION PROTEINS (ARDAAND ARDB) WHOSE EXPRESSION IS CONTROLLED BY HOMOLOGOUS REGULATORY SEQUENCES

The IncN plasmid pKM101 (a derivative of R46) encodes the antirestrict ion protein ArdB (alleviation of restriction of DNA) in addition to an other antirestriction protein, ArdA, described previously. The relevan t gene, ardB, was located in the leading region of pKM101, about 7 kb from oriT. The nucleotide sequence of ardB was determined, and an appr opriate polypeptide was identified in maxicells of Escherichia coli. L ike ArdA, ArdB efficiently inhibits restriction by members of the thre e known families of type I systems of E. coli and only slightly affect s the type II enzyme, EcoRI. However, in contrast to ArdA, ArdB is ine ffective against the modification activity of the type I (EcoK) system . Comparison of deduced amino acid sequences of ArdA and ArdB revealed only one small region of similarity (nine residues), suggesting that this region may be somehow involved in the interaction with the type I restriction systems. We also found that the expression of both ardA a nd ardB genes is controlled jointly by two pKM101-encoded proteins, Ar dK and ArdR, with molecular weights of about 15,000 and 20,000, respec tively. The finding that the sequences immediately upstream of ardA an d ardB share about 94% identity over 218 bp suggests that their expres sion may be controlled by ArdK and ArdR at the transcriptional level. Deletion studies and promoter probe analysis of these sequences reveal ed the regions responsible for the action of ArdK and ArdR as regulato ry proteins. We propose that both types of antirestriction proteins ma y play a pivotal role in overcoming the host restriction barrier by se lf-transmissible broad-host-range plasmids. It seems likely that the a rdKR-dependent regulatory system serves in this case as a genetic swit ch that controls the expression of plasmid-encoded antirestriction fun ctions during mating.