IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions

Abstract

The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.

Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences

“…All three acidic antirestriction proteins are encoded by genes located in leading regions of plasmid and phage genomes, which enter the host cell early during conjugation or infection. Some aspects of regulation of plasmid-encoded Ard functions have been studied in detail (6,32). In this study, we show that plasmid pKM101 encodes a second antirestriction protein, designated ArdB, in addition to ArdA.…”

“…WA2377 (2), HB129 (33), and BZ216 (obtained from T. A. Bickle) are the rA+ mA+, rB+ mB+, and rD+ mD+ derivatives of E. coli K-12, respectively. BA509 and BA2357 are the rRI+ mRI + and rR124+ mR124+ derivatives of AB1157, carrying the EcoRI plasmid (7) and plasmid R124 (6,22), respectively. BA532 is the rpl+ mpl+ derivative of AB1157 lysogenic for P1CMts (7).…”

“…Plasmids pAB1 and pAB7 were described previously (6). pAB36, pAB37, and pAB38 were made by subcloning the appropriate XhoI-BamHI fragments of pKM101::TnSQf13O, pKM101::TnSQf246, and pKM101::TnSQ64 to pBluescriptII KS DNA cut with XhoI and BamHI.…”

“…Recently, we have identified two homologous antirestriction proteins, Ard and ArdA, encoded by unrelated plasmids ColIb-P9 and pKM101, respectively. Like the 0.3 protein of T7 phage, these proteins are very acidic and specifically inhibit both the restriction and the modification activities of type I systems (6,18). All three acidic antirestriction proteins are encoded by genes located in leading regions of plasmid and phage genomes, which enter the host cell early during conjugation or infection.…”

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids

“…ArdB is encoded by the leading region of pKM101 that is transferred early during conjugation and has been shown to possess an antirestriction activity directed primarily against type I restriction endonucleases (7). Like ArdB, the predicted KlcA polypeptide shows no significant similarity to other known proteins with antirestriction activity (ArdA of pKM101 [6], Ard of ColIb-P9 [11,46], and the product of the 0.3 gene of bacteriophage T7 [13]). The regulation of ardB is strikingly similar to the regulation of kicA.…”