Evasion of restriction systems by plasmid ColIb-P9 during bacterial conjugation.
thesisposted on 19.11.2015, 08:53 authored by Timothy Damian. Read
Conjugative transmission of enterobacterial plasmid ColIb was found to be relatively resistant to type I and type II restriction systems specified by the recipient cell. One process contributing to evasion of restriction barriers during conjugation apparently involves transfer of multiple copies of the plasmid. A more specialized anti-restriction mechanism requires the product of the ColIb ard (alleviation of restriction of DNA) gene. The Ard phenotype was discovered from the ability of ColIb to alleviate EcoK (type I) restriction of unmodified infecting the plasmid-harbouring cell. Ard activity alleviated restriction of phage by representatives of all three families of type I R-M system, but not by type II or type in systems. ColIb had no effect on the modification activity of EcoK but this activity was impaired by multicopy recombinant plasmids supporting overexpression of ard. A 498 nucleotide sequence corresponding to ard was identified. The Ard protein (apparent mass of c22 KDa) was visualised following in vitro transcription and translation of ard+ recombinant plasmids. Gene fusion studies indicated that only the C-terminal two-thirds of the Ard protein is necessary for the restriction alleviation phenotype. A ColIbdrd ard mutant was constructed by a gene replacement strategy. Properties of the plasmid showed that Ard protects ColIb from EcoK restriction following conjugative transfer and that protection requires expression of the gene on the immigrant plasmid. The ard gene is located in the leading region, which is the portion of ColIb transferred first in conjugation, and is near to the psiB and ssb loci. The latter two genes are known to be zygotically induced following transfer. However, studies with lacZ reporter probes showed that ard was not subject to zygotic induction; the gene is apparently expressed at very low levels before, and after conjugative transfer. It is proposed that ard facilitates the productive transfer of ColIb between its different natural enterobacterial hosts.