Plasmid-mediated restriction evasion mechanisms
thesisposted on 15.12.2014, 10:38 by Nicola Jayne. Althorpe
Plasmid ColIb-P9 (Incll) encodes mechanisms which allow it to avoid destruction by type I and type II restriction enzymes during transfer by conjugation between strains of Escherichia coli. A genetic system was developed to analyse these mechanisms. The system relied on measuring Collb-mediated rescue of the restriction-sensitive plasmid R751 (IncPp) from destruction by EcoKI (type I) and EcoRI (type II). One Collb mechanism was known to involve a plasmid-encoded antirestriction gene known as ardA, the product of which is active against type I enzymes. Tests for alleviation of EcoKI restriction of R751, showed strong protection by a co-transferring Collb (Ard+) plasmid, slight protection when Collb was resident in the recipient and no effect when Collb was immobilised in the donor by removal of its nic site. Hence, expression of ardA is activated in the recipient cell following transfer no detectable transfer of the ArdA protein occurs from the donor to the recipient. The ardA gene is found in the leading region of Collb, which is defined as the first segment of the plasmid to enter the recipient cell during conjugation. Nucleotide sequencing of 11.7 kb of this region identified ten open reading frames. Furthermore, the region also contains three dispersed repeat sequences homologous to a novel single-stranded DNA promoter described by Masai and Arai (1997, Cell 89, 897-907). It is proposed that these secondary structures form in the transferring T-strand of Collb and function as promoters for transcription of genes encoded on the unique plasmid strand transferred during conjugation. Another mechanism, which acts independently of ardA, alleviates restriction of both type I and type II enzymes in the recipient in second or subsequent rounds of transfer. Two separate mechanisms appear to be operating since alleviation of type I restriction occurs in trans and is constitutive. In contrast, alleviation of type II restriction is by a czs-acting mechanism. The 'substrate saturation' hypothesis, whereby increasing amounts of transferred DNA saturates the restriction system (Read et al. 1992, Mol Microbiol 6, 1933-1941) is ruled out by data presented.