Role of plasmid ColIb-P9 DNA primase.

The sog locus of ColIb-P9 is known to encode a DNA primase that generates RNA primers for DNA synthesis on a variety of DNA templates and it promotes bacterial DNA replication in primase-defective (dnaG3) mutants of Escherichia coli K-12. The thesis reports that the physiological role of the enzyme is in conjugative metabolism of plasmid DNA. Derivatives of ColIb-P9drd-1 carrying defined sog mutations were constructed by vivo recombination. The mutant plasmids were maintained stably, showing that the primase is inessential for vegetative DNA replication, but they were deficient in transconjugant formation during bacterial mating. Amounts of conjugative DNA synthesis in matings involving mutants and complementing plasmids imply that the primase is required for efficient DNA synthesis on the plasmid strand transferred to the recipient cell, and that the enzyme may act in the donor cell to promote synthesis of a replacement strand. Recipient dnaG3 bacteria, treated with rifampicin to inhibit transcription, recovered some ability to synthesise chromosomal DNA during mating with donors of a Sog+ conjugative plasmid. Recovery was dependent on plasmid primase and an active DNA transfer system but it did not require transmission of a functional sog gene to the recipient cell. It is argued that the recovery reflects conjugative transfer of plasmid primase, that the transferred enzyme normally acts to initiate DNA synthesis on the plasmid strand transmitted from the donor cell, and that sog primase is selectively transferred during conjugation, possibly in association with plasmid DNA. Isolation of recombinant plasmids carrying the origin of transfer or an entry exclusion gene(s) of ColIb-P9drd-l is described.