Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance

Abstract

We surveyed plasmids representative of most incompatibility groups for their conferred deoxyribonucleic acid (DNA) primase activity. RP4 (IncP) was one of the few with such activity although, unlike the derepressed IncIalpha plasmids (which also specify a primase), it did not suppress the dnaG mutation. Using deletion and Tn7 derivatives of RP4, we located the presumed primase structural gene (pri) in the 37- to 42-kilobase region. Tn7 insertions in the adjacent Tra1 region also reduced or caused overproduction of primase. We purified the RP4 primase to a single polypeptide of molecular weight 118,000. It is an anisometric molecule and functions as a monomer, initiating complementary strand synthesis on phi X174 DNA in Escherichia coli dnaG cell extracts in the presence of ribonucleotide triphosphates and rifampin. It is immunologically unrelated to either the E. coli dnaG or the IncIalpha plasmid-specified DNA primases. RP4 pri mutants conjugated with a lower efficiency into some bacterial species, including Salmonella typhimurium. Back-transfer experiments showed that this effect was recipient specific. There was also a comparable reduction in mobilization efficiency of R300B by RP4 pri into such recipients. Loss of RP4 primase led to detectable plasmid instability. The RP4-specified primase therefore seems to serve two functions: the single DNA strand transferred during conjugation is primed by it in the recipient cell, and it appears to be necessary for the efficient priming of discontinuous plasmid DNA replication despite the presence of the chromosomal priming system.