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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.
Insights
The RP4 plasmid encodes a DNA primase essential for bacterial conjugation and plasmid replication. This primase initiates DNA synthesis during transfer and replication, ensuring plasmid stability and efficient transfer between bacterial cells.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Plasmids are extrachromosomal DNA elements crucial for bacterial adaptation.
- DNA primase activity is vital for DNA replication initiation.
- The role of plasmid-encoded primases in bacterial conjugation and replication is not fully understood.
Purpose of the Study:
- To investigate the DNA primase activity of plasmids from various incompatibility groups.
- To characterize the RP4 plasmid's primase, including its gene location, purification, and function.
- To determine the biological significance of the RP4 primase in plasmid maintenance and transfer.
Main Methods:
- Survey of incompatibility group plasmids for primase activity.
- Genetic analysis of RP4 using deletion and Tn7 mutagenesis to locate the primase gene (pri).
- Purification and biochemical characterization of the RP4 primase.
- Assay of primase activity in Escherichia coli dnaG mutant extracts.
- Immunological comparison with other DNA primases.
- Conjugation and mobilization experiments with RP4 pri mutants.
Main Results:
- RP4 (IncP) was identified as a plasmid with DNA primase activity.
- The RP4 primase structural gene (pri) was mapped to the 37- to 42-kilobase region.
- The RP4 primase was purified as a 118,000-molecular-weight monomer and shown to initiate DNA synthesis.
- RP4 pri mutants exhibited reduced conjugation efficiency and plasmid instability.
- The RP4 primase is immunologically distinct from E. coli and IncIalpha plasmid primases.
Conclusions:
- The RP4 plasmid encodes a functional DNA primase involved in complementary strand synthesis during conjugation.
- This primase is also necessary for efficient priming of discontinuous plasmid DNA replication.
- The RP4 primase plays a significant role in plasmid stability and inter-bacterial DNA transfer.