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Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication
Abstract:
Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product.
Insights
Replication of mini-F plasmid DNA in Escherichia coli requires the sigma 32 factor for transcription of the repE gene. Sigma 32-mediated repE transcription is negatively controlled by its own product, the E protein.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- F factor and mini-F plasmid replication are crucial in bacterial genetics.
- Heat shock response in Escherichia coli involves the sigma 32 (encoded by rpoH) heat shock factor.
- The sigma 32 factor regulates heat shock gene expression.
Purpose of the Study:
- To investigate the role of the sigma 32 factor in mini-F plasmid replication.
- To elucidate the mechanism of repE gene transcription and its regulation.
Main Methods:
- Operon fusion assays to study gene transcription.
- Direct determination of repE mRNA levels.
- In vitro transcription assays using purified RNA polymerase.
Main Results:
- Mini-F plasmid replication is inhibited in rpoH mutants lacking sigma 32.
- repE mRNA levels increase transiently with temperature upshift in wild-type cells but decrease in rpoH mutants.
- In vitro transcription assays show sigma 32-dependent initiation from the repE promoter.
- E protein negatively regulates sigma 32-mediated repE transcription.
Conclusions:
- Sigma 32 is essential for the transcription of the mini-F repE gene.
- Plasmid replication initiator E protein is synthesized via sigma 32-mediated transcription.
- repE gene expression is autoregulated by its product, E protein, through negative feedback.