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Ability of IncP-9 plasmid pM3 to replicate in Escherichia coli is dependent on both rep and par functions

Yanina R Sevastsyanovich1, Marina A Titok, Renata Krasowiak

  • 1School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

Molecular Microbiology
|July 28, 2005
PubMed

Insights

IncP-9 plasmid replication in E. coli requires sufficient rep expression. The par operon, specifically ParB and its promoter, can restore replication by enhancing rep expression.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • IncP-9 plasmids are prevalent in Pseudomonas species.
  • These plasmids can transfer to other Gram-negative bacteria but often lack stable maintenance outside their native genus.

Purpose of the Study:

  • To investigate the replication mechanism of IncP-9 plasmids in heterologous hosts.
  • To identify factors contributing to the replication defect of IncP-9 plasmids in Escherichia coli.

Main Methods:

  • Functional analysis of the oriV-rep fragment from IncP-9 plasmid pM3 in Pseudomonas putida and E. coli.
  • Reporter gene assays, transcriptional start point mapping, and promoter mutation analysis.
  • Investigating the role of the par operon determinants (ParB and its promoter) in complementing replication deficiency.

Main Results:

  • The oriV-rep fragment replicated autonomously in P. putida but not in E. coli.
  • Replication in E. coli was restored by providing additional rep in trans, indicating insufficient rep expression.
  • Reporter assays and promoter analysis confirmed a promoter deficiency in E. coli.
  • The par operon components, ParB (supplied in trans) and its promoter (cis to oriV-rep), suppressed the replication deficiency.

Conclusions:

  • The IncP-9 plasmid replication defect in E. coli stems from insufficient rep gene expression due to promoter inefficiency.
  • The par operon, through ParB and its promoter, can enhance rep expression, thereby restoring plasmid replication in E. coli.
  • ParB likely facilitates rep expression by altering DNA conformation or interacting with RNA polymerase at the rep promoter.

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