Host factor titration by chromosomal R-loops as a mechanism for runaway plasmid replication in transcription termination-defective mutants of Escherichia coli

J Mol Biol. 2003 Sep 5;332(1):31-46. doi: 10.1016/s0022-2836(03)00753-8.

Abstract

Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 degrees C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 degrees C following overexpression of rnhA(+). Mutant combinations rnhA-nusG or recG-rho were synthetically lethal at 21 degrees C, with the former being suppressed by recG(+) overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 degrees C or 37 degrees C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA(+) or recG(+). Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom(+), and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and delta64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Chromosomes / genetics*
  • DNA Replication*
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Mutation
  • Nucleic Acid Conformation*
  • Peptide Elongation Factors / genetics
  • Peptide Elongation Factors / metabolism
  • Plasmids / genetics*
  • Plasmids / metabolism
  • Rho Factor / genetics
  • Rho Factor / metabolism
  • Ribonuclease H / genetics
  • Ribonuclease H / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic*

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • NusG protein, E coli
  • Peptide Elongation Factors
  • Rho Factor
  • Transcription Factors
  • RecG protein, E coli
  • Ribonuclease H
  • ribonuclease HI