Host factor titration by chromosomal R-loops as a mechanism for runaway plasmid replication in transcription termination-defective mutants of Escherichia coli.
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.
Harinarayanan
R
R
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Gowrishankar
J
J
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics*; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009690:Nucleic Acid Conformation; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D012234:Rho Factor / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C070291:NusG protein, E coli; D010445:Peptide Elongation Factors; D012234:Rho Factor; D014157:Transcription Factors; C071446:RecG protein, E coli; D016914:Ribonuclease H; C078856:ribonuclease HI