Abstract
The ability to clear transcription complexes from DNA is especially important after DNA damage that produces replication stress. Using the bacterium E. coli, we show here that mutations in RNA polymerase that reduce termination, inhibitors of Rho-dependent termination, and inversion of a highly transcribed ribosomal RNA operon both enhance sensitivity to the quinolone ciprofloxacin (CPX); and identify two transcription factors, SspA and RapA, that impact these effects in opposite ways. We demonstrate that the rapA promoter is induced by CPX, independent of the LexA/RecA SOS response but is dependent on DnaA. Previous work has shown that RapA is expressed highest in rapidly growing cells whereas SspA levels respond to starvation. The factors have opposing effects on tolerance to chronic exposure to CPX, with RapA promoting cell growth and SspA inhibiting it. Functional SspA is also required for the CPX toxicity of the rRNA operon inversion; in sspAΔ mutants it has no negative consequence. In otherwise wild-type cells, loss of RapA has little effect except in strains lacking RNase HI, the enzyme that removes RNA/DNA hybrids from DNA. However, in cells lacking SspA, RapA strongly promotes survival, suggesting that SspA must block positive effects of RapA on tolerance. The RapA requirement for CPX tolerance is not relieved by RNase HI overexpression and therefore RapA must be not be merely preventing R-loop formation. RapA also in some way promotes the use of RNA loops to initiate DNA replication in the absence of DnaA. We propose that SspA stabilizes stalled or post-termination RNAP/DNA complexes and that the presence of SspA prevents RapA release of these complexes.