Modulation ofrpoSfitness by loss ofcpdAactivity during stationary-phase inEscherichia coli

Abstract

AbstractExperimental evolution ofEscherichia coliin one month long stationary-phase in lysogeny broth batch cultures repeatedly selected mutations in the genes for the stationary-phase sigma factor RpoS and the cAMP phosphodiesterase CpdA. The founder strain carried a previously identified allele ofrpoS, referred to asrpoS819,a partially functional variant that confers growth advantage in stationary-phase (GASP). The 46 base duplication at the 3’ end ofrpoS819produces a longer protein present at very low levels compared to wild type RpoS. A newrpoSvariantrpoS92, carrying a re-duplication of the original duplication inrpoS819,arose during the first week of our evolution experiment. InrpoS92, an in-frame stop codon truncated RpoS819 creating a shorter RpoS92 whose levels are restored to that of wild type RpoS. Transcription profiling ofrpoS92indicated a shift in gene-expression to that of wild-typerpoS, reversing some of the expression trends ofrpoS819. Δ3cpdA, carrying an in-frame three base deletion, had arisen late in our evolution experiment. It is a loss of function mutation, which elevates cAMP levels. Using mixed culture competition experiments, we demonstrate thatrpoS92confers GASP, whereas Δ3cpdAconfers relatively modest GASP in comparison to the ancestralrpoS819. Δ3cpdAmediates epistatic repression ofrpoS92GASP. The original survivor carrying both rpoS92 and Δ3cpdAbesides other mutations displays robust GASP, highlighting the role of these additional mutations in reversing the epistatic interaction between Δ3cpdAandrpoS92.In 10- and 20-day old spent media, there is a reduction in the competitive fitness ofrpoS92,which is arrested by Δ3cpdA.Thus the activity of RpoS fluctuates via genetic mutations in deep stationary phase, and additional mutations in CpdA helps modulate the competitive fitness of RpoS variants.