Modulation of rpoS fitness by loss of cpdA activity during stationary-phase in Escherichia coli

Abstract

Experimental evolution of Escherichia coli in 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 of rpoS, referred to as rpoS819, a partially functional variant that confers growth advantage in stationary-phase (GASP). The 46 base duplication at the 3 end of rpoS819 produces a longer protein present at very low levels compared to wild type RpoS. A new rpoS variant rpoS92, carrying a re-duplication of the original duplication in rpoS819, arose during the first week of our evolution experiment. In rpoS92, an in-frame stop codon truncated RpoS819 creating a shorter RpoS92 whose levels are restored to that of wild type RpoS. Transcription profiling of rpoS92 indicated a shift in gene-expression to that of wild-type rpoS, reversing some of the expression trends of rpoS819. {Delta}3cpdA, 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 that rpoS92 confers GASP, whereas {Delta}3cpdA confers relatively modest GASP in comparison to the ancestral rpoS819. {Delta}3cpdA mediates epistatic repression of rpoS92 GASP. The original survivor carrying both rpoS92 and {Delta}3cpdA besides other mutations displays robust GASP, highlighting the role of these additional mutations in reversing the epistatic interaction between {Delta}3cpdA and rpoS92. In 10- and 20-day old spent media, there is a reduction in the competitive fitness of rpoS92, which is arrested by {Delta}3cpdA. 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.