Study of the impact of global RNA chaperones on bacterial evolution and adaptabilityMaster 2 internship

Global RNA-binding regulators, like Argonaute in eukaryotes or Hfq in bacteria, play an essential role in cell physiology by organising and empowering post-transcriptional networks. Their inactivation results in fitness loss and pleiotropic phenotypes, such as metabolic and morphological abnormalities, sensitivity to stress, growth retardation, up to lethality. But are such regulatory proteins essential in the long run? Could living organisms learn to live normally without them?
To tackle this question, we performed experimental evolution of E. coli bacteria deprived of two important RNA chaperones, Hfq and ProQ. These proteins interact with >1000 different transcripts, including regulatory small RNAs (sRNAs) and mRNAs. By directly binding these RNAs or by catalysing base-pairing interactions between sRNAs and mRNAs, Hfq and ProQ control the expression of ~25% of E. coli genes. The deletion of the hfq and proQ genes impacts on the bacterial growth, dramatically perturbs the bacterial transcriptome and proteome, and results in multiple stress sensitivities. By evolving such Hfq- and ProQ-deficient bacteria for 1000 generations, we could observe in real time how they adapted to this massive deregulation in their gene expression. Metagenomic sequencing of the evolved populations has identified numerous mutations that underlie the ability of these bacteria to regain fitness. However, the exact molecular mechanisms behind this adaptation remain unclear.
In this Master 2 project, we will leverage the metatranscriptomic analysis of the evolved populations to understand how exactly these Hfq- and ProQ-deficient bacteria have managed to compensate for their initial handicap, which pathways and metabolic processes they chose to rewire in order to restore their fitness. By correlating the detected mutations with transcriptomic and phenotypic changes (growth, stress sensitivity, performance of regulatory circuits), we will dissect the evolutionary dynamics of the evolving post-transcriptional networks and its adaptive logic. This training will employ microbiology and molecular biology techniques, gene expression measurements by northern blotting and/or RT-qPCR, RNA turnover measurements, fluorescent reporters, competitive fitness and mutation rate measurements.
Contact: Alexandre SMIRNOV (alexandresmirnov[at]unistra.fr)