Abstract
The bacterial RelA-dependent stringent response exerts a strong influence over various processes. In this work, the impact of the relA gene mutation in Escherichia coli cells was evaluated by a quantitative proteomics analysis, employing stable-isotope labeling and high-resolution mass spectrometry. Chemostat cultures of E. coli W3110 and ΔrelA mutant strains were performed at two dilution rates (0.1 and 0.2 h−1) to assess the influence of the relA gene mutation in steady-state protein levels. A total of 121 proteins showed significant alterations in their abundance when comparing the proteome of mutant to wild-type cells. The relA gene mutation induced changes on key cellular processes, including the amino acids and nucleotide biosynthesis, the lipid metabolism, transport activities, transcription and translation processes, and responses to stress. Furthermore, some of those changes were more pronounced under specific growth conditions, as the most significant differences in protein ratios were observed at one of the dilution rates. An effect of the relA gene mutation in the acetate overflow was also observed, which confers interesting characteristics to this mutant strain that could be useful in the production of recombinant proteins. Overall, these results provide a valuable insight into the E. coli stringent response under defined steady-state conditions, suggesting that this stress response might influence multiple metabolic processes like the acetate overflow or the catabolite repression.
Highlights
The ribosome-bound RelA enzyme is the main bacterial synthetase for guanosine tetraphosphate, a molecule that is at the core of the stringent response
This response is triggered by the deprivation of intracellular amino acids and is characterized by rapid alterations in transcriptional activities, whereby genes required for amino acid biosynthesis are upregulated and genes associated with cell growth are repressed
The effects of relA gene mutations on the global gene expression have been tested, most studies have been focused on the response of Escherichia coli cells under growth transitions caused by glucose–lactose diauxie (Chang et al, 2002; Traxler et al, 2008), H2O2 treatment (Chang et al, 2002), or the addition of chemical analogs that mimics amino acid starvation, such as serine hydroxymate
Summary
The ribosome-bound RelA enzyme is the main bacterial synthetase for guanosine tetraphosphate (ppGpp), a molecule that is at the core of the stringent response This response is triggered by the deprivation of intracellular amino acids and is characterized by rapid alterations in transcriptional activities, whereby genes required for amino acid biosynthesis are upregulated and genes associated with cell growth are repressed. Proteome Analysis of E. coli ΔrelA Cells (Durfee et al, 2008) These transient shifts give only temporary and usually growth-dependent gene expression changes in response to environmental alterations, which bias the characterization of physiological states.
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