Influence of the RelA Activity on E. coli Metabolism by Metabolite Profiling of Glucose-Limited Chemostat Cultures

Abstract

Metabolite profiling of E. coli W3110 and the isogenic ΔrelA mutant cells was used to characterize the RelA-dependent stringent control of metabolism under different growth conditions. Metabolic profiles were obtained by gas chromatography–mass spectrometry (GC-MS) analysis and revealed significant differences between E. coli strains grown at different conditions. Major differences between the two strains were assessed in the levels of amino acids and fatty acids and their precursor metabolites, especially when growing at the lower dilution rates, demonstrating differences in their metabolic behavior. Despite the fatty acid biosynthesis being the most affected due to the lack of the RelA activity, other metabolic pathways involving succinate, lactate and threonine were also affected. Overall, metabolite profiles indicate that under nutrient-limiting conditions the RelA-dependent stringent response may be elicited and promotes key changes in the E. coli metabolism.

A Comparative Proteome Analysis of Escherichia coli ΔrelA Mutant Cells

“…Although it is not entirely understood how acetate overflow and the RelA-stringent control are connected, lower acetate levels in mutants suggest that the absence of the RelA-stringent control might reduce metabolic bottlenecks that ultimately lead to the accumulation of by-products, such as acetate. These results are also in agreement with metabolomics profiling analyses performed at the same conditions (Carneiro et al, 2012), where the accumulation of acetate and lactate, another metabolic by-product resulting from the metabolic overflow, was lower in mutant cultures. In this instance, we cannot state that alterations detected in protein ratios related to translation processes are caused by the gene mutation or a consequence of the reduced accumulation of acetate, but clearly the lack of the RelA activity induced profound alterations in many growth-related processes.…”

“…Previous work on E. coli chemostat cultures (Carneiro et al, 2012) has shown that the RelA activity is growth rate-dependent and differently affects the metabolism of slow- and fast-growing cultures. As such, it is relevant to understand the influence of the RelA activity in the coordination of metabolic processes under different growth conditions.…”

“…Samples from chemostat cultivations with E. coli strains W3110 and Δ rel A mutants at two dilution rates were tested. The dilution rates of 0.1 and 0.2 h −1 were chosen in this study because they present distinct metabolic phenotypes as previously verified in our laboratory (Carneiro et al, 2011, 2012). In particular, at a dilution rate of 0.2 h −1 , wild-type E. coli cells tend to accumulate acetate, which is characteristic of the overflow metabolism.…”

“…This metabolic behavior is very relevant in recombinant bioprocesses, where the accumulation of acetate hampers protein productivity, decreasing the cost-effectiveness of these processes. Recently, it was detected that strains devoid of RelA activity can decrease the accumulation of acetate (Carneiro et al, 2012), which indicates that recombinant bioprocesses can benefit from the use of Δ rel A mutants strains. In order to have a comprehensive overview of the RelA activity in E. coli cells, we analyzed the set of up and downregulated proteins under different steady-state conditions and explored the influence of the Δ rel A mutation in various cellular processes.…”

MBROLE 2.0—functional enrichment of chemical compounds

“…The first version of MBROLE contained functional (biological) data on compounds from several public databases, including the Kyoto Encyclopedia of Genes and Genomes (KEGG) ( 9 ), the Human Metabolome Database (HMDB) ( 10 ), the Chemical Entities of Biological Interest (ChEBI) database ( 11 ) and the PubChem database (pubchem.ncbi.nlm.nih.gov). MBROLE has been used to analyse metabolomics experiments carried out in organisms such as human ( 12 ), rat ( 13 ), Escherichia coli ( 14 ), Thermobifida fusca ( 15 ), Synechococcus elongates ( 16 ), Phoenix canariensis ( 17 ) or Cordyceps bassiana ( 18 ).…”