We should be aware that there are slight differences in the media CI-1033 molecular weight composition due to auxotrophic requirements (kanamycin and valine were added to the medium used to cultivate the mutant strain). Because we were concerned that the metabolic state of the two cultures would not be comparable if valine was also added to the wild-type culture (i.e., while the mutant strain would use valine essentially to balance the inefficiency to naturally synthesize valine for biosynthetic purposes, in the wild-type, it would be used as a carbon source, which would interfere with the

intracellular carbon distribution and, consequently, with the metabolic state of cells), we considered a Inhibitors,research,lifescience,medical stringent Inhibitors,research,lifescience,medical confidence level (99%) to reduce the influence of these environmental variations and metabolites in the biosynthetic pathway of valine which were not considered to be significant biomarkers. Metabolite profiles that presented statistically significant changes regarding factor A (i.e., bacterial strains) were further used to determine the degree of association between the metabolite profiles produced by the W3110 and ΔrelA E. coli cultures. We have applied a correlation analysis Inhibitors,research,lifescience,medical based on Pearson’s correlation coefficients (r) that measure the strength of the association between two conditions (e.g. bacterial strains), which can vary between 1 and −1 (r > 0 indicates a positive relationship, r = 0 indicates the

absence of a relationship and r < 0 indicates a negative relationship). In this work, metabolite profiles with r < 0.6 Inhibitors,research,lifescience,medical were considered to correspond to a weak relationship between the metabolic behavior of W3110 and ΔrelA E. coli cultures. Enrichment pathways analyses were performed using two bioinformatics tools: the Metabolite

Biological Role (MBRole) a web-server tool for carrying out over-representation analysis of biological and chemical annotations Inhibitors,research,lifescience,medical in metabolomics data [21]; and the Pathway Activity Profiling (PAPi), an algorithm that measures metabolic pathway activities from metabolite profiles at different experimental conditions [22]. 3. Results 3.1. Growth Parameters of E. coli Chemostat Cultures Chemostat cultures of the E. coli W3110 and the isogenic ΔrelA mutant were run at different dilution rates (0.05, 0.1 and 0.2 h−1) L-NAME HCl and the determined growth parameters are shown in Table 1. Table 1 Growth parameters of the W3110 and ΔrelA mutant E. coli strains in aerobic glucose-limited chemostat cultures. Overall, the biomass yields increased with the dilution rate, but the mutant strain exhibited a slightly higher biomass yield than the W3110 strain in the same conditions. In turn, the W3110 strain produced higher amounts of acetate than the mutant strain when growing at a higher dilution rate (0.2 h−1). Residual concentrations of glucose were also detected in the chemostat cultures, but only at higher dilution rates. 3.2.