Under glucose

limiting conditions, the wild type isocitrate lyase activity is enhanced 10 times compared to batch conditions, which is in accordance with previous proteome analysis of glucose limited cultures [37, 38] and enzyme activity levels [22, 38] under similar growth conditions. This is presumably due to different cAMP levels under glucose abundant and limiting conditions, since cAMP binding to Crp is necessary for regulatory activity of Crp. Under high glucose levels, cAMP concentrations are low and the cAMP-Crp complex cannot be formed. Consequently, activation of transcription of glyoxylate pathway genes by Crp cannot occur. If crp is deleted from the genome (i.e. in a Δcrp strain), no major differences in transcript levels of aceA or find more aceB between a culture grown under high and low glucose levels should be noticed, which was confirmed by transcriptome analysis [39].

Furthermore if Crp represses transcription of glyoxylate genes under high glucose levels as alleged in a few studies [25, 39], a difference in aceA and aceB transcript levels should be noticed between the wild type and the crp knockout strain under high glucose concentrations, which was not observed [39]. Under glucose limiting Saracatinib clinical trial conditions however, cAMP levels rise and the cAMP-Crp complex is properly formed, enabling the functioning of the regulator. Now Crp binds the DNA, competes with the binding of the repressor IclR and hereby activates transcription. If under these low glucose concentrations Crp is absent Wilson disease protein (i.e. in a Δcrp strain), the activities of the enzymes

involved in the glyoxylate shunt should drop, since IclR can now fully repress aceBAK transcription. This was confirmed by Nanchen et al. who studied the behavior of a Δcrp strain under glucose limitation [23]. However, the transcription of glyxoylate genes is the result of the regulatory activity of multiple regulators and not only Crp. If the repressors IclR and ArcA are inactive, i.e. in the ΔiclR and the ΔarcA strain, isocitrate lyase levels are increased compared to the wild type (see Table 2). The malate synthase activity in E. coli is the result of the activity of two isoenzymes, malate synthase A (gene: aceB) and G (gene: glcB) [40]. Both genes are members of different operons and the corresponding enzymes are members of different pathways, i.e. malate synthase A is the second enzyme of the glyoxylate pathway, Epoxomicin clinical trial whereas malate synthase G acts in the glycolate pathway. Figure 3B depicts the transcriptional regulation of the glc operons. The obtained malate synthase activities (see Table 2) are somewhat contra-intuitive.