Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement

Abstract

Regulation of the main metabolic pathways of Escherichia coli K12 was investigated based on 2-dimensional electrophoresis (2DE) and the measurement of enzyme activities. The cells were grown aerobically in different carbon sources, such as glucose, acetate, gluconate or glycerol. Microaerobic cultivation was also conducted with glucose as a carbon source. Fifty-two proteins could be identified based on 2DE, and 26 enzyme activities from the main metabolic pathways-including glycolysis, pentose phosphate pathway, TCA cycle, Entner-Doudoroff pathway and fermentative pathway-were assayed. These enzyme activities, together with global and quantitative protein expression, gave us a clear picture of metabolic regulation. The results show that, compared with the control experiment with glucose as a carbon source under aerobic conditions, glycolytic enzymes were slightly up-regulated (<2-fold), TCA cycle enzymes were significantly down-regulated (2- to 10-fold), and fermentative enzymes such as pfl and adhE were highly up-regulated (>10-fold) under microaerobic conditions in glucose medium. When acetate was used as a carbon source, pfkA, pykF, ppc and zwf were down-regulated, while fbp, pckA, ppsA and mez were significantly up-regulated. Glyoxylate enzymes such as aceA and aceB were strongly up-regulated (>10-fold) and TCA-cycle-related enzymes were also up-regulated to some extent. With gluconate as a carbon source, edd, eda, fbp and TCA cycle enzymes were up-regulated. With glycerol as a carbon source, fbp and TCA cycle enzymes were up-regulated, while ackA was significantly down-regulated. Protein abundance obtained by 2DE correlated well with enzyme activity, with a few exceptions (e.g., isocitrate dehydrogenase), during aerobic growth on acetate.