Aerobic regulation of isocitrate dehydrogenase gene (icd) expression in Escherichia coli by the arcA and fnr gene products

Abstract

Isocitrate dehydrogenase, the icd gene product, has been studied extensively regarding the regulation of enzymatic activity and its relationship to the metabolic flux between the tricarboxylic acid cycle and the glyoxylate bypass. In this study, the transcriptional regulation of icd gene expression was monitored by using an icd-lacZ gene fusion and shown to vary over a 15-fold range in response to changes in oxygen and carbon availability. Anaerobic cell growth resulted in fivefold-lower icd-lacZ expression than during aerobic growth. This negative control is mediated by the arcA and fnr gene products. When different carbon compounds were used for cell growth, icd-lacZ expression varied threefold. The results of continuous cell culture studies indicated that this control may be due to variations in cell growth rate rather than to catabolite repression. DNase I footprinting at the icd promoter revealed a 42-bp ArcA-phosphate-protected region that overlaps the start site of icd transcription. Phosphorylation of ArcA considerably enhanced its binding to DNA, while ArcA-phosphate exhibited an apparent dissociation value of approximately 0.1 M. Based on these studies, ArcA appears to function as a classical repressor of transcription by binding at a site overlapping the icd promoter during anaerobic cell growth conditions.The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the conversion of isocitrate to ␣-ketoglutarate, with concomitant production of NADH and carbon dioxide (3, 15). In Escherichia coli, ICDH activity is regulated by posttranslational modification involving a phosphorylation of serine 113 within the homodimeric protein (16,17,34,36). This reaction is catalyzed by the AceK protein, isocitrate dehydrogenase kinase/phosphatase, to inactivate ICDH under conditions when the cell is grown on acetate or fatty acids. AceK also serves as a protein phosphatase that restores ICDH activity under alternative cell growth conditions when glucose or its saccharide precursors are present. The modulation of ICDH enzyme activity in the cell aids in maintaining the optimal amounts of TCA cycle intermediates, since this enzyme is at the branch point for carbon flow to the glyoxylate bypass pathway (3,14). In the competing reactions, isocitrate lyase converts part of the isocitrate pool to glyoxylate and succinate, while malate synthase then combines glyoxylate with acetyl coenzyme A to form malate. Thus, when E. coli is grown on acetate or its direct precursors, the glyoxylate bypass reactions makes it possible for the cell to generate four-carbon compounds needed for biosynthetic reactions while also balancing its needs for energy via TCA cycle-derived NADH and FADH.Little is known about the control of isocitrate dehydrogenase (icd) gene expression in E. coli under different cell growth conditions. Early studies by Gray et al. demonstrated that ICDH enzyme activity varied over a 10-fold range depending on the availability of oxygen and the composition of the cell growth mediu...

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

“…Similar observations were made for several other TCA cycle genes, including gltA, mdh, fumA and icd Woods and Guest, 1994;Park and Gunsalus, 1995;Park et al, 1995b;Chao et al, 1997), as well as for some respiratory pathway genes, frdABCD, dmsABC and narGHJI (Jones and Gunsalus, 1987;Li and DeMoss, 1988;Cotter and Gunsalus, 1989;reviewed in Gunsalus and Park, 1994). The molecular basis for this ArcA-and Fnr-independent control is not well understood, but it may be related to minor effects of DNA supercoiling (Axley and Stadtman, 1988).…”

Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

“…3B), as shown previously (16,60,63). Although expression of genes encoding TCA cycle enzymes was shown to be increased 6-to 70-fold in ArcA mutants during anaerobic growth (9,(44)(45)(46)(47)(48) and in vitro TCA cycle enzyme activities were indeed increased in the ArcA mutant (Table 4), the in vivo TCA flux could not be higher in the mutant because the concomitantly generated NADH could not be reoxidized. In the presence of exogenous electron acceptors, however, this was feasible.…”

“…Generally, the Arc (anoxic redox control) two-component system is considered to be a global transcriptional regulator in response to the redox conditions and in particular to deprivation of oxygen under microaerobic conditions (2,22,28,37). Although the expression of the TCA cycle genes gltA, icdA, sdhCDAB, fumA, and mdh was known to double in fully aerated ArcA mutants (9,(45)(46)(47)(48), previous flux (2) and phosphorylation data for the sensor kinase ArcB (22) suggested that ArcA regulation did not affect aerobic glucose catabolism. While ArcA is clearly active under fully anaerobic conditions (36), Arc-dependent regulation can modulate anaerobic TCA cycle fluxes only in the presence of electron acceptors that reoxidze the concomitantly generated reducing equivalents.…”

Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

“…We used the isocitrate dehydrogenase promoter (P icd ) to control the expression of glpK. This promoter would express glpK strongly during the aerobic preculture stages and then reduce glpK expression during the microaerobic fermentation through the arcA and fnr gene products (21). The use of these promoters gave us the expression profiles that we wanted without the need for inducers.…”