In Escherichia coli, expression of the glyoxylate bypass operon appears to be controlled, in part, by the product of icIR+. Mutations (17,19). This bypass is essential for growth on acetate, since it yields C4 acids while avoiding the net loss of the acetate carbons as carbon dioxide in the Krebs cycle (Fig. 1). After induction, the flow of isocitrate through the glyoxylate bypass is regulated, in part, by the phosphorylation of isocitrate dehydrogenase (IDH), the Krebs cycle enzyme that competes with isocitrate lyase (8,12,24). During growth on acetate, ca. 70% of the IDH is maintained in the inactive phosphorylated form (22,23,32), reducing the activity of this enzyme and so forcing isocitrate through the bypass (24, 32). The phosphorylation and dephosphorylation of IDH are catalyzed by a single bifunctional enzyme, IDH kinase/phosphatase (20, 21).The metabolic and regulatory proteins of the glyoxylate bypass reside in the same operon, which maps at 91 min on the E. coli chromosome (4,5,23,26 When required, ampicillin (200 ,ug/ml) tetracycline (12.5 ,ug/ml), or kanamycin (50 ,ug/ml) was included in the growth media.Measurement of enzymatic activities. Cultures were grown at 37°C in a gyratory incubator to mid-log phase and were then harvested by centrifugation at 4,000 x g for 10 min. The cells were suspended in 10 ml of extraction buffer (25 mM N-morpholinepropanesulfonate [pH 7.5], 2 mM ,-mercaptoethanol, 1 mM EDTA) and then pelleted again by centrifugation. Cell pellets were stored at -80°C. For assay, the samples were thawed, suspended in 5 ml of extraction buffer, and disrupted by sonication. Cellular debris was removed by centrifuged at 22,000 x g for 20 min, and the samples were assayed for IDH phosphatase activity. Samples derived from cultures harboring plasmid were also assayed for ,-lactamase activity to ensure that the plasmid had not been lost during growth.The activity of IDH phosphatase was measured by monitoring the release of [32P]phosphate from [32P]phospho-IDH,
The control of the glyoxylate bypass operon (aceBAK) of Escherichia coli is mediated by two regulatory proteins, IclR and FadR. IclR is a repressor protein which has previously been shown to bind to a site which overlaps the aceBAK promoter.
The aceBAK operon was partially induced by a multicopy plasmid which carried the promoter region of the gene which encodes its repressor, iclR. Gel shift and DNase I analyses demonstrated that IclR binds to its own promoter. Disruption of iclR increased the expression of an iclR::lacZ operon fusion. Although aceBAK and iclR are both regulated by IclR, aceBAK expression responds to the carbon source, while expression of iclR does not.
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