Regulation of the glyoxylate bypass operon: cloning and characterization of iclR

Abstract

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,

Role of Glyoxylate Shunt in Oxidative Stress Response

“…In P. aeruginosa, aceA (PA2634) and glcB (PA0482, malate synthase) are present at different genomic locations (18), and our global genomic analysis demonstrated that this is also the case in many other species of bacteria. In E. coli, IclR negatively regulates the aceBAK operon and appears to be directly regulated by glyoxylate and pyruvate, which have been shown to bind to the C-terminal domain of IclR (39,40). IclR is also indirectly regulated by FadR, a fatty acid biosynthesis regulator (41).…”

Preparation and properties of pure, full‐length Ic1R protein of escherichia coli. Use of time‐of‐flight mass spectrometry to investigate the problems encountered

“…We identified a clone containing this Clal fragment, designated XGLY 1, by probing an E. coli X genomic library with two hybridization probes, generated by amplification of portions of the aceB and iclR regions. Primers were designed by reference to the known sequences of these genes (Byrne et al, 1988;Sunnarborg et al, 1990), and the sequences were amplified using Taq DNA polymerase according to Saiki et al (1988). The smaller of the two ClaI-Hind I l l fragments derived from the hGLYl clone, which contains all of the coding region for the IclR protein, including its natural promoter, was subcloned into pBR322, giving plasmid pCHicl.…”

“…et al., 1988;Rieul et al, 1988), along with the nearby structural gene for an apparent repressor protein, iclR (Sunnarborg et al, 1990;Nkgre et al, 1991). It has been demonstrated that IclR protein expressed from the cloned iclR gene interacts specifically with a small operator-like region of DNA in the promoter region of the aceBAK operon (Cortay et al, 1991;Nkgre et al, 1991Nkgre et al, , 1992.…”

Identification and Characterization ofglxR, a Gene Involved in Regulation of Glyoxylate Bypass inCorynebacterium glutamicum

“…Thus, expression of the glyoxylate bypass enzymes is regulated transcriptionally by the available carbon sources (43), but the mechanism by which this is achieved is not known, although Wendisch et al suggested the involvement of acetyl-coenzyme A as a signaling molecule (43). In Escherichia coli, the IclR repressor controls the regulation of the aceBAK operon (6,38) and hence bypasses expression. The expression of iclR is regulated by FadR, which is known to regulate the expression of genes involved in fatty acid metabolism.…”