Analytical Sedimentation of the IIAChb and IIBChb Proteins of the Escherichia coli N,N′-Diacetylchitobiose Phosphotransferase System

Abstract

The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates. The accompanying papers describe the proteins and product of the Escherichia coli N,N-diacetylchitobiose ((GlcNAc) 2 ) PTS-mediated permease. The phosphoenolpyruvate:glycose phosphotransferase system (PTS) 1 consists of two soluble general proteins, Enzyme I and HPr, required for the uptake of all PTS sugars. These proteins are coupled to substrate-specific permeases or transporters that are ultimately responsible for the concomitant uptake and phosphorylation of carbohydrate substrates. Phosphorylation proceeds sequentially, beginning with the autophosphorylation of Enzyme I by phosphoenolpyruvate. A frequent sequence is as follows: phospho-Enzyme I donates its phosphate to HPr, phospho-HPr to sugar-specific IIA proteins, phospho-IIA to IIB, and phospho-IIB in conjunction with the membrane receptor, IIC, phosphorylates and transports the substrate. The structure and oligomeric state(s) of both Enzyme I and HPr have been extensively studied (for reviews see Refs. 6 -8). Several sugar-specific PTS transporters have also been well characterized, including the Escherichia coli glucose and mannitol transporters. To date, however, there have been few reports on the oligomeric states of the sugar-specific proteins, particularly in their phosphorylated states. Furthermore, although phosphate is transferred from one protein to another as summarized above, there have been no reports describing the isolation of a transition state intermediate or stable complex between two reacting PTS proteins.In the accompanying papers (1-3), 2 we characterize the transport kinetics, product, and two of the sugar-specific transport proteins of the (GlcNAc) 2 (N,NЈ-diacetylchitobiose) or chb catabolic operon of E. coli. The phosphoryl transfer reaction sequence resulting in the uptake of (GlcNAc) 2 was studied, and the soluble proteins IIA Chb , IIB Chb , and an active site mutant (C10S), of IIB Chb were purified to apparent homogeneity. A phosphoryl group is transferred from phospho-HPr to IIA Chb and from phospho-IIA Chb to IIB Chb . Phospho-IIA Chb was found to be 5-10-fold more stable than homologous phospho-IIA proteins. This stability, as well as that of phospho-IIB Chb , enabled us to use analytical ultracentrifugation to determine the oligomeric states of IIA Chb and IIB Chb in both their unphosphorylated and phosphorylated forms and of an analogue of a potential transition state intermediate in the phosphotransfer reaction between the proteins. EXPERIMENTAL PROCEDURESMaterials-Buffers and reagents were of the highest purity available. The solvent used in all analytical ultracentrifuge experiments was 25 mM sodium phosphate buffer, pH 8, unless otherwise specified. The values of solvent density and viscosity were calculated from composition as described in Laue et al. (9) Chb complex were prepared as described in the accompanying reports (2, 3). Native gel ...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…70,2006 PHOSPHOTRANSFERASE SYSTEM-RELATED PHOSPHORYLATION 945 during gel filtration (786) and in crystals (224). Dimeric EIIA Chb and PϳEIIA Chb were observed when ultracentrifugation was carried out (398). EIIAB Man was found in the dimeric form both in the cytoplasm and linked to the membrane (216,826).…”

The Chitin Disaccharide,N,N′-Diacetylchitobiose, Is Catabolized byEscherichia coli and Is Transported/Phosphorylated by the Phosphoenolpyruvate:Glycose Phosphotransferase System

“…[ 3 , and Me-TCB-E. coli strain XL1-Blue was grown in minimal M9 media containing 0.5% lactate supplemented with (GlcNAc) 2 , (GlcNAc) 3 , or Me-TCB. Fig.…”

Chitin Catabolism in the Marine Bacterium Vibrio furnissii

“…To appropriately modify the plasmids, the V. furnissii methylase gene was cloned into E. coli strain S17-1, and plasmids were propagated in this strain prior to their use for transforming V. furnissii. 4 …”

“…The deduced amino acid sequence reveals that there are two potential start sites (methionine residues) separated by two amino acid residues. 2 ; lane E, (GlcNAc) 3 ; lane F, (GlcNAc) 4 ; lane G, (GlcNAc) 5 ; lane H, (GlcNAc) 6 ChiP Expression in E. coli-The primary sequence of the chitoporin gene determined as described above was used to subclone the chitoporin gene into the overexpression vector, pET21a under the control of the T7 promoter ("Experimental Procedures"). Outer membrane proteins were analyzed by SDS-PAGE using Method 1 (Fig.…”

“…At this wavelength, 1.0 optical density unit corresponds to 0.5 mg of cell protein/ml. V. furnissii strains were grown either in high salt LB (LMB, Luria broth supplemented with an additional 10 g/liter NaCl) or in minimal media containing HEPES (50 mM, pH 7.5), 50% artificial sea water (ASW), 0.1% NH 4 Cl, 0.001% K 2 HPO 4 , and 0.5% DL-lactate (lactate-ASW) (8). The minimal medium was supplemented with other carbon sources as indicated.…”