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

Abstract

3 H]Me-TCB uptake system was induced only by (GlcNAc) n , n ‫؍‬ 2 or 3. The apparent K m of transport was 50 -100 M, and effective inhibitors of uptake included (GlcNAc) n , n ‫؍‬ 2 or 3, cellobiose, and other PTS sugars, i.e. glucose and GlcNAc. Presumably the PTS sugars inhibit by competing for PTS components. Kinetic properties of the transport system are described.We have reported that wild type Escherichia coli and a mutant unable to transport GlcNAc can utilize the chitin disaccharide, N,NЈ-diacetylchitobiose or (GlcNAc) 2 1 as the sole source of carbon for growth (6).2 An E. coli transposon mutant was selected that was unable to grow on (GlcNAc) 2 but behaved normally on GlcNAc, and the mutant was used to clone the (GlcNAc) 2 catabolic operon. Sequence analysis of the genes in the operon compared with the sequence of the complete E. coli genome (7) showed that the (GlcNAc) 2 operon corresponded to the previously described "cryptic" cellobiose operon (8, 9) In the preliminary report (6) we demonstrated that the wild type allele of this operon encoded for (GlcNAc) 2 but not cellobiose utilization; the cel genes were therefore renamed and constitute the chb (N,NЈ-diacetylchito-biose) operon. In earlier work (10), a (GlcNAc) 2 permease was described in the Gram-negative, chitinolytic marine bacterium Vibrio furnissii. For these experiments, we employed a nonhydrolyzable (GlcNAc) 2 analogue, methyl ␤-N,NЈ- H]Me-TCB). In V. furnissii, the transport product was characterized as unmodified [3 H]Me-TCB, and its apparent K m for uptake was Ͻ1 M.In the present studies, we show that transport of (GlcNAc) 2 / [ 3 H]Me-TCB in E. coli is mediated via the phosphoenolpyruvate:glycose phosphotransferase system (PTS), with the sugar accumulated as its phosphorylated derivative. The phosphoryl group is linked to the C-6 position of the nonreducing GlcNAc. In E. coli, the apparent K m for [ 3 H]Me-TCB uptake is 50 -100 M. Thus, the chitin disaccharide, (GlcNAc) 2 , is transported by different mechanisms in these two closely related organisms. This is in sharp contrast to other sugars, as discussed below. EXPERIMENTAL PROCEDURESMaterials-Reagents for bacterial media were obtained from Difco Laboratories (Detroit, MI), J. T. Baker (Phillipsburg, NJ), and BBL Microbiology Systems (Cockeysville, MD). Molecular biology reagents were obtained from New England Biolabs (Beverly, MA), U.S. Biochemical Corp. (Cleveland, OH), and Stratagene (La Jolla, CA). Radioisotopes were purchased from Life Science Products. Whatman GF/F glass microfiber filters, and thin layer chromatography plates (Silica-Gel 60) were purchased from EM Science (Cherry Hill, NJ). (GlcNAc) 2 was prepared as described (11) or by modifications to be described elsewhere. Other buffers and reagents were of the highest purity commercially available. PTS proteins, such as homogeneous Enzyme I, HPr, and IIA Glc , were kind gifts from Drs. Norman Meadow, Regina Savtchenko, and Roshan Mattoo.Bacterial Strains-E. coli strain XL1-Blue MR (D(mcrA)183 ⌬(mcrCB-hsdSMR-mrr)173 endA...

Uptake of N , N ′-Diacetylchitobiose [(GlcNAc) 2 ] via the Phosphotransferase System Is Essential for Chitinase Production by Serratia marcescens 2170

“…Therefore, to study whether S. marcescens uses the PTS for (GlcNAc) 2 , a homolog of the E. coli chb operon was searched for in S. marcescens. It has been recently reported that in E. coli the uptake of (GlcNAc) 2 is mediated by the PTS, and the (GlcNAc) 2 -specific enzyme II permease is encoded in the chb operon (14,15). The operon consists of chbB (encodes the IIB domain of enzyme II permease), chbC (IIC domain), chbA (IIA domain), chbR (possibly a repressor for the chb operon), chbF (possibly phosphochitobiase), and chbG (function unknown).…”

Isolation and Characterization of IIAChb, a Soluble Protein of the Enzyme II Complex Required for the Transport/Phosphorylation ofN,N′-Diacetylchitobiose in Escherichia coli

“…Mg 2ϩ ( Fig. 8 and (1). The chb or chitobiose operon contains three genes originally thought to be required for the uptake and phosphorylation of cellobiose but now known to encode the three (GlcNAc) 2 -specific PTS transport proteins, IIA Chb , IIB Chb , and IIC Chb , respectively (7).…”

Characterization of an Exo-β- d -Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

“…␤-N-Acetylglucosaminidase (GlcNAcase; reaction 3) further hydrolyzes the dimer to form GlcNAc or releases GlcNAc from chitooligosaccharides (6). Some organisms degrade GlcNAc 2 to GlcNAc and GlcNAc-1-phosphate by GlcNAc 2 phosphorylase (reaction 4) (28) or convert the dimer to GlcNAc-6-phosphate-GlcNAc by a GlcNAc 2 phosphotransferase system (reaction 5) followed by degradation to GlcNAc and GlcNAc-6-phosphate by 6-phospho-␤-glucosaminidase (reaction 6) (15,16). Another pathway for chitin degradation is proposed to occur through deacetylation of chitin by chitin deacetylase (reaction 7).…”

“…Alternatively, chitin can be degraded by chitosanase and GlcNase after the initial deacetylation of chitin (Fig. 1A) (6,15,16,28). In T. kodakaraensis KOD1, as in many organisms, chitin is first degraded into GlcNAc 2 by ChiA Tk (32,33), but the pathway downstream from GlcNAc 2 was suggested to follow a distinct pathway, that is, deacetylation of GlcNAc 2 by an uncharacterized deacetylase and successive hydrolysis to GlcN by GlmA Tk (Fig.…”