MALTOSE TRANSPORT IN AEROMONAS-HYDROPHILA - PURIFICATION, BIOCHEMICAL-CHARACTERIZATION AND PARTIAL PROTEIN-SEQUENCE ANALYSIS OF A PERIPLASMIC MALTOSE-BINDING PROTEIN

Abstract

A clinical isolate of Aeromonas hydrophila was demonstrated to transport [C-14]maltose with similar kinetics to enteric bacteria (K-m: 0.3 mu M; V-max: 22 nmol min(-1) per 10(9) cells). The uptake of [C-14]maltose was completely inhibited in the presence of unlabelled maltose or maltodextrins, whereas other mono- and disaccharides, such as glucose, galactose, sucrose, lactose or melibiose, had no effect. A protein with an apparent molecular mass of 39 kDa (maltose-binding protein; MBP) was identified in osmotic-shock fluid of maltose-grown cells by SDS-gel electrophoresis, and was purified to homogeneity by either amylose affinity chromatography or ion-exchange chromatography. Equilibrium dialysis experiments revealed the ability of the purified protein to bind [C-14]maltose with high affinity (K-D = 1.6 mu M). Unlabelled maltose and maltodextrins competed for the binding site. In a reconstitution experiment, A. hydrophila MBP poorly restored the transport activity of a binding-protein-deficient Escherichia coli (Delta malE) mutant. N-terminal sequence analyses of the purified native protein and of peptides generated by cleavage with CNBr and subsequently separated by HPLC revealed about 56% identical amino acid residues, as compared to enterobacterial MBPs. We conclude that maltose is transported into A. hydrophila via a binding-protein-dependent transport system.