Maltose transport in Aeromonas hydrophila: purification, biochemical characterization and partial protein sequence analysis of a periplasmic maltose-binding protein.

A clinical isolate of Aeromonas hydrophila was demonstrated to transport [14C]maltose with similar kinetics to enteric bacteria (Km: 0.3 microM; Vmax: 22 nmol min-1 per 10(9) cells). The uptake of [14C]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 [14C]maltose with high affinity (KD = 1.6 microM). 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.