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

Universitat Osna brack, Fach bereich BiologieKhemie, Abtei I ung Mikrobiologie, D-49069 A clinical isolate of Aeromonas hydrophila was demonstrated to transport [14C]maltose with similar kinetics to enteric bacteria (K,,,: 0.3 pM; VM: 22 nmol min-l per lo9 cells). The uptake of [14C]maltose was completely inhibited in the OsnabrUck,-FRG presence of unlabelled maltose or maltodextrins, whereas other monoand 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 pM). Unlabelled maltose and maltodextrins competed for the binding site. In a reconstitution experiment, A. hydrophila MBP poorly restored the transport activity of a binding-proteindeficient Escherichia coli (AmalE) 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 O/ O 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.