Binding-protein-dependent arginine transport in Pasteurella haemolytica

Baylor College of Medicine, One Baylor Plaza, Department of Microbiology and Immunology, Houston, Texas 77030, USA 2 Author for correspondence: Sarah K. Highlander. Tel: +1 713 798 6311. Fax: +1 713 798 7375. e-mail sarahh@bcm.tmc.edu ABSTRACT A periplasmic arginine transport system that is a member of the ATP-dependent transport superfamily was identified in Pasteurella haemolytica. The gene encoding the periplasmic binding protein ( lapT ) was cloned and the protein overexpressed in Escherichia coli. LapT was purified to homogeneity using a modified osmotic shock procedure and anion-exchange column chromatography. Filter-binding assays established that LapT is an t-arginine-binding protein. Various amino acids were tested for their ability to inhibit L-arginine binding to LapT. When present in 100-fold excess, only L-arginine, D-arginine and citrulline competed with L-arginine for binding. Arginine transport in P. haemolytica whole cells was competitively inhibited by the same amino acids, suggesting that the LapT permease specifically transports L-arginine. The dissociation constant for the L-arginine-LapT complex was 170 nM and the stoichiometry of binding was approximately 0.8 mol L-arginine (mol LapT) -1 . A polyclonal antibody raised against the purified protein permitted detection of LapT in P. haemolytica periplasmic fractions. Keywords: Pasteurella haemolytica , arginine transport, periplasmic arginine-binding protein, permease, ABC transporters