Giles E. M. Martin

Sugar—Cation Symport Systems in Bacteria

Publisher Summary This chapter describes sugar–cation symport systems in bacteria and presents the properties of the bacterial sugar–H + transport proteins, their substrate specificities, effects of certain inhibitors, their genes, and the amino acid sequences of the proteins. An individual transport system catalyzes the simultaneous translocation of protons with a sugar molecule, symport, or the experimentally indistinguishable antiport of hydroxyl ions. Energy derived from respiration or ATP hydrolysis is stored as the electrochemical gradient of protons and can, therefore, drive the accumulation of the nutrient against a concentration gradient. Sugar–H + symport proteins can also catalyze a facilitated diffusion of radioisotope-labeled sugar across the membrane, either into or out of the cells or vesicles. An important step in cloning and sequencing a transport protein of Escherichia coli ( E. coli ) is to establish the location of the corresponding gene on a chromosome. This can be achieved by first isolating a mutant impaired in the relevant transport activity. The structural and functional roles of individual amino acids may be evaluated by modification with reagents and in vivo and in vitro mutagenesis.

Dissection of discrete kinetic events in the binding of antibiotics and substrates to the galactose-H+ symport protein, GalP, of Escherichia coli.

GalP is the membrane protein responsible for H+-driven uptake of D-galactose intoEscherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35–55% of the total inner membrane protein ofE. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the interaction between the ligands and GalP. The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-1-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The ease of genetical manipulation of thegalP gene inE. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.