SINGLE AMINO-ACID SUBSTITUTIONS AFFECTING THE SUBSTRATE-SPECIFICITY OF THE ESCHERICHIA-COLI K-12 NUCLEOSIDE-SPECIFIC TSX CHANNEL

The Tsx protein from the Escherichia coli outer membrane is a channel- forming protein containing a nucleoside-specific binding site. The ant ibiotic albicidin enters the cell via this substrate-specific channel. Because albicidin is toxic for E. coli at a very low external substra te concentration, the Tsx channel is likely to contain a binding site for this antibiotic. To identify residues involved in the Tsx substrat e-specific channel activity, we devised a selection scheme to isolate albicidin-resistant tsx mutants synthesizing Tsx proteins with defects in their nucleoside uptake function. We recovered seven distinct albi cidin-resistant tsx alleles, six point mutations and a 39-base pair du plication. The mutants with a duplication of residues 21-33 of Tsx or with single amino acid substitutions of residue Gly28 (to Arg) and Ser 217 (to Arg) are completely deficient in nucleoside uptake at a low su bstrate concentration. Substitutions of Phe27 to Leu, Gly28 to Glu, Gl y239 to Asp, and Gly240 to Asp result in a Tsx protein partially defec tive in nucleoside transport. These mutant proteins still permit nonsp ecific diffusion of serine indicating that the mutations do not result in a block of the Tsx channel. Our results are discussed in terms of a model for the topological organization of the Tsx protein within the outer membrane of E. coli.