PMID:8349629
| Citation |
Fsihi, H, Kottwitz, B and Bremer, E (1993) Single amino acid substitutions affecting the substrate specificity of the Escherichia coli K-12 nucleoside-specific Tsx channel. J. Biol. Chem. 268:17495-503 |
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| Abstract |
The Tsx protein from the Escherichia coli outer membrane is a channel-forming protein containing a nucleoside-specific binding site. The antibiotic albicidin enters the cell via this substrate-specific channel. Because albicidin is toxic for E. coli at a very low external substrate concentration, the Tsx channel is likely to contain a binding site for this antibiotic. To identify residues involved in the Tsx substrate-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 albicidin-resistant tsx alleles, six point mutations and a 39-base pair duplication. The mutants with a duplication of residues 21-33 of Tsx or with single amino acid substitutions of residue Gly28 (to Arg) and Ser217 (to Arg) are completely deficient in nucleoside uptake at a low substrate concentration. Substitutions of Phe27 to Leu, Gly28 to Glu, Gly239 to Asp, and Gly240 to Asp result in a Tsx protein partially defective in nucleoside transport. These mutant proteins still permit nonspecific 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. |
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| Keywords |
Amino Acids/metabolism; Anti-Bacterial Agents/pharmacology; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Base Sequence; Coliphages/metabolism; DNA, Bacterial; Drug Resistance, Microbial/genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli/drug effects; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Kinetics; Molecular Sequence Data; Mutation; Nucleosides/genetics; Nucleosides/metabolism; Organic Chemicals; Receptors, Virus/metabolism; Xanthomonas |
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