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 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.
Fsihi
H
H
Department of Biology, University of Konstanz, Germany.
Kottwitz
B
B
Bremer
E
E
D000596:Amino Acids / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003090:Coliphages / Q000378:metabolism; D004269:DNA, Bacterial; D004352:Drug Resistance, Microbial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009705:Nucleosides / Q000235:genetics / Q000378:metabolism*; D009930:Organic Chemicals; D011991:Receptors, Virus / Q000378:metabolism; D014974:Xanthomonas
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009705:Nucleosides; D009930:Organic Chemicals; D011991:Receptors, Virus; C066743:Tsx protein, E coli; C046156:albicidin