The Escherichia coli Yej transporter is required for the uptake of translation inhibitor microcin C

Abstract

Microcin C (McC), a peptide-nucleotide antibiotic, targets aspartyl-tRNA synthetase. By analyzing a random transposon library, we identified Escherichia coli mutants resistant to McC. Transposon insertions were localized to a single locus, yejABEF, which encodes components of a putative inner membrane ABC transporter. Analysis of site-specific mutants established that all four components of the transporter are required for McC sensitivity. Since aspartyl-tRNA synthetase in yej mutant extracts was fully sensitive to McC, we conclude that yej mutations interfere with McC uptake and that YejABEF is the only inner membrane transporter responsible for McC uptake in E. coli. Other substrates of YejABEF remain to be identified.

FIG. 1.

The mechanism of action of McC. (A) The structure of the mcc locus. See text for details. (B) The Trojan-horse mechanism of McC's action is schematically illustrated. Intact McC, a peptide-nucleotide, is taken up by a bacterial cell, where it is processed by peptidases. Processed McC is structurally similar to aspartyl-adenylate, an intermediate of the tRNA^Asp aminoacylation reaction catalyzed by Asp-RS. Unlike aspartyl-adenylate, processed McC is nonhydrolyzable. It binds to and inhibits Asp-RS, leading to inhibition of translation.

FIG. 2.

Cells with mutations in yejABEF are McC resistant. The growth of the indicated wild-type and mutant strains in the presence or in the absence of 10 μg/ml of McC is shown. The results shown are representative of those obtained in three independent experiments. OD₆₀₀, optical density at 600 nm; AU, absorption units; wt, wild type.

FIG. 3.

McC processing and Asp-RS inhibition in extracts of yejABEF mutants. S30 extracts of the indicated cells were prepared and incubated with or without 2 μg/ml McC to allow processing, and the tRNA^Asp aminoacylation reaction was carried out. The amounts of aminoacylated tRNA^Asp (measured as the incorporation of [C¹⁴]Asp in trichloroacetic acid-precipitable material) are shown. Error bars show standard deviations. See Metlitskaya et al. (14) for experimental details.

FIG. 4.

Phylogenetic tree of periplasmic components of oligopeptide ABC transporting systems. YejA and its close homologs are marked in red, nickel-oligopeptide transporters are marked in blue, experimentally verified proteins are marked in green, and experimentally uncharacterized proteins are in black. Accession numbers of proteins from NCBI RefSeq database (http://www.ncbi.nlm.nih.gov/RefSeq/) are indicated. The tree was created using the NEIGHBOR program from the PHYLIP package (8).