Many Escherichia coli K-12 strains display an intrinsic resistance to the peptide antibiotic microcin J25. In this study, we present results showing that the leucine-responsive regulatory protein, Lrp, is involved in this phenotype by acting as a positive regulator of YojI, a chromosomally encoded efflux pump which expels microcin out of cells. Exogenous leucine antagonizes the effect of Lrp, leading to a diminished expression of the pump and an increased susceptibility to microcin J25.Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid antibacterial peptide produced by Escherichia coli (3,30). It is active against E. coli, Salmonella, and Shigella strains. MccJ25 intake is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA (31, 32). E. coli RNA polymerase is the target of antibiotic action (12, 37). The binding site for MccJ25 is located in the secondary channel of the enzyme (1, 21), which provides a route by which the nucleotide substrates reach the catalytic site. Thus, MccJ25 inhibits transcription by clogging the channel and blocking the access of substrates to the active center (1, 21).Four genes (mcjA, mcjB, mcjC, and mcjD) are required for MccJ25 synthesis, export, and immunity (34, 35). The mcjA gene encodes the primary structure of MccJ25 as a 58-aminoacid precursor, from which a 37-amino-acid N-terminal leader is removed. The 21-residue mature peptide has a compact, extraordinary structure, which consists of an eight-residue lariat ring and a C-terminal tail which folds on itself and passes through the ring, where it is sterically trapped (2, 29, 36). The mcjB and mcjC gene products are involved in this assembly process (14). The mcjD product has a dual role. It works as a dedicated ATP-binding cassette exporter of MccJ25 and, at the same time, provides immunity against both the endogenous MccJ25 synthesized in producer cells and the exogenous microcin that gains entry (11,35).We have observed that in twofold serial dilution assays on LB medium or M9 minimal medium plates with several E. coli K-12 strains as indicators (including the widely used strain MC4100), MccJ25 gives rise to turbid zones of growth inhibition. These observations suggested that E. coli strains have an intrinsic partial resistance to MccJ25. However, the polyauxotrophic strain AB1133 gives completely clear halos and is routinely used in our laboratory as an indicator strain. We reasoned that a study of this unusual sensitivity could give us a clue to understanding the origin of the natural resistance of other E. coli strains. In the present work, we demonstrate that the increased sensitivity of AB1133 depends on the leucine used to supplement the AB1133 auxotrophy for this amino acid. The leucine effect seems to be mediated by the global regulator Lrp, which acts as a positive regulator of an endogenous chromosome-encoded MccJ25 efflux pump, YojI. Exogenous leucine would antagonize the effect of Lrp, leading to a diminished expression of the pump and to the accumulation o...