The Leucine-Responsive Regulatory Protein, Lrp, Modulates Microcin J25 Intrinsic Resistance inEscherichia coliby Regulating Expression of the YojI Microcin Exporter

Abstract

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...

The Ile13residue of microcin J25 is essential for recognition by the receptor FhuA, but not by the inner membrane transporter SbmA

“…We have previously shown that yojI , an E. coli ORF with an unknown function, encodes an efflux pump that can use MccJ25 as a substrate (Delgado et al , 2005). In fact, YojI seems to be responsible for part of the intrinsic resistance shown by most E. coli strains to MccJ25 (Socías et al , 2009). Although the most likely explanation for the low activity of I13K, as compared with that of native MccJ25, would be a deficient uptake of the antibiotic, we investigated the possibility that both peptides enter cells to the same extent, but, once in the cytoplasm, the mutant peptide could be more efficiently pumped out than MccJ25 by YojI.…”

Autoinducer-2 Quorum Sensing Contributes to Regulation of Microcin PDI in Escherichia coli

“…Microcin production by Gram-negative bacteria is typically triggered by environmental and nutritional factors (Duquesne et al, 2007 ). Examples include microcin B17, C, E492, and J25 that are regulated by a global regulator (e.g., OmpR and sigma factors), or in response to depletion of nutrient, carbon, and or nitrogen source (de Lorenzo, 1985 ; Moreno et al, 2002 ; Socias et al, 2009 ). Unlike bacteriocins from lactic-acid producing bacteria and for which quorum sensing (QS) is known to play a regulatory role (Drider et al, 2006 ), to date there have been no reports about the contribution of QS to the regulation of Class I, IIa, or IIb microcin expression.…”

Efflux-Mediated Drug Resistance in Bacteria

“…[148, 149] YojI, an ABC exporter, functions with TolC and mediates resistance to microcin J25[150] and its expression is modulated by the leucine-responsive regulatory protein (Lrp). [151] Preincubation of FloR pump-producing florfenicol-resistant strains with anti-FloR antibody, in the presence of lysozyme and ethylenediaminotetraacetic acid, increased the intracellular accumulation of florfenicol. [152] A paired SMR pump, MdtJI, exports spermidine.…”