TABLE 2.

Contribution of oprM and opmH to erythromycin and triclosan efflux

Strain/plasmidOMP gene deletion/complementationEfflux components expressedMIC (μg/ml)a
TriEry
PAO327bΔoprJ ΔoprMMexJK, OpmH12816
PAO367ΔoprJ ΔoprM ΔopmH362MexJK168
PAO367/pVLT35cΔoprJ ΔoprM ΔopmH362MexJK168
PAO367/pPS1313cΔoprJ ΔoprMMexJK, OpmH12816
ΔopmH362/opmH+
PAO397/pBSP II-KSΔoprM ΔoprJ ΔoprN ΔopmH362None28
PAO397/pPS1370dΔoprM ΔoprJ ΔoprN ΔopmH362MexJK28
PAO397/pPS1371ΔoprM ΔoprJ ΔoprN ΔopmH362/oprM+MexJK, OprM232
PAO397/pPS1372ΔoprM ΔoprJ ΔoprN ΔopmH362/opmH+MexJK, OpmH>1,0248
PAO397/pPS1373ΔoprM ΔoprJ ΔoprN ΔopmH362/oprM+ opmH+MexJK, OprM, OpmH51264
PAO397/pPS1424ΔoprM ΔoprJ ΔoprN ΔopmH362/opmH+OpmH88
PAO397/pPS1425ΔoprM ΔoprJ ΔoprN ΔopmH362/oprM+OprM28
  • a Erythromycin (Ery) MICs were determined using the broth microdilution method, and triclosan (Tri) MICs were determined using the agar incorporation method, except for PAO327 and the PAO367 series of strains, for which the microdilution method was used.

  • b The genotype of PAO327 is Δ(mexAB-oprM) nfxB Δ(mexCD-oprJ) Δ(mexXY) mexL (MexJK constitutively expressed) and MexEF-OprN uninducible due to a mexT insertion (18).

  • c In pVLT35 and pPS1313, opmH transcription is driven from the lacI repressor-controlled Ptac promoter; media used for MIC determinations with cells harboring these two plasmids were therefore supplemented with 1 mM isopropyl-β-d-thiogalactopyranoside (Gold Biotechnology, St. Louis, MO).

  • d Hybrid mexJK (pPS1370), mexJK-oprM (pPS1371), mexJK-opmH (pPS1372), and mexJK-oprM-opmH (pPS1373) operons were assembled on pBSP II KS(−). pPS1371 contains oprM, 52 bp of its upstream region and 13 bp of its downstream region without transcriptional terminator. pPS1372 and pPS1373 contain opmH, 72 bp of its upstream region and 136 bp of its downstream region, including the transcriptional terminator. The recombinant plasmids were transformed into Δ(mexAB-oprM) Δ(mexCD-oprJ) Δ(mexEF-oprN) Δ(mexLJK) Δ(mexXY) ΔopmH362 strain PAO397, and efflux of the indicated antimicrobials was assessed by MIC determinations. On pPS1370-pPS1373, constitutive transcription of the cloned genes originates from the mexJK operon promoter (PJK). pPS1424 and pPS1425 are based on pVLT35, and transcription of cloned genes is driven from the lacI repressor-controlled Plac promoter; media used for MIC determinations with cells harboring these two plasmids were therefore supplemented with 1 mM IPTG.