The Down-Regulation of E. coli OmpF in Response to Sub-Inhibitory Concentrations of Kanamycin Is Not Mediated by MarA

Abstract

Previous studies have shown that transient resistance to aminoglycosides involved the downregulation of OmpF porins, a type of porin expressed during osmoregulation in E. coli. The MarA protein from the marRAB operon, implicated in maintaining a multi-antibiotic resistance phenotype, during an antibiotic challenge, was also found to be involved in OmpF downregulation. In order to determine the regulation of OmpF levels by MarA during an aminoglycoside challenge, OmpF protein levels within the BW25113 (wild type), JW5249 ( Δ marA ) and JW0912 ( Δ ompF ) after exposure to different concentrations of antibiotic were compared. The kanamycin cassette was removed in Δ ompF and Δ marA strains via pCP20 plasmid transformation, MICs were then determined using the microtitre broth dilution method and each strain was grown under 0x, 1/2x and 3x MIC kanamycin. Membrane protein isolation was then performed followed by SDS-PAGE and Western Blot band quantification of OmpF. After the successful removal of the kanamycin cassette, Δ ompF showed the lowest MIC at 4.5ug/ml kanamycin while WT and Δ marA both had MIC at 9.1µg/ml kanamycin. However, Δ ompF showed the most rapid growth under 1/2x MIC kanamycin, followed by WT and Δ marA strains. Western Blotting showed that Δ ompF had the highest OmpF protein levels. Meanwhile, Δ marA, Δ ompF and WT strains showed 88%, 73% and 68% decreases in OmpF protein levels, respectively, during the 1/2xMIC kanamycin challenge. Hence, these results indicated that MarA does not mediate OmpF downregulation during subinhibitory Kanamycin challenge.