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. 2006 Jan;188(1):115–123. doi: 10.1128/JB.188.1.115-123.2006

TABLE 1.

Effects of mutations of the efflux protein EmhB on polycyclic aromatic hydrocarbon transport, antibiotic sensitivity, and toluene tolerance in P. fluorescens cLP6a-1

Strain or mutationa Hydrocarbon transport (ratio of uninhibited/inhibited)b
Antibiotic sensitivity (MIC, μg ml−1)c
Toluene toleranced
Phenanthrene Anthracene Chl Nal R6G Dq Cip Str Growth Recovery
Strains
    cLP6a (wild type) 0.40 ± 0.20 (3) 0.33 ± 0.11 (3) 16 16 >128 4 0.031 8 − +
    cLP6a-1 (disrupted EmhB) 0.86 ± 0.09 (3) 0.75 ± 0.16 (3) 2 1 4 0.5 0.004 4 − −
    cLP6a-1 (pUCP26) (negative control) 1.06 ± 0.05 (9) 0.90 ± 0.11 (6) 0.5 1 8 1 0.004 1 − −
    EmhBHis 0.53 ± 0.07 (9) 0.48 ± 0.13 (6) 8 32 128 8 0.031 2 − +
    EmhB (complemented control) 0.56 ± 0.08 (3) 0.56 ± 0.07 (3) 32 128 >128 8 0.063 1 + +
Central cavity mutations
    A384P/A385Y 0.61 0.67 4 16 32 2 0.016 2 − +
    F386A 0.52 0.50 8 32 >128 2 0.016 1 − +
    F458A 0.55 0.56 8 16 64 4 0.016 1 − +
    F459A 0.60 0.56 8 16 64 4 0.016 1 − +
    N99A 0.53 0.49 4 32 64 2 0.031 1 − +
    D101A 0.84 0.96 1 16 16 1 0.016 2 − +
    D101N 0.80 0.70 2 16 32 2 0.016 1 − +
    D101E 0.50 0.47 8 32 >128 4 0.016 4 − +
Pore domain mutations
    F316A 0.52 0.50 16 32 32 4 0.016 1 − +
    F317A 0.46 0.48 4 16 64 4 0.016 2 − +
    F682A 0.46 0.44 8 32 >128 4 0.016 2 − +
    F281A 0.90 0.83 <0.5 8 32 2 0.008 4 − −
    F325A 0.88 0.76 2 8 16 2 0.008 2 − −
    N282A 0.71 0.74 1 8 32 2 0.016 4 − +
Outer membrane protein docking domain mutation
    A206S 0.48 0.53 32 128 >128 16 0.125 2 + +
a

Except for the wild type, all EmhB pumps were expressed in the efflux-deficient mutant P. fluorescens cLP6a-1 on the broad-host-range plasmid pUCP26. The negative control was P. fluorescens cLP6a-1 carrying the vector (pUCP26) only.

b

For the strains expressing the EmhBHis (pBH5-EmhBHis) and EmhB (pBH5) proteins and for the negative-control strain (pUCP26), the averages ± standard deviations are given, and the numbers in parentheses indicate the number of independent experiments performed. All other transport data are the averages of duplicate samples taken from single experiments.

c

MICs were determined using the microtiter broth dilution method (11). Two independent experiments were done, and representative data are shown. Abbreviations: Chl, chloramphenicol; Nal, nalidixic acid; R6G, rhodamine 6G; Dq, dequalinium chloride; Cip, ciprofloxacin; and Str, streptomycin.

d

Toluene tolerance was assessed by monitoring growth on LB agar plates in the presence of vapor phase toluene. Growth in the presence of toluene was scored as + (positive for growth) or − (negative for growth). The ability of the strains to recover from toluene exposure was scored as + (growth upon removal of toluene) or − (no growth upon removal of toluene). Two independent experiments were done.