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. 2019 May 22;201(12):e00134-19. doi: 10.1128/JB.00134-19

FIG 7.

FIG 7

EipB has an internal disulfide bond. (A) Cysteines C69 and C278 are spatially proximal in the EipB structure and form a disulfide bond. C278 is present at the EipB C terminus that follows β14, and C69 is present in a loop connecting β2 and β3. (B) His-tagged wild-type EipB and EipB cysteine mutant proteins (C69S, C278S, and C69S+C278S) were purified and mixed with a protein loading buffer with (+) or without (−) 1 mM DTT. Protein samples were resolved by SDS–12% PAGE. This experiment was performed three times. The picture of a representative gel is presented. (C) Growth on SBA plates containing 3 μg/ml of carbenicillin with (+) or without (–) 2 mM IPTG of a serially diluted (10-fold dilution) B. ovis ΔeipB strain ectopically expressing wild-type EipB (Plac-eipB), C69S mutant (Plac-eipBC69S), C278S mutant (Plac-eipBC278S), or C69S+C278S mutant (Plac-eipBC69S+C278S). B. ovis wild-type (WT) and ΔeipB carrying the pSRK empty vector (EV) were used as a control. The days of growth at 37°C and 5% CO2 are reported for each plate. A representative picture of the different plates is presented. (D) Enumerated CFU after growth on SBA plates containing 3 μg/ml of carbenicillin with (+) or without (–) 2 mM IPTG of serially diluted (10-fold dilution) B. ovis ΔeipB strains expressing different versions of eipB from a plasmid (wild type and cysteine mutants; see panel C legend). EV strains and SBA plates with no carbenicillin, with or without IPTG, were used as controls. This experiment was independently performed twice, with two different clones each time, and all plate assays were done in triplicate. Each data point indicates the mean ± the standard error of the mean. One-way ANOVA, followed by Dunnett’s posttest (to the wild type), supports the conclusion that eipB-dependent protection against the cell wall antibiotic, carbenicillin, is significantly diminished when disulfide-forming residues C69 (**, P < 0.005) and C278 (**, P < 0.003) are individually or both (*, P < 0.01) mutated to serine. This is effect is evident with leaky eipB expression from Plac but diminished when the expression of wild-type and mutant eipB alleles is induced by IPTG.