Involvement of outer membrane protein TolC, a possible member of the mar-sox regulon, in maintenance and improvement of organic solvent tolerance of Escherichia coli K-12

Abstract

Escherichia coli mutants with improved organic solvent tolerance levels showed high levels of outer membrane protein TolC and inner membrane protein AcrA. The TolC level was regulated positively by MarA, Rob, or SoxS. A possible mar-rob-sox box sequence was found upstream of the tolC gene. These findings suggest that tolC is a member of the mar-sox regulon responsive to stress conditions. When a defective tolC gene was transferred to n-hexane- or cyclohexane-tolerant strains by P1 transduction, the organic solvent tolerance level was lowered dramatically to the decane-tolerant and nonane-sensitive level. The tolerance level was restored by transformation of the transductants with a wild-type tolC gene. Therefore, it is evident that TolC is essential for E. coli to maintain organic solvent tolerance.

FIG. 1

SDS-polyacrylamide gel electrophoresis of envelope proteins from the organic solvent-tolerant mutants. The organisms were aerobically grown in LBGMg at 37°C. Envelopes were prepared from cells in the exponential growth phase and extracted with 0.5% sarcosyl. Whole envelopes containing 40 μg of protein (A), the sarcosyl-soluble fraction containing 30 μg of protein (B), and the sarcosyl-insoluble fraction containing 20 μg of protein (C) were electrophoresed on a 0.1% SDS–12% polyacrylamide gel. Protein was stained with Coomassie brilliant blue R-250. Lanes: M, molecular mass markers; 1, JA300; 2, OST3408; 3, OST3410. Arrows indicate the proteins discussed in the text.

FIG. 2

SDS-polyacrylamide gel electrophoresis of outer membrane protein. The sarcosyl-insoluble envelope protein fraction was prepared as described in the legend to Fig. 1. Samples were electrophoresed on a 0.1% SDS–12.5% polyacrylamide gel. Protein was detected by staining with Coomassie brilliant blue R-250 (A) or by using antiserum against TolC (B). Lanes: M, molecular mass markers; 1, JA300; 2, OST3408; 3, OST3410; 4, JA300(pBluescript II); 5, JA300(pHA105); 6, JA300(pOST42BR); 7, JA300(pHc3R); 8, JA300T; 9, OST3408T; 10, JA300S; 11, JA300R. Arrows indicate the proteins discussed in the text.

FIG. 3

Detection of AcrA in the cyclohexane-tolerant mutants. Sarcosyl-soluble membrane protein fractions were electrophoresed as described in the legend to Fig. 1. Protein was detected with antiserum against AcrA protein. A block containing AcrA is shown. Lanes: 1, JA300; 2, OST3408; 3, OST3410; 4, JA300(pBluescript II); 5, JA300(pHA105); 6, JA300(pOST42BR); 7, JA300(pHc3R); 8, JA300T; 9, OST3408T; 10, JA300S; 11, JA300R.