Sodium dodecyl sulfate hypersensitivity of clpP and clpB mutants of Escherichia coli

Abstract

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.

FIG. 1.

Western blot for ClpP levels in wild-type (MC4100) E. coli grown in the presence of SDS. Cells were grown at 37°C in LB with 0, 0.5, 1.0, 2.0, and 4.0% SDS. Cellular proteins were detected with anti-ClpP antibody (17). Lane 1, prestained molecular weight markers. The ClpP band intensities (pixels/mm²) from left to right were as follows: in lane 2, 20,450 (0% SDS); in lane 3, 14,660 (0.5% SDS); in lane 4, 58,190 (1% SDS); in lane 5, 144,550 (2% SDS); and in lane 6, 92,030 (4% SDS). The latter four values correspond to ClpP increases of 0.7-, 3-, 7-, and 5-fold, respectively.

FIG. 2.

Increase in ClpP and capsular material for wild-type (MC4100) E. coli grown at 37°C in the presence of 0 to 10% SDS. ▪, ClpP levels determined by intensity of Western blots; ♦, capsule levels determined by spectrophotometric assay for the uronic acids in colanic acid (5). To make sure that increasing levels of SDS did not interfere with the colanic acid assays, SDS was removed prior to the assay by the addition of KCl to a final concentration of 5% (13). Also, controls with 0 to 10% SDS but no colanic acid gave no color development. All points represent averages of three or more measurements. The data shown in Fig. 1 are one set of the ClpP measurements used here.