FIGURE 1.

Effect of Ffh depletion on growth, Ffh levels, integrity of the cytoplasmic membrane, OmpA translocation, and SecA levels/distribution. A, effect of Ffh depletion on cell growth. Growth of WAM121 cells cultured in the presence (control) and absence (Ffh depletion) of 0.2% arabinose was monitored by measuring the A₆₀₀. After 4 h of depletion, 0.2% arabinose was added to part of the culture (readdition). B, Ffh levels in WAM121 cells grown in the presence (control) and absence (Ffh depl) of arabinose and in MC4100 cells from which WAM121 was derived. Whole cells (0.02 A₆₀₀ units) were subjected to SDS-PAGE followed by immunoblot analysis with antibodies to Ffh. C, histograms representing the fluorescence of cultures stained with the fluorophore PI, whose uptake is an indicator for the integrity of the cytoplasmic membrane. D, effect of Ffh depletion on OmpA translocation. Ffh-depleted and control cells were labeled with [³⁵S]methionine for 30 s. OmpA was immunoprecipitated and subjected to standard SDS-PAGE analysis, and labeled material was detected by phosphorimaging. To calibrate the system with an OmpA precursor, CM124 cells (SecE depletion strain, in which the secE gene is under control of the araBAD promotor) grown in the absence of arabinose (SecE depl) was used. E, SecA levels and distribution. Whole cells (WC; 0.1 A₆₀₀ unit per lane) and cytoplasmic membranes (CM; 5 μg of protein) were separated by means of SDS-PAGE and subsequently subjected to immunoblot analysis with antibodies to SecA.