| Abstrakt: |
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of Escherichia coliK-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [35S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other traproducts in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and traTproduct synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of traTproduct and membrane F-pilin, as assessed in an Flac tramutant strain which is incapable of elaborating pili, was not rapid. Both traTproduct and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of traTproduct did not decrease even after the culture was incubated for 24 h. When wild-type Flacpiliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the Flac tramutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the Flacstrain compared with the Flac trastrain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces. |
