FIG. 2.

Levels of BkdR in wild type and in the crc mutant of P. putida. These cultures were grown and harvested as in Fig. 1, and 200 μg of each cell extract was loaded onto an SDS–12% PAGE gel, blotted to Hybond-P membrane, and treated with anti-BkdR (14). More protein was used than in the experiment of Fig. 1 because of the low copy number of BkdR per cell. Hybond-polyvinylidene difluoride (PVDF) membranes (Amersham) were used in this experiment because PVDF has a better binding capacity for low-molecular-weight proteins. Proteins were separated by electrophoresis in an SDS–12% PAGE gel. Lane 1, P. putida grown in 2xYT plus 0.3% valine–0.1% isoleucine; lane 2, P. putida JS394 grown in 2xYT plus 0.3% valine–0.1% isoleucine; lane 3, P. putida JS394(pJRS196) grown in 2xYT plus 0.3% valine–0.1% isoleucine; lane 4, P. putida PpG2 grown in 0.3% valine–0.1% isoleucine synthetic medium; lane 5, P. putida PpG2 grown in 0.3% valine–0.1% isoleucine plus 40 mM succinate; lane 6, P. putida JS386 grown in 2xYT plus 0.3% valine–0.1% isoleucine; lane 7, 20 ng of purified BkdR.