FIG. 1.

Western blot with anti-E1α serum (10) of crc mutants grown in 2xYT plus valine-isoleucine. The extracts in lanes 1 to 6 were from cultures grown in 2xYT plus 0.3% valine and 0.1% isoleucine (wt/vol); the extract in lane 7 was from a culture grown in glucose minimal medium. Each lane contained 5 μg of protein. Lane 1, P. putida PpG2; lane 2, P. putida JS394; lane 3, P. putida JS394(pJRS196); lane 4, P. aeruginosa PAO1; lane 5, P. aeruginosa PAO8020; lane 6, P. aeruginosa PA8020(pJRS196); lane 7, P. putida PpG2 grown in glucose. All cultures were grown to an A₆₆₀ of between 0.6 and 0.8, and cell extracts were prepared as described before (16). Electrophoresis was done in an SDS–8.5% PAGE gel. Western blots were screened by using the ECL-Western blotting analysis system (Amersham Pharmacia Biotech) with Hybond-ECL nitrocellulose membranes according to the manufacturer's instructions. To determine whether the ECL detection method was quantitative, increasing amounts of PpG2 grown in valine-isoleucine-lactate medium were loaded on a gel and used for Western blotting with anti-E1α serum. The blot was scanned with a Molecular Dynamics densitometer, and pixel values versus micrograms of protein were graphed and shown to be a linear plot (data not shown).