FIG. 5.

Amounts of NtrC protein expressed from a single chromosomal ntrC⁺ allele or from plasmid pJES311, which overexpresses NtrC^WT. Cells were cultivated under nitrogen-limiting conditions as for assays of glnA′-‵lacZ expression (see Materials and Methods), and 0.5 ml of culture at an OD₆₅₀ of 0.5 was concentrated and loaded on an SDS–10% polyacrylamide gel. Amounts of NtrC protein in crude lysates were determined by Western blotting with mouse polyclonal antiserum raised against the carboxy-terminal domain of NtrC. (A) Crude lysates of SK3424 (ntrC) (lane 1) and SK1490 (ntrC⁺) (lane 2) were compared with dilutions of lysates of SK3425(pJES311): lane 3, 1:1,250; lane 4, 1:625; lane 5, 1:125; lane 6, 1:25; lane 7, undiluted. (B) Crude lysates of strain SK1490 (lanes 2, 4, 6, and 8) were compared with different amounts of purified NtrC^WT added to crude lysates of SK3424: lane 1, 0 ng; lane 3, 12.5 ng; lane 5, 25 ng; lane 7, 50 ng. MW, BenchMark molecular weight standards (Gibco BRL).