Figure 3. Characterization of the wild-type strain and the nasT and nasS mutants of P. denitrificans with different nitrogen sources.

(A) NADH-dependent nitrate reductase activity and NasG polypeptide detection by 2D-PAGE analysis in the wild-type strain of P. denitrificans. The 2D-PAGE analyses were carried out in cytoplasmic fraction from cells grown with different nitrogen sources obtained by subcellular fractionations, as indicated in Materials and Methods. The different nitrogen sources were nitrate, ammonium, ammonium plus nitrate, glutamate, or glutamate plus nitrate (10 mM each). Isoelectric focusing was performed from IPG strips (range 4–7) and second dimension was carried out onto 12% polyacrylamide gels. The nitrate reductase activity (NR) was assayed in cytoplasmic fractions and expressed as nmol NO₂⁻ formed min⁻¹ mg⁻¹ (n.d., not detected). (B) RT-PCR analysis of structural nas genes in the wild-type and nasT and nasS mutant strains of P. denitrificans. To isolate RNA, wild-type and nasT and nasS strains were cultured in minimal medium with different N-sources, as described in the Materials and Methods section and harvested at an A₆₀₀ of ∼0.4. The rpoB gene was used as housekeeping. −/+, without/with reverse transcriptase. (C) NasG polypeptide detection by 2D-PAGE analysis and NADH-nitrate reductase activity in cytoplasmic fractions from nasT and nasS mutant strains of P. denitrificans. The 2D-PAGE analysis and the NR activity assays were performed as indicated in (A) for the wild-type strain.