FIG 3.

Transcription studies of MMA-dependent gene expression in M. extorquens DM4 by PCR on reverse transcriptase-produced total cDNA. RNA was isolated from cultures grown with MMA (lines 1 and 2) or with (NH₄)₂SO₄ as a nitrogen source with either methanol (lines 3 and 4) or succinate (lines 5 and 6) as a carbon source and reverse transcribed, and the cDNA was quantified before PCR amplification. Controls were performed for each primer pair with M. extorquens DM4 genomic DNA (1.5 ng) (lane +), water (lane −), and samples from which RT was omitted prior to PCR amplification (lanes 2, 4, and 6). (A) PCR amplification of cDNA (4 ng) was performed with primer pairs spanning intergenic regions (black triangles). Thick arrows denote experimentally confirmed operonic structures. (B) PCR amplification from cDNA of individual genes (primer pairs are indicated by white triangles) of the NMG pathway (left) and of other genes investigated in this study (right), starting from 8 ng total cDNA, except for the transcriptional regulator (16 ng cDNA).