Figure 2

Gene expression studies with reverse transcription linked absolute quantification Real-Time PCR and immunoblotting. A. Relative transcription level of fdh-mhy operon in cells grown on different carbon sources (M and P denotes for the maltose and peptide (casein hydrolysate) content of the medium, respectively, see Materials and Methods). The transcription level of the fdh-mhy operon in cells grown on DP medium was taken as 100 %. For reverse transcription RNA was purified from three parallel cultures, cDNA was made using the fhl8frC1R primer designed to the 3' end of mhyH. In the Real-Time PCR a primer pair (fhl10N-fhl05R) designed to the mhyF gene was used. B. Effect of sulfur on the transcription level of fdh-mhy operon (M, P and S denotes for the maltose, peptides (casein hydrolysate) and sulfur content of the medium, respectively, see Materials and Methods). For reverse transcription RNA was purified from three parallel cultures, cDNA was made with fhl805R primer designed to the 3' end of mhyC. In the Real-Time PCR a primer pair (fhl805R-fhl806N) designed to the mhyC was used. C. D. Western blotting of the same samples as in A (C) and B (D). In the immunoblotting experiments 20 μg total cellular protein, derived from the same cultures as above, was separated on 12% SDS polyacrylamide gel after boiling in SDS-loading buffer for 10 min. Proteins were blotted on nitrocellulose membrane and the FdhB protein was detected using anti-FdhB antibody.