Observation of blue-pigment overproduction by the null mutants and transcriptional assay of actII-orf4 of the actinorhodin biosynthetic gene cluster. (A) Blue-pigment over-production by the null mutants of cmdB or cmdA-F and complementation by introduction of the corresponding functional genes. Strains were grown on MS for 3 days at 30°C. The back of the plate is shown. (B) Transcription of actII-orf4 in null mutant of cmdA-F. Total RNA was isolated from solid MS cultures grown for 14, 24, 50, 62, and 74 h, and reverse-transcribed into cDNA for PCR amplification. The 16S rRNA gene of the mutant was used as an internal control. PCR products were electrophoresed in 2% agarose gel at 100 v for 0.5 h.