e. cmdA, C and F) and wild type M145. Introduction of additional copies of the functional cmdB or cmdA-F into the mutants could reduce GSK2126458 mw the production of blue pigment to the wild-type level (Figure 6A), confirming that blue pigment over-production was caused by mutation of the genes, and also suggesting that these genes are involved in repression of blue pigment production in M145. Figure 6 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. Initiating transcription of the pathway-specific regulatory gene actII-orf4 of actinorhodin biosynthesis at an earlier growth stage in the cmdA-F null mutant In S. coelicolor, pathway-specific regulatory gene actII-orf4 is essential for initiating transcription of the
whole biosynthetic gene cluster of blue-pigment actinorhodin . To study transcription of actII-orf4 in the cmdA-F RG-7388 mw null mutant, we harvested spores/mycelium from MS plates after different growth periods and isolated RNA for RT-PCR. As seen in Figure 6B, transcription of actII-orf4
in the null mutant started as early as 16 h and then reached a maximum at 40 h, ~24 and 34 h earlier than was observed in M145. Discussion Here, we report that an operon of six genes cmdABCDEF (SCO4126-4131) of S. coelicolor, encoding five membrane proteins and one membrane-located ATP/GTP-binding protein, affects differentiation and causes increased production of an antibiotic, actinorhodin. The ΔcmdABCDEF strains reveal aberrant branches and short Dynein aerial hyphae. Expression of cmdB, and therefore presumably of the whole operon, was detectable during vegetative growth, but increased substantially as soon as aerial growth was detectable. Similar conserved gene clusters are also found in other Streptomyces species, e.g. S. avermitilis (SAV4098-4103; ), S. griseus (SGR3915-3920; ) and S. lividans (Our unpublished data). Serious block in forming aerial hyphae in S. lividans and in the development of coiled aerial hyphae in S. avermitilis were observed when their cmd operons were disrupted. Together, these results indicate that CmdABCDEF proteins mainly affect Streptomyces differentiation early in aerial hyphae formation.