lividans ZX7 [29] and S. avermitilis NRRL8165 [22] were hosts for studying functions of cmdABCDEF genes. Streptomyces were cultivated on Mannitol Soya flour Selleck Volasertib medium (MS; 30). A cellophane sheet was placed over the agar medium when it was necessary to collect mycelium/spores or when cultures were to be examined by scanning electron microscopy [31]. Manipulation of Streptomyces DNA and RNA followed Kieser et al. [30]. E. coli strain DH5α (Life Technologies Inc) was used as cloning host. Plasmid isolation, transformation and PCR amplification

followed Sambrook et al. [32]. DNA fragments were purified from agarose gels with the Gel Extraction Master kit (Watson). Construction and complementation of Streptomyces null mutants Cosmid SCD72A of S. coelicolor containing cmdABCDEF selleck chemicals llc genes was kindly provided by Professor David Hopwood. Cosmid SAV3-17 of S. avermitilis containing the SAV4098-4103 genes was constructed in our laboratory. PCR-targeted mutagenesis was used PLX3397 to replace precisely the cmdABCDEF or SAV4098-4103 genes with an antibiotic resistant gene and then remove the marker but leaving an 81-bp “”scar”" sequence

when necessary [20]. Derivatives of the Streptomyces chromosomal-integrating plasmid pSET152 [33] or pFX101 containing the functional cmdABCDEF genes were employed for complementing the mutated genes. PCR primers for construction and complementation of Streptomyces null mutants are listed in Additional file 1. Scanning electron microscopy (SEM) Streptomyces cultures were grown on MS medium covered with cellophane disks. After 7 days incubation at 30°C, the cells were fixed with fresh 2% glutaraldehyde (pH7.2) and 1% osmium tetroxide. After dehydration, ethanol

was replaced by amyl acetate. The samples were then dried with the supercritical drying method in HCP-2 (Hitachi), coated with gold by Fine Coater JFC-1600 (Jeol), and examined with a JSM-6360LV scanning electron microscopy (Jeol). Light microscopy Streptomyces spores were evenly spread onto MS medium, into which cover-slips were then inserted at an angle of approximate 60°C. After 4 days incubation at 30°C, cells attached to cover-slips were fixed with methanol followed by washing with phosphate-buffered saline. Samples were then stained with 4′,6-diamidino-2-phenylindole (DAPI, 25 μg/ml) at room temperature for 30 minutes. After that, samples were observed Methocarbamol by laser scanning confocal microscope Fluoview FV1000 (Olympus). Images were processed with Image-Pro Plus 6.0. Reverse-transcription (RT) PCR assay S. coelicolor were cultured on MS medium covered with cellophane disks, and RNA was isolated from cultures at a series of incubation times. The RNA samples were treated with DNase (RNase-free, Takara) to remove possible contaminating DNA and, after quantification, reverse-transcribed into cDNA by using “”Revert Acid First Strand cDNA Synthesis”" kit (MBI Fermentas). Then equal 25-ng products were subjected to PCR amplification (25 cycles).