Studies On Bi-exponential Growth And The WhiG Gene Of Streptomyces Griseus

Streptomyces are mycelial multicellular soil actinomycetes in which adverse environmental conditions induce a developmental program involving complex morphological differentiation coordinated with synthesis of an enormous variety of interesting and useful secondary metabolites. Development system in some Streptomyces spp. is characterized by an unique multiphasic growth kinetics in liquid medium. After spore germination, first logarithmic growth is followed by a transient cessation of cell growth, which potentially serves as a key developmental switch in regulatory system, leading to initiation of second logarithmic growth. Physiological stresses induce expression of specialized RNA polymerase sigma factors, which direct transcription of adaptive regulatory proteins. A signal transduction via not only two-component regulatory system but also serine/threonine kinases generally regulates morphological and physiological differentiation in Streptomyces. Streptomyces whiG gene is a key gene encoding a developmentally important RNA polymerase sigma factor(awhiG), which specifically initiates development program and determines the developmental fate of the cell.In this study, growth of Streptomyces griseus in liquid YMPD medium was determined by measuring the OD560 at the interval of 1 hour. The data confirmed that there was a bi-exponential growth in the S. griseus. Transient cessation of cell growth was detected. It began reproducibly at19 hour after inoculation and lasted 3 hours.A 1-Kb PCR product was isolated by means of PCR using the total DNA of S. griseus as template. The product was ligated into pGEM-3Zf(+), leading to construct a plasmid, designated pLZl. Sequence data showed that the DNA fragment containing a complete ORF was 975bp, 68.3% GC content. Its amino acid product was a putative RNA polymerase sigma factor, suggesting the ORF was the true whiG hologue. An internal segment of the whiG gene of S. griseus was amplified from pLZl through PCR. The 304bp DNA fragment was inserted into the EcoRI/BamHI site of E. coli-Streptomyces shuttle plasmid pKC1139, generating pKC1139::A whiG, named pLZ107, for gene disruption. pLZ107 then was transformed into methylation-deficient E. coli ET12567.Parameters affecting protoplast formation and regeneration were investigated. The culture was shake-cultured at 30℃ for 40 hours in liquid medium YMPD supplemented with 0.5% glycine. The washed mycelia with 10.3% sucrose were incubated in P buffer containing Img/ml lysozyme at 30癈 for 1 hour. Under the conditions, the amount of prepared protoplasts was up to 109 /ml, estimated by microscopic counts. Several kinds of regeneration medium were tested to identify the potential effects of regeneration medium on protoplast regeneration. The observation showed that the regeneration medium RIM was superior against R2YE and SpMR. The regeneration frequency was 10%.Many attempts to transform S. griseus protoplast with plasmid pLZ107 were unsuccessful. The possible reasons were discussed.