Clonging And Identification Of RsigB In Streptomyces Rimosus

Streptomyces rimosus is a kind of soil-dwelling bacteria with a complex lifecycle and abundant secondary metabolic products. Because of its ability of producting oxytetracycline(OTC), Streptomyces rimosus has become commercial producer of OTC. As other soil-dwelling bacteria, it is challenged with diverse environmental stresses in soil condition and during its developmental process. During its long-term evolution, it has formed a complete set of morphological differentiation and metabolic regulation mechanism. In this study, rsigB was selected as research target, and the results are as follows:1:Cloning and identification of sigB family regulatory factor rsigBRed pingment was observed when M4018was grown on plate under stress conditions (42℃, alkaline), while brown pigment, which is produced with oxytetracycline biosynthesis was disappear. It suggests the secondary metabolic pathway should be shifted. The genomic DNA sequencing of S. rimosus type strain M4018has been accomplished by the cooperation work of our team and Professor Iain Hunter’s team(unpublished). A new sigma factor was found based on genomic DNA information. It inferred that it belongs to one member of sigB family because it has a high homologous identity (about75%) with other sigB-like factors from Streptomyces coelicolor and Streptomyces griseus, so it was named rsigB.2:Expression of RsigB in E.coliBased on pET system, recombinant plasmid pET28a-rsigB was constructed, then transformed into E.coli BL21(DE3) cells. The recombinant E.coli cells were cultured under different stress conditions, and RsigB was expressed by induction of IPTG to analyze the stress tolerance abilities of RsigB. The results indicate that the expression of RsigB in E.coli BL21(DE3) could increase viability under different stress conditions, such as high temperature, osmotic or oxidative stresses.3:Functional analysis of rsigB in M4018Firstly, rsigB was introduced into M4018chromosome. Compared with M4018(control), the mutant’s tolerance to osmotic and oxidative stress increase. However overexpression of rsigB can’t activate carotenoid biosynthesic in M4018.In the case of that rsigB was blocked in M4108, rsigB deleted mutant produced aerial hyphae and spores eatlier than those observed in M4018. However, the mutant with extra rsigB copy delayed its growth when they were grown in Emerson plates. Quantitative PCR analysis showed that rsigB could associated with the aerial hyphae differentiation indication gene chpE based on their transcriptional levels. The experimental results indicate that rsigB deletion can promote morphological differentiation when grown on plates, and multi-copy rsigB mutants can enhance the tolerant abilities to osmotic stress or oxidative stress when they were submerged in shaking flask culture.