Establishing intergeneric conjugation system in Streptomyces carneohygroscopicus SH121

YANG Zhuo;LI Jiali;XIAO Le;HE Jing;State Key Laboratory of Agricultural Microbiology,Huazhong Agricuitural University;

Streptomyces carneohygroscopicus SH-121;;intergeneric conjugation;;genetic manipulation;;gene knockout

Streptomyces carneohygroscopicus SH-121 isolated during selecting biocontrol agent of citrus diseases exhibits strong inhibition towards Gram positive bacteria and many pathogenic fungi in plant.Establishment of a highly efficient genetic manipulation system is critically required for functionally analysing genes and genetic engineering in Streptomyces.In this study,the effects of heat treatment,pre-germination time,donor hosts,ratio of donor and recipient and MgCl_2 concentration in medium on conjugation efficiency were studied by single factor experiments.Under heat treatment at 50 ℃ for 10 min,pre-germination at 37℃for 1h,using Escherichia coli DCDA/pUZ8002 as donor host,with the ratio of donor to recipient being 100 to 1,supplementing M-ISP4 medium with 40mmol/L MgCl_2,a highly efficient intergeneric conjugation method for SH-121 was developed with highest efficiency of 1.03×10~(-3)conjugants/recipient.The integrative plasmid pSET152 and the plasmid pYZ09 for gene knockout was successfully transferred into SH-121 with the method newly established.It is indicated that this genetic manipulation system could be used to discover bioactive natural products and study their biosynthetic gene clusters in SH-121.