| Streptomyces is a gram-positive bacteria that is widely distributed in nature.During the complex growth and development process,it can produce secondary metabolites,such as antibiotics,active enzymes,immunosuppressants,etc.They have a wide variety and high activity.They are widely used in the fields of industry and agriculture,medicine,and biological control.They have important application and research value.The model strain of streptomyces is Streptomyces coelicolor.The whole genome was sequenced in 2002.The genome contains not only multiple secondary metabolite biosynthetic gene clusters,but also multiple regulatory genes which include 67 pairs of genes.A variety of regulatory factors including the typical two-component signal transduction system(TCSs).TCSs are usually composed of histidine kinases and response regulatory proteins,and are a transmembrane signaling system.At present,only part of the functions of TCSs have been explained,and they play important regulatory roles in the growth and development,protein secretion,and secondary metabolism of Streptomyces,but the functions of most TCSs are still unknown.SCO4155/SCO4156 is a pair of typical TCS in Streptomyces coelicolor,in which SCO4155 is a histidine kinase and SCO4156 is a response regulatory protein of the OmpR family.This TCS has high sequence identity with MprAB TCS in Mycobacterium tuberculosisThis study conducted a preliminary study on the biological function of SCO4155/SCO4156 TCS in Streptomyces coelicolor M145,and explored the biological role of this system in StreptomycesFirst,a plasmid for the mutation of sco4155/56 was constructed,and the gene deletion was carried out by homologous recombination to obtain the deletion mutant strain?4155/56 of sco4155/56.By comparing the phenotypes of M145 and ?4155/56,it was found that the formation of aerial hyphae of ?4155/56 and the synthesis of actinomycin were both advanced.The phenotype of the complementary strain C-?4155/56 is close to that of the wild-type strain,indicating that SCO4155/SCO4156 affects the growth and development of the strain and the biosynthesis of actinomycin.Real-time PCR analysis showed that the expression level of ?-ketoacyl synthase gene sco5087(actl-1)in the mutant strain was significantly up-regulated.Using bioinformatics analysis to predict the recognition site of the regulatory protein SCO4156.In order to verify the binding of SCO4156 to the predicted site,His-SCO4156 protein was expressed and purified heterologously.In vitro binding experiments showed that SCO4156 directly binds to the predicted sites of the promoter sequences of sco4157,sco6287,sco 7075,sco0574 and other genes,indicating that these genes are the target genes of SCO4156.sco4157 encodes a HtrA-like serine protease.There are four HtrA-like proteases in Streptomyces coelicolor.We used Real-time PCR to quantitatively analyze the expression of some of the target genes of SCO4156 and the HtrA-like protease gene,and determined that the expression levels of each gene in the mutant strains were different,and the expression level of sco4157 was significantly up-regulated In addition,this study explored the expression of four HtrA-like protease genes and four regulatory protein genes under SDS conditions.Under SDS conditions,the expression level of the sco4157 gene in the mutant strain was significantly increased,and the target genes also had certain expression differences under stress conditions,indicating that SDS can be used as a stress signal to cause changes in bacterial gene expressionBecause SCO4156 and SCO7075 have high sequence identity,and sco7075 is the target gene of SCO4156,in order to study the function of sco 7075,this study constructed the sco7075 gene-deleted strain A7075 and the supplementary strain C-?7075.Compared with M145,the phenotype change of ?7075 is not obvious.We constructed the strains F-?4155/56 and F-?7075 expressing Flag tag fusion proteins to prepare for the next step of searching for the binding sites of SCO4156 and SCO7075 in vivo In summary,the SCO4155/SCO4156 two-component system has a certain degree of influence on the growth and development of Streptomyces and the synthesis of actinomycin.This study predicted and verified some of the target genes of SCO4156,and determined that SCO41 56 directly regulates the expression of sco4157 and sco 7075,indicating that SCO4155/SCO4156 is an important regulatory factor. |
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