The Antimicrobial Activity Of The Endophytic Streptomyces Sp.SAT1 And The Regulatory Function Of WblA

Medicinal plant are unique habitat for endophytes,in which endophytes have great potential for the synthesis of novel natural product through long-term co-evolution with plants.The laboratory preserved 94 endophytic strains isolated from 19 medicinal plants.In this paper,multi-steps activity detection combined with bioinformatics analysis to predict their antibacterial mechanism and verified via genetic engineering and qPCR.The paper mainly includes the following four parts:1.Multi-step activity detections to determine the target strain SAT1.Eight strains with strong bacteriostatic effect(inhibition bandwidth>1.0 cm)on MRSA were screened from 94 strains of antagonistic endophytes.After inhibition spectrum detection,fermentation broth detection and in vitro shoot prevention experiments,Streptomyces sp.SAT1 showed inhibitory effects on eight indicator strains such as Cryphonectria parasitica and Candida albicans,and showed biocontrol effect on poplar canker and chestnut blight in vitro shoots experiments.2.Predicting bacteriostatic active substances by analyzing SAT1 secondary metabolic gene clusters(SMGCs).A total of 29 SMGCs were predicted by analyzing the whole genome of SAT1 by antiSMASH.Among them,Cluster24 and Cluster25 have 52%and 88%similarity with hygromycin B and moenomycin gene cluster,respectively,which may play a role in the antibacterial activity.After homology alignment analysis,Cluster24 contains 23 genes and only hygT,hygH,hygG and hygZ showed lower similarity(only 32-40%),and moeR5 and S5 were only 28%and 45%in Cluster25.So we speculate that Cluster24 and Cluster25 are likely to synthesize new substances like hygromycin B and moenomycin.3.The antibacterial activity of wblA enhanced expression mutants.As a global negative regulatory gene,several studies have shown that wblA can affect the biosynthesis of a variety of secondary metabolites and Streptomyces sporulation.In this study,two fragments W1(241 1 bp)and W2(854 bp)containing wblA gene were constructed into pSET152 to get mutants SAT1-W1 and SAT1-W2,respectively.The bacteriostatic experiments showed that the antibacterial activity of the two mutants was significantly lower than that of the wild type,which showing wblA has an inhibitory effect on bacteriostatic related genes.4.The analysis of wbIA 's function on the expression of genes in Cluster24 and Cluster25 via qPCR.The housekeeping genes rpoA and recA were used as common internal references to detect the expression level of the target genes.The relative quantitative results showed that the expression of wblA was increased by 2.45 and 2.54 times in SAT1-W1 and SAT1-W2,respectively.In SAT1-W1,the three genes hygM,hygO and hygP of Cluster24 were almost no longer expressed,while reduced 72-88%in SAT1-W2,and the expression levels of moeA5,moeP5 and moeGT2 in Cluster25 reduced 29-60%in SAT1-W1,while increased by 1.9,1.19 and 1.95 times in SAT1-W2.Therefore,we suspect that the substance synthesized by Cluster24 has fungi-inhibiting activity,and the substance produced by Cluster25 needs further verification.In this paper,the antibacterial activity characteristics of endophytic Streptomyces sp.SAT1 and the SMGCs with bacteriostatic activity were studied in detail.This study provides theoretical support for the subsequent in-depth study of the bacteriostatic mechanism,active substances identification and the construction of high-yield antibiotic-functional strains.