| Streptomyces 5406 was widely utilized as biofertilizer in 1970s but the widespread usage encountered many different problems that eventually put to the end of biofertilizer at that time.Nevertheless,its use as a fertilizer recently resumed especially in Shandong provice in the production of vegetables.At present,our knowledge of Streptomyces 5406strain itself,as well as the application mechanism remains to be investigated.In this study,Streptomyces 5406 was characterized for polyphasic taxonomy,genome sequencing and bioinformatics prediction of secondary metabolite.A few antimicrobial substances were purified and characterized.The main results were summarized below.1.Classification of Streptomyces 5406.The 16S r RNA gene of XY 25Twas determined and the gene showed a high similarity to those of Streptomyces species including Streptomyces silaceus DSM 41861（99.11%）,Streptomyces flavofungini DSM40366（98.49%）and Streptomyces intermedius DSM 40372（98.43%）.DNA-DNA hydridization studies revealed that XY 25Tshowed relatedness values of 40.42±0%,35.20±1.66%40.16±0.16%,separately,which were below the threshold of 70%for classification of a new species in Streptomyces recommended by Wayne.On the basis of a combination of phylogentic distinctness and difference in the morphological,physiological,chemotaxonomic,genotypic,we consider strain XY 25Tto be a novel species in the genus of Streptomyces and submit research report to Schink Bernhard,the professor of Microbiology classification expert in University of Konstanz,and the strain was named as Streptomyces alfalfae XY 25T.2.The genomic DNA of S.alfalfa XY 25Twas extracted and sequenced.The size of genomic was 8 M with a G+C content of 71.8%.The yielded genome sequence contained68 contigs,3 repeat sequences,85 t RNA genes,7174 CDS,1 tm RNA and 626 signal peptides through the gene annotation.The genome contains a large number of genes coding for enzymes that produce bioactive substances,such as acid phosphatase.3.Genes function in improving plant growth have been identified in S.alfalfae XY25T,such as those produce and secret plant hormones.Bioinformatics study showed that genome of S.alfalfae XY 25Thas an acid phosphatase gene,which predicts that it has a function of degrade phosphorus that can release phosphorus from organic phosphorus for plant growth.During the fermentation,it is found that S.alfalfae XY 25Tcould produce some acids that can release potassium and phosphorus by the corrosion.At the same time,genome annotation results showed that S.alfalfae XY 25Thas indole-3-glycerol phosphatese,tryptophan regulation related enyzmes.4.Secondary metabolites produced by S.alfalfae XY 25Twere predicted by the antismash.It has 37 kinds of secondary metabolites:6 PKS gene clusters encoding alkyresorcinol,calicheamicin,BE-14106,tetron and borrdlidin;7 NRPS gene clusters encoding griseoviridin,compledtatin,mirubactin respectively and some unknown gene cluster;6 Terpene gene cluster encoding isorenieratnen,pentalenolactone,hopene and some unknown gene clusters;5 PKS-NRPS gene clusters encoding flaviolin,GE81112,feglymycin,oxazolomycin and some unknown gene clusters.The similarity of gene clusters with Coelichelin,Alkylresorcinol,Albaflavenone and Ectoine were 100%,respectively.It is possible that there are various specific antibacterial substances in the secondary metabolites of the strain,such as Oxazolomycin,Pentalenolactone,Feglymycin and so forth.5.The antibacterial activity produced by S.alfalfae XY 25Tis a specifically against Gram-positive bacteria,such as Bacillus subtilis,Staphylococcus aureus,lactic acid bacteria.The antibacterial material shows a highly efficient antibacterial activity,especially for Bacillus subtilis and the antibiotic is still active at low concentration（2μg/m L）.For separation,using Amberlite XAD-7HP adsorption column fixed in active material from fermentation liquid;eluting active substances with 80%of alcohol which 3times the volume of the pillars;The crude sample of strain XY 25Twas loaded into Agilent HC-C18（2）column（5μm;4.6×250 mm）at the condition of 1.0 m L/min flow rate and 276 nm wavelength monitored.The mobile phase consisted of acetonitrile and HPLC-grade water containing 0.1%trifluoroacetic acid（TFA）.The fractions were manually collected for the antibacterial activity assay against Bacillus subtilis.At last,the pure sample has been got.6.Active fractions were collected and analyzed using ESI-MS,infrared spectrometer,1H NMR,13C NMR,1H-1H COSY and1H-1H NEOSY data.Comprehensive analysis of antimicrobial spectrum and genomic information showed that the secondary metabolite may be a new isomer of oxazolomycin.This corresponds to the results of bioinformatics prediction.The above studies clarified the status of Streptomyces 5406,and yielded the whole genome sequencing information by using new technnique and new method.Genes of new functions were idnetified in S.alfalfae XY 25T,such as plant growth-improving gene,plant hormone secretion gene and antibacterial genes predicted by bioinformatics analysis.These data provided a theoretical basis for the better application of Streptomyces 5406 in agriculture.At the same time,it promoted the second time development and utilization of Streptomyces 5406 to prevent soil-borne diseases. |
