The Polyphasic Taxonomy Of A New Species Of Glycomyces And Analyses Of CRISPR/Cas System Diversity In Glycomyces

Southern Xinjiang sufficient duration of sunshine,high temperature,drought and dry climate,forming a typical extreme salinity environment,which is rich in actinomycetes new species.In this study,new species of Glycomyces from saline environment were multiphase taxonomic identification and to determine its classification status;at the same time,Glycomyces strains were used as research materials,the structure of CRISPR/Cas system loci was analyzed through the whole genome sequencing,CRISPR structure and the diversity and homology of Cas protein through bioinformatic analysis.In order to explore the mechanism of the immune system of CRISPR/Cas in Glycomyces,a Streptomyces phage was isolated in the environment of Xinjiang.It provided a theoretical basis for finding phages to infect Glycomyces.The main research results as follows:1.Multiple classification identification of a new species of TRM 41368Optimum growth salt concentration of strain TRM 41368 was 5%,optimum growth pH was pH 8,optimal growth temperature was 37?.Negative for decomposition of urea and cellulose,melanin production,starch hydrolysis,oxidase reaction.Positive for gelatin liquefaction,H₂S production,milk coagulation and peptonization,catalase production,nitrate reduction.Chitosan,melezitose are utilized as sole carbon sources.The whole-cell hydrolysates contain meso-diaminopimelic acid as the cell-wall diamino acid,and xylose,glucose,ribose,as the major whole-cell sugars.The diagnostic phospholipids are PC,DPG,PI,PIM,PG,PE.The predominant menaquinone is MK-9?H₆?.The major cellular fatty acids are C_18:1?9c,iso-C_16:0,C_16:0,anteiso-C_17:0,anteiso-C_15:0.The G+C content of the genomic DNA of the type strain is 69.9 mol%.It had a relatively low DNA-DNA relatedness value with G.fuscus TRM 49117^T?ANI=70.59%?.On the basis of the polyphasic evidence,the strain TRM41368^T should be designated as a novel species of the genus Glycomyces,for which the name Glycomyces xiaoerkulensis sp.nov.is proposed.2.Bioinformatic analysis of CRISPR system structures in GlycomycesThe CRISPR system loci in the genomes of 15 Glycomyces strains were analyzed by BLAST searches,multiple sequence alignment,conservation analysis,RNA structure prediction,and sequence similarity searches using HMMER.CRISPR systems were identified in all the analyzed Glycomyces genomes and were divided into two classes,CRISPR1 and CRISPR2.In CRISPR1,intact CRISPR/Cas systems were found in nine of the Glycomyces strains and were classified as four types,type I-A,type I-E,type III-A,and type III-B.In CRISPR2,no intact CRISPR/Cas system was found in the other six strains.The leader sequences of the CRISPR1 loci in the nine strains had high GC content and no obvious palindromes.The Cas proteins were found to be strain-specific through structure prediction and analysis.In conclusion,the structural characteristics of CRISPRs in Glycomyces were elucidated,and their CRISPR/Cas systems were grouped for the first time.The results will provide evidence for the study of Glycomyces-related immunologic mechanisms and CRISPR-induced biological functions,and establish a foundation for uncovering the relationship between host and phage or plasmids.3.Isolation,identification and physiological property of phage TRMP45540In order to explore the function and mechanism of CRISPR/Cas system in Glycomyces,we selected the Streptomyces species from actinomycetes as the host bacteria.We used Streptomyces luteus?TRM 45540?as the host to isolate phage from soil from the typical salt environment of Xinjiang,and to study on its biological characteristics.Furthermore some physiological properties such pH stability,thermal stability,multiplicity of infection?MOI?and one step curve were also reported.Phage TRMP45540 with a head diameter of about 5mm.TRMP45540 of the head was about 71 nm and the tail length was about 285 nm,which belonged to the long tail phage.It has a strong endurance of temperature 50°C,its optimum pH value and MOI were about 6.0-7.0 and 0.001 respectively.The phage growth cycle with a detected latent period of 20 min with lytic period of 130 min.TRMP45540 specially infected Streptomyces,it is probably a newly phage.