Identification Of Novel Species Of The Genus Sphingomonas And Micromonospora From The Glacial Frontiers And The Radiation-resistant Mechanisms Of Micromonospora Sp.LH3U1

The damages caused by radiation to human beings as well as the environment cannot be ignored,and the problems of radiation protection and radiation damage repair are receiving more and more attention.Microorganisms are the most radiation resistant organisms in the earth,and the high altitude glacial frontiers can receive more intense ultraviolet radiation and cosmic rays,and microorganisms living in this environment have a strong resistance to radiation.In addition,the discovery and identification of novel species is important for microbial taxonomy,novel species of microorganisms with radiation resistance may have special survival mechanisms and metabolites,and the study of radiation resistance properties and mechanisms of novel species of microorganisms can provide strain resources and research basis for radiation damage protection studies.In this study,two strains of bacteria with radiation resistance were isolated from Laohugou Glacier No.12 in Qilian Mountains and the East Rongbuk Glacier on the north slope of Mount Everest,and identified as potential novel bacterial species by phylogenetic analysis.On the basis of conducting Polyphasic taxonomic analysis for novel species identification,the strains with higher radiation resistance were selected based on the assessment of radiation resistance,and their radiation resistance mechanisms were investigated by genomic-based radiation resistance characterization and metabolomic analysis after radiation stress.The main findings are as follows:1.Strain S2-65 was isolated from the moraine at the glacial frontiers of the East Rongbuk Glacier on the north slope of Mount Everest,with yellow colonies,rod-shaped cells,negative Gram stain and no spore formation.The results of chemical taxonomic characterization showed that strain S2-65 was consistent with the genus Sphingomonas.The genomic DNA G+C content was 66.14%.Based on the 16S r RNA gene sequence and genomic analysis,strain S2-65 clustered with the reference model strain in the phylogenetic tree with ANI,AAI and d DDH values below the prokaryotic species delimitation threshold.Therefore,strain S2-65 was identified as a novel species of the genus Sphingomonas and was named Sphingomonas everestensis.2.Strain LH3U1 was isolated from the moraine at the glacial frontiers of the Laohugou Glacier No.12 in Qilian Mountains,with positive Gram stain,orange colony,dry and raised surface,branched mycelium,and black ellipsoidal spores formed at the end.Strain LH3U1The results of chemical taxonomic characterization indicated that it was consistent with the genus Micromonospora.Based on 16S r RNA gene sequences and genomic analysis,strain LH3U1 clustered with the reference model strain in an evolutionary developmental tree with ANI,AAI and d DDH values below the prokaryotic species delimitation threshold.The genomic DNA G+C content was 70.90%.Therefore,LH3U1 was identified as a novel species of the genus Micromonospora and was named Micromonospora radiodurans.3.The radiation resistance of strains S2-65 and LH3U1 were compared by the survival rate under UVC irradiation,X-ray irradiation and hydrogen peroxide stress.It was found that the survival rate of strain LH3U1 was 11.5%and that of strain S2-65 was 5.5%at UVC irradiation dose of 90 J/m~2.The survival rate of strain LH3U1 was 70.2%and that of strain S2-65 was only 4.7%at X-ray irradiation dose of 300 Gy.Under hydrogen peroxide stress,strain S2-65 survived only at a concentration of 14 m M or less,while strain LH3U1 survived at a concentration of 150 m M or less.The results showed that strain LH3U1 was more resistant to radiation than S2-65 and had stronger resistance to radiation.4.Genomic-based radiation resistance characterization of strain LH3U1,which has high radiation resistance,revealed through functional gene annotation,pangenome analysis,and secondary metabolite prediction that many genes capable of repairing radiation damage exist in strain LH3U1,including genes related to DNA repair(e.g.rec F,rec N,uvr C,etc.)and genes encoding antioxidant enzymes(e.g.kat G,sod N,etc.).Secondary metabolites predicted four clusters of genes encoding terpenoids.It is suggested that the anti-radiation function of this strain works through a combination with repair for DNA damage and antioxidant repair of scavenging the ROS.5.Metabolomic study of strain LH3U1 after radiation stress was performed to analyze the metabolic pathways and differential metabolites before and after irradiation.The cellular metabolism after radiation stress was mainly based on the metabolism of lipids and organic acids and their derivatives,with lipids being components of the protoplasm layer and serving as a defense against radiation,and organic acids being intermediates for the synthesis of terpenoids.Fifty-nine named differential metabolites were screened,among which oxidized lipids such as oxidized phosphatidylserine(PS)and oxidized phosphatidic acid(PA)and some terpenoids(Spirolide E,5-Hydroxycapsanthin-5,6-epoxide,etc.)were significantly different and up-regulated in expression,indicating that ionizing radiation caused peroxidation of lipids on membranes.The terpenoids were able to scavenge the ROS and synthesize pigments capable of absorbing radiation energy,so they play an important role in the radiation-resistant function of strain LH3U1.