Taxonomic And Biological Analyses Of Luteimonas Yindakuii Sp.nov.and Nocardioides Dongxiaopingii Sp.nov.isolated From Plants On Qinghai-tibetan Plateau

Objective:Due to the special climatic conditions and geomorphic characteristics of the Qinghai Tibet Plateau,a special alpine ecosystem has been formed.The plant resources are typical and unique.The microorganisms existing in the extreme environment may have spec ial functions.Studying the wild plant microorganisms in the Qinghai Tibet Plateau can not only expand the understanding of the microbial community,but also provide a source for the development of new structures and new functional bioactive substances.In this study,according to the strategy of culturomics,we analyzed the flora of wild plants in the Qinghai Tibet Plateau,found out the potential human pathogens and animal/plant pathogens,isolated and identified the unknown bacteria,analyzed their possible biological functions,and provided the basis for the future research of biological agents and industrial and agricultural applications.Methods:?1?The leaves of plants on the Qinghai Tibet Plateau were ground into uniform suspension,and then the suspension was diluted by gradient.The appropriate gradient diluent was applied to different kinds of culture media under different conditions by the method of isolation and culture.According to the colony morphological characteristics,single colony was selected and purified.Then the 16S rRNA gene sequence was amplified and sequenced.The obtained 16S rRNA gene sequences of the strains were aligned by GeneBank and EzBioCloud databases to complete the preliminary identification.?2?For the known species of bacteria,the diversity of strains was analyzed.?3?For the potential new species of bacteria,the physiological and biochemical characteristics were analyzed,including cell shape,colony morphology,optimal growth conditions,cell chemical composition etc.Genomic analyses were also applied to identify the taxonomic position of the new species,such as phylogenetic trees,nucleic acid molecular hybridization.The function of protein was predicted by GO,COG,KEGG and Swiss prot databases.The new species of bacteria were named and the type strains were sent to China General Microbiology Culture Collection Center and Japan Collection of Microorganisms for preservation,and then the preservation numbers were obtained.Results:?1?In this study,1068 strains of bacteria were isolated from plant leaf samples of the Qinghai Tibet Plateau.They are distributed in 4 phyla,6 classes,18 orders,37 families and86 genera.The dominant genera are Rhodococcus,Clavibacter,Microbacterium and Curtobacterium.The potential human pathogens and animal/plant pathogens are as follows:Curtobacterium flaccumfaciens?73 strains?,Enterococcus mundtii?3 strains?,Micrococcus endophyticus?1 strain?,Pantoea agglomerans?3 strains?,Sanguibacter inulinus?19 strains?,Sanguibacter keddieii?9 strains?,Xylophilus ampelinus?1 strain?.There are 50 potential new species of bacteria?71 strains?,which can be divided into 24 genera.At present,two new species of bacteria,Luteimonas?S-1072^T and 1626?and Nocardioids?S-1144^T and 4053?,have been identified and named.?2?Two novel bacterial strains?S-1072^T and 1626?were identified by phenotype,physiology and biochemistry,phylogenetic analysis of 16S rRNA gene sequences,genomic characteristics and genomic evolution analysis.The results showed that strains S-1072^T and1626 were a new species of Luteimonas,named as Luteimonas yindakuii sp.nov.,and the type strain was S-1072^T?=CGMCC 1.13927^T=JCM 33487^T?.Strains S-1072^T and 1626 were isolated from the leaves of dandelion?Taraxacum officinale?in the Tibetan Plateau.Dandelion can be used as a Chinese herbal medicine and food.Strains S-1072T and 1626were aerobic,gram-negative,rod-shaped,oxidase and catalase positive,motile with a single flagellum.The optimum growth condition was 28?,pH 7.0,0.5%?w/V?NaCl.Phylogenetic analysis based on 16S rRNA gene sequences showed that strains S-1072T and1626 belong to the genus Luteimonas,which were most similar to Luteimonas arsenica CCTCC AB 2014326^T?97.0%?,Luteimonas terricola CGMCC 1.8985^T?96.9%?and Luteimonas aestuarii KCTC 22048T?96.6%?.The 16S rDNA sequences similarity between our two strains was 99.9%showing that they were in the same species.Phylogenetic analysis of the whole genome revealed that S-1072T was closest to Luteimonas abyssi CGMCC1.12611T.The DNA G+C contents of S-1072T and 1626 were 69.2mol%and 69.3mol%,respectively.The value of digital DNA-DNA hybridization between S-1072T and 1626 was83.1%?higher than the threshold of bacterial species?,and the values were lower than the threshold of 70%.Q-8?98.7%?was the predominant respiratory quinone;the main polar lipids included diphospholidylglycerol,phosphatidylglycerol,phosphatidylethanolamine;the major fatty acids?>10%?included iso-C_16:0,iso-C_15:0,and summed feature 9(10-methyl C_16:06:0 and/or iso-C_17:1?9c).?3?Two novel bacterial strains?S-1144^T and 4053?were identified by phenotypic,physiological and biochemical analysis,phylogenetic analysis of 16S rRNA gene sequences,genomic characteristics and genomic evolution analysis.The results showed that strains S-1144^T and 4053 were a new species of Nocardioides,named as Nocardioides dongxiaopingii sp.nov.,and the type strain was S-1144^T?=CGMCC 4.7568^T=JCM 33469^T?.Strains S-1144^T and 4053 were isolated from the leaves of Lamiophlomis rotata,an important medicinal plant in the Tibetan Plateau.Strains S-1144^T and 4053 were aerobic,gram-positive,catalase positive,oxidase negative,irregular rod-shaped bacteria.After cultured on R2A medium for2 days,the colony morphology was light yellow,transparent,round,smooth and convex.The optimum growth condition was 28?,pH 7.5,0%?w/V?NaCl.The results of polyphase analyses showed that strains S-1144^T and 4053 belong to the genus Nocardioides,which were most similar to Nocardioides litoris DSM 103718^T?98.4%?,Nocardioides rubriscoriae DSM 23986^T?98.2%?and Nocardioides plantarum DSM 11054^T?97.8%?.The DNA G+C contents of the isolates were both 73.5 mol%.The value of digital DNA-DNA hybridization between strains S-1144^T and 4053 was 90.2%?higher than the threshold of bacterial species?,and the values were lower than the threshold of 70%._LL-2,6-diaminopimelic acid was the diagnostic amino acid in cell wall peptidoglycan;the main respiratory quinone was MK-8?H₄?;the polar lipid was composed of diphospholidylglycerol,phosphatidylglycerol,three unidentified phospholipids,one unidentified glycolipid and one unidentified lipid;the main fatty acids included iso-C_16:0,C_17:1?8c,C_17:0 and C_18:1?9c.?4?According to the genome analysis results,the genome size of strains S-1072^T and S-1144^T were 3,040,302 bp and 4,427,648 bp with 2,685 and 4,011 coding genes respectively.Through functional clustering analysis compared with COG,KEGG and Swiss prot databases,it was found that there were genes related to cold adaptation in the chromosome of strains S-1072^T and S-1144^T[genes encoding heat shock protein,cold shock protein and molecular chaperone?DnaJ-DnaK-GrpE?GroESL?,DEAD-box RNA helicase gene?dead?,etc.];radiation resistant genes?homologous recombination related genes:recABCDFGJOR,ruvABC,ssb,priA;excision repair related genes:uvrABCD,mfd,genes encoding glycosylase and endonuclease;base mismatch repair related genes?.In addition,genes for the biosynthesis of betaine,isoflavones,zeatin and siderophore group nonribosomal peptides were also annotated in KEGG database.Conclusion:?1?In this study,1068 strains of bacteria were isolated from plant leaves of the Qinghai Tibet Plateau.They are distributed in 4 phyla,6 classes,18 orders,37 families and 86 genera.There are 109 possible human pathogens and animal/plant pathogens.71 strains are potential new species,which can be divided into 24 genera and 50 species.?2?Four suspected new strains were identified.Strains S-1072^T and 1626 were identified as a new bacterial species of the genus Luteimonas,named as Luteimonas yindakuii sp.nov.,and type strain is S-1072^T?=CGMCC 1.13927^T=JCM 33487^T?.Strains S-1144^T and 4053 were identified as a new species of the genus Nocardioides.They were named as Nocardioides dongxiaopingii sp.nov.,and the type strain is S-1144^T?=CGMCC4.7568^T=JCM 33469^T?.The genome analyses of strains S-1072^T and S-1144^T revealed the existence of genes related to low temperature adaptation,radiation resistance and the synthesis of secondary metabolites with physiological functions,but the function of these genes and the application of coding products still needed further study.