Identification And Genomic Analysis Of Three Novel Bacterial Species From The Intestinal Tract Of Marmota Himalayana

Objective:To adapt to the extremely harsh ecological environment of the Qinghai-Tibetan Plateau,the Marmota Himalayana had a unique natural adaptive microflora structure.This project used the strategy of culturomics to isolate and culture bacteria from the intestinal tract of Marmota himalayana to understand the structural characteristics of the bacterial community.Meanwhile,identification and genome analysis of novel species were carried out to evaluate potential biological implication and provide an important material basis for further exploration of biological functions.Methods:（1）Bacteria from the intestinal samples of Marmota himalayana were isolated under diverse culture conditions.By observing the colony characteristics,individual colonies were selected from the medium plate for purification.The 16S r RNA gene sequences of all purified strains were obtained,which were compared online with the NCBI and Ez Bio Cloud databases to complete the initial identification of the species.All the isolates were stored in glycerol tubes.（2）Based on the initial identification results,suspected novel species were identified and their genomes analyzed.Results include:morphological observations,optimal growth conditions,biochemical reactions,compositional analysis,phylogenetic trees construction,and genome characterization analysis.Functional genome analysis was then performed using the KEGG database and the anti SMASH online tool,combined with phenotyping experiments to explore the potential biological implications.The novel strains that completed the above identification were named,and the type strains were sent to the Guangdong Microbial Culture Collection Center and the Japan Collection of Microorganisms for getting storage numbers.Results:（1）A total of 768 strains of bacteria were isolated from the intestinal samples of Marmota himalayana,belonging to 3 phyla,6 classes,15 orders,31 families and 46 genera.The dominant genus was the Arthrobacter,followed by the genus Enterococcus and Microbacterium.The 768 strains contained 90 known species（591 strains,77%）and 26suspected novel species（177 strains,23%）.Among the 90 known species,25 had biological significance.At present,three suspected novel species of the Arthrobacter have been identified and named.（2）Based on the results of morphological observations,culture characteristics,physiological and biochemical characteristics,bacterial composition detections,phylogenetic relationship and genome analysis,it was supported that the strain pairs zg-Y453^T/zg-Y324,zg-Y462^T/zg-Y411,zg-Y809^T/zg-Y786 were three novel species of the genus Arthrobacter.They were named as Arthrobacter caoxuetaonis sp.nov.（type strain zg-Y453^T）,Arthrobacter zhangbolii sp.nov.（type strain zg-Y462^T）and Arthrobacter gengyunqii sp.nov.（type strain zg-Y809^T）.The characteristics of the Arthrobacter caoxuetaonis sp.nov.（zg-Y453^T/zg-Y324）were as follows:Gram-stain-positive,aerobic or anaerobic,catalase-positive,oxidase-negative,rod-coccus growth cycle in morphology,single bacterial colonies on the BHIB plate was pale yellow,rounded,smooth and moist in texture;the temperature range was 4-37℃（28℃was optimum）,the salt concentration range was 0-8.5%（w/v）（0.5-1.5%was optimum）,and the p H range was 6.0-11.0（p H 9.0 was optimum）;the results of 16S r RNA gene sequence comparison showed that strains zg-Y453^T and zg-Y324 belonged to the genus Arthrobacter,and were most similar to Arthrobacter luteolus（99.45%）,Arthrobacter gandavensis（98.89%）and Arthrobacter koreensis（98.69%）were followed;the ANI and DDH values between strains zg-Y453^T and zg-Y324 were 99.1%and 91.9%,respectively,but lower than95-96%of ANI value and 70%of DDH value when compared with other members of the genus Arthrobacter;the main fatty acid components are anteiso-C_15:0 and iso-C_15:0;the main respiratory quinone is MK-9（H₂）and MK-8（H₂）;the main polar lipid components include diphosphatidylglycerol,phosphatidylglycerol,phosphatidylinositol,four unknown glycolipids,two unknown phospholipids,and two unknown lipids;cell wall sugar includes ribose,mannose,glucose and galactose;the main peptidoglycan amino acids include alanine,glutamic acid,threonine and lysine.The characteristics of the Arthrobacter zhangbolii sp.nov.（zg-Y462^T/zg-Y411）were as follows:Gram-stain-positive,aerobic or anaerobic,catalase-positive,oxidase-negative,rod-coccus growth cycle in morphology,single bacterial colonies on the BHIB plate was yellow,rounded,smooth and moist in texture;the temperature range was 4-37℃（28℃was optimum）,the salt concentration range was 0-8.5%（w/v）（0.5%was optimum）,and the p H range was 6.0-10.0（p H 8.0 was optimum）;the results of 16S r RNA gene sequence comparison showed that strains zg-Y462^T and zg-Y411 belonged to the genus Arthrobacter,and were most similar to Arthrobacter luteolus（99.17%）,Arthrobacter gandavensis（99.03%）and Arthrobacter yangruifuii（98.69%）were followed;the ANI and DDH values between strains zg-Y462^T and zg-Y411 were 98.7%and 88.9%,respectively,but lower than95-96%of ANI value and 70%of DDH value when compared with other members of the genus Arthrobacter;the main fatty acid components was anteiso-C_15:0;the main respiratory quinone is MK-8（H₂）;the main polar lipid components include diphosphatidylglycerol,phosphatidylglycerol,phosphatidylinositol,five unknown glycolipids,two unknown phospholipids,and three unknown lipids;cell wall sugar includes rhamnose,ribose,mannose,glucose and galactose;the main peptidoglycan amino acids include alanine,glutamic acid,threonine and lysine.The characteristics of the Arthrobacter gengyunqii sp.nov.（zg-Y809^T/zg-Y786）were as follows:Gram-stain-positive,aerobic,catalase-positive,oxidase-negative,rod-coccus growth cycle in morphology,single bacterial colonies on the BHIB plate was yellow,rounded,smooth and moist in texture;the temperature range was 4-37℃（28℃was optimum）,the salt concentration range was 0-8.5%（w/v）（0.5%was optimum）,and the p H range was 6.0-11.0（p H 7.0 was optimum）;the results of 16S r RNA gene sequence comparison showed that strains zg-Y809^T and zg-Y786 belonged to the genus Arthrobacter,and were most similar to Arthrobacter citreus（99.52%）,Arthrobacter luteolus（98.48%）and Arthrobacter yangruifuii（98.34%）were followed;the ANI and DDH values between strains zg-Y809^T and zg-Y786 were 98.3%and 85.0%,respectively,but lower than 95-96%of ANI value and 70%of DDH value when compared with other members of the genus Arthrobacter;the main fatty acid components was anteiso-C_15:0 and iso-C_15:0;the main respiratory quinone is MK-8（H₂）;the main polar lipid components include diphosphatidylglycerol,phosphatidylglycerol,phosphatidylinositol,three unknown glycolipids and four unknown phospholipids;cell wall sugar includes ribose,mannose,glucose and galactose;the main peptidoglycan amino acids include alanine,glutamic acid,threonine and lysine.（3）The whole genomes of type strains zg-Y453^T,zg-Y462^T and zg-Y809^T were sequenced with a genome size of 3,555,050 bp,3,464,001 bp and 3,691,056 bp,respectively.The GC contents was 65.4%,65.6%and 64.7%,respectively.We found that the genomes of the three type strains all contained flagellar genes（fli DEFGJKMNOPQRS,flg BCEKLN,Flh A and Mot AB）.Flagellar constructures were observed under transmission electron microscopy,and motility phenotype tests were positive.There are genes associated with low temperature adaptation,which are involved in three metabolic pathways（ko00940:phenylpropanoid biosynthesis,ko00040:pentose and glucuronate interconversions and ko00500:starch and sucrose metabolism）,which were consistent with their phenotypes that could grow at 4℃.The anti SMASH was used to predict the secondary metabolites of the genome sequence,and the results showed that the three type strains had carotenoid synthesis gene clusters,and the expression of carotenoid components was confirmed by bacterial carotenoid ELISA kit.Conclusion:（1）768 strains of bacteria were isolated from the intestinal samples of Marmota himalayana,and 25 of the 90 known species had biological significance such as potential pathogenic,prebiotic or biodegradable functions;the 177 suspected novel species could be divided into 26 genera,and the dominant genus was the Arthrobacter.（2）This study completed the identification and naming of three novel species of the genus Arthrobacter.The strain pair zg-Y453^T/zg-Y324 named Arthrobacter caoxuetaonis sp.nov.,the type strain was zg-Y453^T（=GDMCC 1.2809^T=JCM 35173^T）.The strain pair zg-Y462^T/zg-Y411 named Arthrobacter zhangbolii sp.nov.,the type strain was zg-Y462^T（=GDMCC 1.2880^T=JCM 35170^T）.The strain pair zg-Y809^T/zg-Y786 named Arthrobacter gengyunqii sp.nov.,the type strain was zg-Y809^T（=GDMCC 1.2808^T=JCM 35168^T）.The genomic analysis of strains zg-Y453^T,zg-Y462^T and zg-Y809^T revealed the existence of genes related to dynamic,low temperature growth tolerance and carotenoid synthesis,but their specific functions and applications need to be further studied.