Diversity Of Cold-adapted Microorganism In The Pamir Plateau And Preliminary Study On The Cold Tolerance Mechanism Of A Psychrotrophs TRM 85608

Cold-adapted microorganisms are widely distributed in many cold environments around the earth with unique cold tolerance mechanisms.It not only contributed to the circulation of natural elements,but also produced all kinds of cold-adapted enzymes and biosurfactant that have been widely used in food,detergent,feed and environmental biological treatment.The Pamir Plateau is a cold environment with infertile fields and exposed to high levels of radiation,in which cold-adapted microorganisms may be unusual and abundant.Exploring the diversity of cold-adapted microorganisms in the Pamir Plateau and the cold tolerance mechanisms of them will contribute to the targeted exploration and mining of cold-adapted microorganisms in the Pamir Plateau,improving the cold-adapted tolerance of microorganisms,and realizing the full utilization of cold-adapted microbial resources.Based on culture-independent method of 16S r RNA gene amplicon sequencing technology and culture-dependent technology of isolation and culture under low temperature,72 soil samples from four altitudes(1000-2000 m?2000-3000 m?3000-4000 m?4000-5000 m)in the Pamir Plateau were investigated for microbial diversity.The results showed that 11 051 494 effective sequences and 186 059 OTUs were obtained,which were assigned to 1 176 genera of bacteria and 39 genera of archaea.The dominant genus in bacterial domain was Ralstonia?Flavobacterium?Tychonema and Eubacterium when the elevation gradient from low to high.In addition,Candidatus Nitrososphaera was the dominant genera of the archaea domain in four elevations.Among the culture-independent result,we found that Nocardioides and Solirubrobacter were positively correlated with altitude,while Arenimonas were negatively correlated with it.At the same time,p H and the content of P were related to the community composition of bacteria,and p H and the content of NO₃^-were associated with the community composition of archaea in the Pamir Plateau soil.Based on culture-dependent method,a total of 487 strains of cold-adapted bacteria and 25 strains of archaea were isolated,which belonging to 61 genera and 148 species,including 37 potential new species.Moreover,the results indicated that TSB and R2A medium had a good isolation effect of cold-adapted bacteria,in especial for the genus of Pseudomonas.NOM?F6 and 12%MGM medium have lower isolation efficiency for archaea.We only obtained two genera of the archaea,which belonging to Natrinema and Haloterrigena.The results of culture-independent and culture-dependent showed that there were abundant microbial resources in the Pamir Plateau,and the composition of microbial community might be affected by altitude and environmental physicochemical factors among four elevation gradients.Based on RNA-Seq and SMRT-Seq,the cold tolerance mechanisms of strain Exiguobacterium undae TRM 85608 was studied.The results showed that not only the change of metabolic pathways,as material transport?energy metabolism and quorum sensing played a major role in the formation of cold tolerance of strain E.undae TRM 85608 under cold stress.In addition,the correlation analysis between DNA methylation levels and m RNA expression levels indicated that there were 475 genes whose expression levels were up-regulated and unmethylated,and RT-q PCR validation results showed that expression levels of10 cold response correlated genes(fad M,crt Nb,sig V,met I,gro S,IRC3,csp E,btr,cue R and ahp C)were all up-regulated.Besides,the down-regulated expression results of DNA adenine methyltransferase gene illustrated that it might affected the DNA methylation level of the genome and the expression level of genes by weakening the catalytic ability.The results manifested that the DNA adenine methyltransferase played a key regulatory role at low temperature.In a word,DNA methylation is associating with the cold tolerance of E.undae TRM 85608.By exploring the composition and relative abundance of cold-adapted microbial community in the Pamir Plateau,preliminary studying the changes of microbial metabolic pathways and the relationship between DNA methylation and cold tolerance of cold-adapted microorganisms.This study provided theoretical support for the development and utilization of low temperature microbial resources and ecological function research in the Pamir Plateau,and laid a theoretical foundation for the comprehensive analysis of cold tolerance mechanisms for cold-adapted microorganisms.