Bacterial Community Diversity In The Vertical Climatic Zones Of Mingyong Glacier And Retreat Region

Mingyong Glacier located in the northwestern of Yunnan Province belongs to the Meili Snow Mountain which is a branch of Hengduan Mountains near Qinghai-Tibet Plateau where is famous for its higher biological diversity in the world. It is the only monsoon glacier with low latitude and high altitude in China. In a very narrow area without any geographical isolation, the altitude dropped down nearly 5,000 m but horizontal distance is less than 15 km. This area is composed of a series climatic zone of glacier, forests and dry warm valleys, and all the biological diversities are gathered in this very narrow area. In this paper, the ecological distribution of microbes in these vertical climatic zones of Mingyong Glacier and its retreat region was studied; especially the composition and structure of bacterial community was investigated.Firstly, based on traditional microbial pure culture, total 37,513 strains of bacteria were isolated from the samples of Mingyong Glacier by using four different media. According to colony morphology.391 strains were identified, most of them can grow at 4? to 37?, part of them can only growth at 4? to 25?. PYGV medium was better than eutrophic medium of LB and Organnic in isolating bacteria in this area, which indicated that oligotrophic culture medium, should be the best medium for isolation and identification of bacteria in glacier region. Most of the isolates were Gram-negative bacteria. In additional, all of them fall into four pylums of Proteobacteria. Actinobacteria. Firmicutes. Bacteroidetes, four classes of y-Proteobacteria, Actinomycetes, Bacillus, Flavobacteria, seven orders of Pseudomonas. Enterobacteriaceae, Xanthomonas, Streptomyces, Micrococcus, Bacillus, Flavobacterium, seven families of Pseudomonas, Enterobacteriaceae, Xanthomonas, Streptomyces, Brevibacteraceae, Bacillus, Flavobacterium and seven genus of Pseudomonas, Yersinia, Stenotrophomonas, Streptomyces Brevibacterium,Bacillus and Flavobacterium. Among them, more than 35% of identified bacteria were Pseudomonas, and the rest 65% identified bacteria were distributed into 6 genara. At the end, six cold-active bacteriophages were isolated and characterized by using isolated bacteria as host cell at low-temperatures. All isolated phages are cold-active phages and they can proliferate at 4?.In order to explore new suitable classification and identification methods for low temperature bacteria in this area, a GC-MS method based on composition of unsaturated fatty acids in membrane was used to identify cold-adapted bacteria. Total of 12 low-temperature bacteria from three different climatic zones were analyzed. The clustering analysis results showed that the most of the bacterial classification were consistent with the results of 16S rRNA gene construction. It demonstrated that this method can be used to classify low temperature bacteria scientifically. Furthermore, the analysis of unsaturated fatty acids in membrane could provide more precise taxonomic information than 16S rRNA gene analysis, which is suitable to distinguish the low temperature bacteria at the same genus in phylogenetic relationship. The results also demonstrated that the proportion of unsaturated fatty acids in those cold-adapted bacterial cell membranes was inversely proportional to temperatures, the lower the temperature, the higher content of unsaturated fatty acids. Although most of the unsaturated fatty acid composition was similar to other reported low temperature bacteria, but the fatty acid composition of some low temperature bacteria is not the same, which deserves further study in future.In order to investigate the composition and structure of bacterial communities in different climatic zones in Mingyong glacier region, the 16S rRNA gene V6 hypervariable region sequencing was used to analyze the unculturable bacteria in 11 soil samples from this area. Four representative samples distributed in three vertical climatic zones were analyzed. The results showed that all bacteriacan be divided into 21 phyla.42 classes and 79 orders. The dominant bacteria included five phyla such as Proteobacteria, Deinococcus-Thermus, Firmicutes, Actinobacteria and Nitrospirae. The Verrucomicrobia was the unique phylum in Mingyong glacier and have not been reported in other glacial studies yet. The bacterial community composition in different climatic zones was analyzed by 16S rRNA gene V6 hypervariable region sequencing. Significant differences in the bacterial composition at different climatic zones were observed. These differences can be identified even at the level of phylum or class, which showed that each climatic zone has its unique bacterial community in this area. It is hard to classify the bacterial communities exactly at the level of family, due to a large number of species and genus included at this level. At the genus level, Corynebacterium, Propionibacterium and Streptococcus are dominant bacterial communities among the top ten genera, which indicates that they were the most widely distribution in this region and had better adaptability to the environment. Pseudomonas can be found in all samples, but it occupied one position among the top ten genera only in samples from cold-temperate zone and near Frigid Zone. These results showed that Pseudomonas is one of the most important populations in this area. This result was consistent with the results of culturable bacterial community.At the end of this study, a metagenomic method was used to analyze the soil sample from the cold temperate zone which was located between the mid-temperate and the frigid zones. After analysis of 30G sequencing data by this method, total of 44 at level of phylum and 49 classes bacteria have been identified, which indicated a very high bacterial diversity in this region. In terms of community composition, compared whit the result from V6 hypervariable region sequencing method,6 kind of bacteria has been found at the phylum level,5 kind of bacteria at classes level,3 kind of bacteria at order level can be identified with these two methods, but the proportion of each bacteria was different, and at the family or under the family level, the composition of species is signficantly different. The reason lead to these differences can be concluded below:Firstly, a PCR amplification process is involved in 16S rRNA sequencing analysis, and different bacterial template DNA and optimal conditions will give different yield of product. Secondly, two analyses used different methods in species annotation. In general, metagenomic coverage of annotated species should be far greater than that achieved by 16S rRNA gene V6 hypervariable region sequencing analysis, so metagenomic method should be better inanalysisof microbial community in complex ecological environmental samples than other methods, especially be beneficial to investigate the composition of microorganism in a special ecological environment. But this technology is significantly limited by its high price. In the study of metabolic pathways using metagenome sequencing data, we found that the hot-spot signaling pathways in bacteria in this region were concentrated in three aspects:environment information processing, genetic information processing and metabolism. The genes involved in metabolism and transport of amino acids, signal transduction and production of energy are listed ont the top three among all functional genes, which is consistent with the hot-spots in the signal transduction pathway.All in all, our study can be summarized as below:in traditional pure culture, all isolated bacteria from 11 samples can are divided into 4 phyla and 4 classes. In 16S rRNA gene V6 hypervariable region sequencing analysis all bacteria from 4 samples can be divided into 21 phyla and 42 classes by 16S rRNA gene V6 hypervariable region sequencing. Meanwhile, in metagenome sequencing analysis, all bacteria from sample of cold temperate zone can be divided into 44 phyla and 49 classes. From the above results we can concluded that three methods have their own characteristics:pure culture method is mainly suitable for collection of culturable microorganisms but limited in providing information in structure of bacterial communities. However,16S rRNA gene V6 hypervariabie region analysis can provide a large of bacterial community information quickly and economically, but it cannot provide any bacterial-related metabolic pathway and functional genes information. All in all, the metagenomics method can provide more information in bacterial communities, metabolic pathways and functional genes, and the method of species annotation can also be extended to bacteria other than the microbial population, but it is also be limited by its high operation price. Furthermore, the complexity from samples, bioinformatics analysis technique and depth of the sequencing also are important factors in obtaining the good results. Of course, with the continuous development of the technology, we believe that metagenomic analysis method will be an important mean for community and metabolic analysis in future.In this paper, the bacterial community distribution in different climatic zones of Mingyong glacier was systemically investigated and analyzed by pure culture, culture-free methods, membrane unsaturated fatty acid composition analysis and metagenomic analysis. This is the first report about the composition and diversity of bacterial community in Mingyong Glacier region. The distribution of bacterial communities among different vertical climatic zones was studied by using a variety methods. It's a systematic microbial investigation and resource collection in this area, which provide a good chance to allow human beings to comprehensive understanding the unique bacterial community structure in this area.The results showed that the GC-MS method based on composition of unsaturated fatty acids in membrane was used to identify the cold-adapted bacteria can provide more detailed scale taxonomic information than 16S rRNA gene sequence analysis. This method should be an important method for the classification of microbes, especially low temperature bacteria. The method is also expected to be used for fine classification of low temperature bacteria in other areas.In this study, a batch of culturable cold-active bacteriophages and their host bacteria were isolated and collected, which could be used to study cold-adapted mechanism of bacteria, phage-host interaction and phage-host co-evolution. The results of this study not only provide comprehensive understanding of microbial ecology in this unique geographical area, but also provide valuable information and resources for further studies in biogeochemistry, microbial ecology and application of microbial resources in this area.