| Hot spring was a kind of hot environment on earth and many thermophilic microbes survived in hot springs around the world. Thermophilies in hot springs varied a lot because of the locations. So research of thermophilies in hot spring among different areas is important for further understanding of this kind of ecosystem。Geobacillus spp. were often predominant bacteria among cultural hot spring bacteria and also widely distributed in other hot environment such as compost. Recent years more and more Geobacillus strains were isolated from cool temperature soils, researchers begin to pay more attention to this question that which internal mechanism give this genus ability to survive on low temperature. In this study, we first researched the thermophilies diversity of three hot spring in Fuzhou, then we did some work to explain how the Geobacillus adapting low temperature.Thermophilies is a kind of microbes with high importance and more and more attentions were attracted for their survive mechanisms and broad applications. There are many hot springs around Fuzhou areas and they are suitable habitat for thermophiles. Illumina MiSeq platform was applied to identify the bacteria diversity of the three hot spring waters:DaTang, TangCheng and ShuangLong. The main thermophilic bacteria in three hot spring waters were Proteobacteria, Firmicutes, Aquificae, Nitrospirae, Armatimonadetes, Thermi, GAL15, Acidobacteria, Chlorobi, Actinobacteria, Chloroflexi, OP1, OD1, Bacteroidetes and EM3. The thermophilic bacteria diversity was rich in the three waters but showed much variation among them. Pearson correlation and RDA analysis show that may result from the differences of their geographic position, water temperature and water chemical composition. Cultural thermophiles were studied of three hot springs Datang, Tangcheng and Shuanglong. Results showed that there were more thermophiles in Dangtang than Tangcheng, and lest in Shuanglong. Further research showed that diversity of thermophiles was closely related with pH, effective nitrogen and available phosphorus levels of the hot springs. Thremophiles diversity was richer in hot springs with Lower pH, higher level effective nitrogen and available phosphorus. A total of 32 different strains were isolated from the three hot springs and Genus Geobacillus and Genus Anoxybacillus were the main thermophiles. Compared with the cultural results, we found only strains of Firmicutes were cultured and other strains couldn’t be cultured in laboratory. To further study the function of the strains in these hot spring, optimization of cultural conditions or metagenomics may be used in the future.A thermophiles strains Geobacillus sp. was identified and sequenced. This strain had a growth termperature range from 47℃ to 74℃, and the optimum growth temperature was 60℃.16S rDNA gene sequences of CHB1 was 99.26% similar with that of Geobacillus thermoglucosidasius DSM2542T. G+C value of this strain is 42%. Genomic hybridization value with type strain Geobacillus thermoglucosidasius DSM2542T was 75%. But there was some differences on physiological characteristics and fatty acids. So this strain was identified as a subsp of Geobacillus thermoglucosidasius. Genome size of this strain was about 3.22 M and there were 4122 open reading frames in this genome.To research the mechanism of Geobacillus sp. CHB1 adapting lower temperatures, experimental evolution method was used to make CHB1 growing at temperatures lower than its minimum required temperature for a long time. After six month of adaption, three evolved populations of Geobacillus sp. CHB1 could grow at 43℃ which was much higher than the ancestor strain 47℃. Evolved strains showed high growth rate compared with ancestor strain A. Fatty acid of evolved strain Ec altered a significantly when cultured at low temperaturel, especially for increasing of 15:00 iso. Proteomics resulted showed that proteins associated to nucleotide metabolism were higher expressed and that associated with fatty acid metabolism were down regulated in evolved strain Ec. The results were in accordance with the high growth rate and fatty acid alternation of evolved strain. On the basis of the first six months adaption evolution to lower temperature, Ec was selected to be evolved to lower temperatures for another 12 months. The results showed that, when cultured in liquid medium three evolved populations Fl, F2 and F3 all could grow at temperature from 38℃ to 39℃. When cultured on solid agar at 39℃, strain F3d from population F3 could grow at form colonies. These demonstrated that the minimum required temperature of Geobacillus sp. B1 decreased at least 4℃ on the basis of strain Ec after 12 months of adaption evolution to lower temperatures.We re-sequenced the population EC. F2 and F3 and found there were 15 genes mutated in both of the two populations and seven of them had different mutant position on the genes., such as MFS、DNA polymerase submit β,AB hydrolases and peptidase. They were The common mutant genes might played key roles during the process of lower temperature adaption. Two colonies F3b and Fd were selected from population F3 for re-sequencing, homozygosity SNPs were much more than population F3, which indicated that when evolved to lower temperature there were many mutations in individual strains of the population. When the strain with specific mutation hasn’t become prevalent, the mutation position of the population exhibits heterozygosity. In this evolution process there was a mutant strain with cell division was inhibited, genome re-sequencing showed a mutant in gene FtsK of this strain. These founds promoted our knowledge about how thermophilic strains adaption low temperatures.Three evolved populations all showed high catalase activity which was at least 20 folds that of ancestor strain A. This catalase was identified as monofunctional heme catalase by TOF-MS-MS and qPCR also proved high expression of this gene in evolved strains. Fermentation broth and recombinant catalase both showed above 40% relative activity at 0℃ to 40℃, so this catalase was in form of activity when evolved strains grew at low temperature. Some prior studies found catalase could improve growth ability of many mesophilic bacteria, so we think catalase high expression in this study may also enhanced Geobacillus sp. CHB1 growth ability under lower temperature.In summary, low temperature populations of Geobacillus sp. CHB1 showed enhanced ability on low temperature adaptation. For example, the minimum required growth temperature decreased about 9℃, growth rate increased significantly, alternation ability of fatty acids changed, higher catalase expression level of evolved populations and proteomics changes in nucleotide and fatty acids metabolism. Mutation genes involved in low temperature adaptation course were identified by comparative genomics. MFS transporter, DNA polymerase submit β, AB hydrolases, peptidase and others genes mutated in low temperature adaptation populations; how these genes mutation affect the growth parameters, fatty acids composition and protein expression need further study. |
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