The Isolation And Identification Of Halophilic Bacteria And The Proteomic Analysis Of The Adaption To Salinity Of Natranaerobius Thermophiles

Halophile is the first extremophile found by human beings. Because of its unique mechanism of adaptation to salt environment, it has potential application value in many fields.Therefore, it has great significance to carry on the research of halophiles.In this study, two halophilic bacteria BZ-SZ-XJ18 and BZ-SZ-XJ27 were isolated and purified from saline alkali Lake in Xinjiang province. The polyphasic taxonomical method was used to analysis and identify the two strains. Results from 16 S rRNA gene sequence analysis, DNA G+C mol% determination, DNA-DNA hybridization test, multilocus sequence analysis, phenotypic and growth characteristics detection and physiological and biochemical characteristics detection show that BZ-SZ-XJ18 and BZ-SZ-XJ27 were two new species belonging to genus Bacillus and genus Halomonas respectively. BZ-SZ-XJ18 was designated as Bacillus urumqiensis(=CGMCC1.12916T=DSM 29145T=JCM 30195T). BZ-SZ-XJ27 was designated as Halomonas urumqiensis(=CGMCC 1.12917 T =JCM 30202 T).The mechanism of adaptation to salt environment of Natranaerobius thermophiles JW/NM-WN-LFT(DSM 18059T) was researched by using isobaric tags for relative and absolute quantitation. 1492 proteins were detected, in which 114 proteins were up-regulated proteins and 128 proteins were down-regulated proteins. The isoelectric point of differential proteins had a wide range.The isoelectric point of the proteins in 3-6 accounted for 74%, which is related with high negative charge of the proteins. And this is the main mechanism of the halophiles’ protein to function in the salt environment. Proteins related to glycine betaine synthesis were detected. Glycine betaine is the main compatible solute of halophiles which could maintain the osmotic pressure of cells. This was consistent with the results of the determination of compatible solutes in the previous research, indicating that the bacteria can adapt to the high salt environment by using compatible solutes. Proteins related to the transportation of Na+ and K+ were also detected, which was consistent with the predicted ion pump from the genome sequencing indicating that N. thermophiles could also use "salt-in" strategy to adapt to the survival environment of the high salinity.109 up-regulated proteins were validated by using Droplet Digital PCR. According to the comparison of gene expression ratio between salinity 2.5 M and salinity 3.7 M Na+ condition, there were about 90% of the genes with more than 1.5 fold change in which 93% of genes up-regulated significantly. The results showed the consistence between the verification results in transcription level and the results in translation level. Genes with more than 10 fold change accounted for 37% indicating that these genes might have special mechanism for N. thermophiles to adapt to high salinity environment. The verification results in transcription level laid a foundation for further research on the halophilic mechanism of this strain.