Cloning And Expression Of Ectoine Biosynthesis Genes Of A Moderately Halophilic Bacterium Chromhalobacter Sp. NJS-2 From Antarctica Deep-sea And Preliminary Study On Biological Functions Of Ectoine

Due to its specific geographical environment and seafloor topography features, Prydz Bay in Antarctica maintains a high-salt, low-temperature and high-density characteristics. The specific ecological environment offers rich microbial resources and most of them are still unknown. They often have specific physiology to adapt to the specific circumstances. Thus, They are good materials for the study of adaptation mechanism in moderate halophile.The moderate halophile strain NJS-2 was selected from Prydz Bay deep-sea sediment and identified by 16S rDNA as Chromohalobacter sp. NJS-2. The types of compatible solutes accumulated by this strain were analyzed and identified. Data shows that it accumulates ectoine and glutamate as the main compatible solutes. To clone the genes encoding enzymes for the biosynthesis of ectoine, a number of primers were designed according to the conservative amino acid sequences. Thus, partial sequences of ectABC were cloned by PCR using the genomic DNA of this strain. After many times of PCR and inverse PCR amplification, we eventually got a 2379bp DNA fragment. Software analysis shows that this fragment contains three genes encoding enzymes for ectoine biosynthesis, ectA, ectB, ectC and their upstream and downstream sequences.The three encoding genes, ectA, ectB and ectC, were cloned and expressed in E.coli BL21(DE3). The enzymatic activities of the purified recombinant proteins, EctA, EctB and EctC, were detected by adding their specific substrates. By this means, we can verify the reaction equation and elucidate the biosynthetic pathway for ectoine in vitro. Data shows that the three recombinant proteins have similar enzymatic property.Ectoine is one of the most effective compatible solutes in protection of biological macromolecules, such as DNA or proteins. Therefore, we study the effect of ectoine secreted by NJS-2 strain on the protection of Cu-Zn superoxide dismutase against heating, the protection of Ectoine on enzyme is the better than that of proline , glycerol, trehalose , glutamic acid and betaine.We also analysed the ectoine effect on heat resistance of microorganism under high osmotic pressure by NJS-2.It has been found the ectoine accumulation in NJS-2 in the presence of 10% NaCl which increase the heat resistance of NJS-2 itself. It has been found that the Extraneous ectoine can raise the heat resistance of the protection on the living microorganisms, under specific condition , the concentration of which is related to the different microorganisms...