Cloning And Expression Of EctABC Gene Cluser From Halomonas Sp. QHL1 Isolated From Qinghai Lake

The halophiles are microorganisms surviving in the high salt environment. In the long-term evolution, halotolerant and halophilic prokaryotes living in high salt environments have developed two principal mechanisms, the salt-in-cytoplasm mechanism and the organic-osmolyte mechanism, to avoid the loss of water from the cell and achieve a cytoplasm osmotic strength equal to that of the surrounding environments. Archaea and extreme halophiles balance the external osmotic pressure by accumulation of K+and Cl’; moderate halophiles mainly adopt the organic-osmolyte mechanism. Ectoine is the most common osmolytes in aerobic moderate halophilic bacterium. As the protective agent of cell, DNA and protein structure, ectoine helps cells to resist heat stress, freezing, drying, high salinity, radiation and oxygen free radical. It gains much attention in the broden application prospects of ectoine in enzymes, PCR stabilizers, cosmetics, chemicals and pharmaceutical industry. Recently, some ectABC clusters have been cloned from many species in order to study the regulation of the salt tolerance mechanisms of halophilic bacteria in the molecular level. Salt tolerance associated genes are transformed into the plants to build genetically engineered strains and new varieties of crops. It is of great significance for improving the the saline dry soil in China. It also has an important economic value that genetically engineered salt-tolerant strains are appling to biological treatment of high-salt organic wastewater.In this study, thirty-five strains of halophiles were isolated from twenty water samples collected in Qinghai Lake by a RM medium with high salinity. The moderate halophile QHL1 was selected and studied. On the basis of its morphological and physiological characteristics, the isolate exhibited regularly round, transparent yellowish brown and medium size with a prominent center and smooth edge. Short rod, gram-negative and arranged singularly or in clusters in microscopic morphology. Strain QHL1 utilized fructose, sucrose, glucose, maltose, yeast extract, peptone, ammonium salt, urea and nitrate as the carbon and nitrogen sources, but could not use melibiose, melezitose, xylitol and sodium nitrite. MR, gelatin, H2S and indole test were negative, but oxidase test and halophilic test were positive. Strain QHL1 lived in the salinity ranging from 0.04 to 2.74 mol/L and the optimum salinity was 0.86 mol/L. QHL1 growed in the temperature of 10℃ to 45 ℃ and the optimum temperature was 37℃1. QHL1 growed in the pH of 4.5 to 10 and the optimum pH was 8.5. Based on the 16S rDNA gene sequence analysis,16S rDNA gene of strain QHL1 was 1420bp and the halophile QHL1 had a 99% similarity with Halomonas sp. TNB158. According to its morphological, physiological and biochemical characteristics combined with the sequence analysis of the 16S rDNA, strain QHL1 was preliminarily identified as a member of the genus Holomonas. The intracellular compatible solute of ectoine accumulated in QHL1 was detected by thin-layer chromatography based on the similar Rf, and the content of ectoine was determined as 269.9 μg/mL by high-performance liquid chromatography. The conserved gene of ectB from strain QHL1 was amplified by PCR. The ectABC gene cluster for biosynthesis of ectoine was further cloned by genome walking methods with specific primers. Analysis of the DNAStar software showed that ectA, ectB and ectC genes organized as an operon and the size were 579bp,1269bp and 390bp which were predicted to encode peptides of 192,422 and 129 amino acids, respectively. The deduced amino acid sequences of EctA, EctB and EctC share 57%,96% and 85% identity to 2,4-diaminobutyric acid acetyltransferase,2,4-diaminobutyric acid transaminase and ectoine synthase of Halomonas sp. Nj223 respectively according to homology analysis. Promoter sequences and position of ectABC gene cluster were predicted through online software. The ectB gene was subcloned into pET-28a to construct the recombinant expression vector pET-ectB by molecular cloning. The recombinant plasmid was transformed into E. coli BL21(DE3) and induced by IPTG, a specific protein band (about 46kDa) was observed in SDS-PAGE analysis.