The Identification Of Chromohalobacter Sp. DSM 22428~T And The Study Of Its Betaine Aldehyde Dehydrogenase

Strain DSM 22428T was a endophytic moderately halophilic bacterium isolated by our lab. The organism grew at NaCl concentrations in the range of 0.6-20%(w/v), which had a wider salt tolerance and had better adaptability to the violent change of outside salt concentration than halophilic archaea. The previous study of our lab confirmed that strain DSM 22428T can resist high osmotic stress through the accumulation of osmoprotectant, glycine betaine in the cell. Glycine betaine can function as the most effective osmoprotectant accumulated by bacteria, plants and animals. The betB of plants had been studied deeply and was the focus of plant resistance engineering. The gene sequences of BADH are quite different in different species, which results in the different osmotic adjustment ability of bacteria. In bacteria, glycine betaine is synthesized by choline dehydrogenase and betaine aldehyde dehydrogenase,and the latter is the key enzyme of glycine betaine synthesis. In addition, BADH also plays an important role in pathogen invasion. Therefore, the properties study on BADH of strain DSM 22428T will help understand its characteristics deeply and have important significance for clarify salt tolerance mechanism of strain DSM 22428T. The previous study of our lab found that strain DSM 22428T was a new species of Chromohalobacter, and relevant identification work had been started. However, as molecular identification plays a more and more important role in new species identification, DNA hybridization, fatty acids, polar lipid analysis has become the necessary items.The betB of Chromohalobacter DSM 22428T was heterologously expressed in E. coli and its function was verified. Then we conducted enzyme purification, properties study and Chromohalobacter DSM 22428T identification, results are as follows:1. According to the gene sequence of betB of Chromohalobacter DSM 22428T, we designed a pair of primers and get the full sequence of betB of Chromohalobacter DSM 22428T. After double enzyme digestion, the betB was inserted into expression vector and transformed into E. coli BL21 (DE3). We successfully carried out the heterologous expression of BADH in recombinant Escherichia coli. We added 1mM choline to basic medium of different NaCl concentration gradient, then inculated Chromohalobacter DSM 22428T, and found that compared to the control, the salt tolerance of wild strain can be significantly improved if outsides offered choline of a certain concentration. The result indicated that the BADH of wild strain had significant osmotic adjustment ability. To further verify whether the BADH of heterologous expression had osmotic adjustment ability, we added 1mM choline to LB medium of different NaCl concentration gradient, then inculated recombinant Escherichia coli, induced by IPTG, and found that the growth rate of recombinant Escherichia coli was much higher than the control. The result indicated that the BADH of heterologous expression also had significant osmotic adjustment ability.2. We used 0.3mM IPTG to induce recombinant E. coli overexpressing BADH protein, broke cells by high-pressure homogenizer machine, and purified protein by a few steps. Finally we get pure protein. The protein concentration was about 1.5mg/ml, the monomer molecular weight was about 61KDa which was consistent with the molecular weight analysed by bioinformatics.3. We studied the properties of the purified BADH and found that the optimum pH was 7.5, the specific activity was 3.6×10-2 U/mg. The enzyme has salt tolerance. When the salt concentration is 2M, the enzyme remained about 40% of maximum activity, indicating that the enzyme has a strong resistance to high salt concentration.4. We identified strain DSM 224281 according to the identification standards. The organism can grow at NaCl of 0.6-20% (w/v) (optimum 5-6%, w/v), at temperatures of 5-45℃ (optimum 35℃) and at pH 5-9 (optimum pH 7-8). It accumulated poly-β-hydroxybutyric acid and produced exopolysaccharides. The major fatty acids were summed feature 3 (C16:1ω7c and/or C15:0iso 2-OH,23.1%), C18:1 ω7c (22.6%) and C16:0 (22.3%). The major respiratory ubiquinone was Q-9 (86%), The polar lipids was Lipid, Aminolipid, Phosphatidylglycerol, Phosphatidylethanolamine, Glycoaminolipid, Diphosphatidylglycerol, Phosphoglycoaminolipid. Phylogenetic analysis of 16S rRNA and concatenated 16S rRNA, gyrB, rpoD genes sequences revealed that the strain DSM 22428T represents a member of the genus Chromohalobacter. The closest relative type strain was Chromohalobacter israelensis ATCC 43985T. The DNA-DNA relatedness values between strain DSM22428T and C. israelensis DSM67687, C. salexigens DSM 3043T, C. marismortui CGMCC 1.2321T, C. beijerinckii DSM 7218T, C. canadensis DSM6769T, C. nigrandesensis DSM14323T, C. sarecensis DSM15547T ranged from 15%±2% to 43%±1%,. respectively. Based on its morphological and physiological characteristics, as well as on its phylogenetic position prove that strain DSM22428T represents a novel species of the genus Chromohalobacter.