Cloning And Characterization Of Glycine Betaine Transporter Genes From Gram-Positive Moderately Halophilic Bacteria

Halobacillus trueperi was isolated from hypersaline sediments of the Great Salt Lake in U.S.A, which could endure salt concentration from 0.5% to 25% NaCl (w/v), and grow optimally in media containing 5% ~ 10 % NaCl.Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity. A partial fragment of the glycine betaine transporter betH gene was obtained from the genome of H, trueperi with degenerate primers. Through Southern blot hybridization and inverse PCR , a 5.1 kb EcoRI fragment containing the betH gene was sequenced. The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembranes regions. BetH showed 56% identity to the OpuD of Bacillus subtilis and belonged to BCCT family. Its putative promoter region was highly homologous to B-dependent promoter of B. subtilis. A 2.6 kb fragment including the betH gene was subcloned into pUC18 and transformed into the E. coli MKH13, colonies appeared on the plate of the selective M9 medium. The accumulation of glycine betaine in E. coli MKH13 was examined using 13C nuclear magnetic resonance spectroscopy.Depending on the conserved amino acids of glycine betaine ABC transporter, a pair of degenerate primers were designed. With H. trueperi genomic DNA and degenerate primers, a 560 bp PCR fragment was obtained and labeled as a probe. After H. trueperi genomic DNA was digested with different endonucleases, Southern blot result showed a 2.6 kb positive fragment digested by EcoRI and IPCR was carried out to obtain the flanking sequence. BLAST result showed the fragment containing the opuAA and its 5' upsteam sequence was obtained. Then, a piece of degenerate primer was designed according to the conserved amino acids of glycine betaine ABC transporter system glycine betaine-binding protein as a reversed primer. Combined with the opuAA-up, a 2.3 kb fragment was obtained through PCR. BLAST result showed a fragment which contained the partial opuAA, the whole opuAB and partial opuAC sequences were obtained. By Southern hybridization and IPCR, a 5.2 kb positive signal was found at the lane digested by AflII and the fragments were cloned and sequenced. BLAST results showed the whole glycine betaine transporter opuA gene sequences had been obtained successfully.Halobacillus sp.D8 is a spore-forming, Gram-positive moderately halophilic bacterium, which was isolated from Xinjiang in China. Its genomic DNA was partially digested by SauiAl. DNA fragments from 4 to 16 kb were collected after electrophoresis and ligated with BamHI-digested pUC18 to construct genomic library. The total number of recombinant plasmids is about 9000. 13C-NMR method has detected the accumulation of glycine betaine in the ethanol extracts of Halobacillus sp. D8, which grows on the media of SWYE(10% NaCl). Depending on the degenerate primers which were designed according to the conserved amino acids of glycine betaine secondary transporter, a fragment about 1 kb was obtained by PCR. The PCR fragment was purified and labeled with DIG as a probe. Through in situ hybridization and colony PCR, a positive recombinant plasmid was isolated from the genomic libraryand sequenced. The inserted DNA fragment was about 4.3 kb. Analysis of the DNA sequence revealed the existence of three ORFs. One of them was identified as the betH gene of Halobacillus sp. D8. A hydrophobicity plot of BetH revealed an alteration of hydrophobic and hydrophilic segments that was characteristic for integral membrane protein, suggesting the presence of 12 transmembrane-spanning segments and belonged to BCCT family.