Cloning And Characterization Of Ectoine Synthesis Genes And Cloning Of Glutamine Transporter Genes From Halobacillus Dabanensis D-8～T

Halobacillus dabanensis D-8, isolated from Daban salt lake in Xinjiang region of China, is a spore-forming moderately halophilic bacterium. To isolate a fragment of the ectABC from H. dabanensis D-8^T, a PCR strategy with degenerate primers was used. Primer AP1 and BP2, BP1 and BP2 were designed. Southern hybridization experiments were performed using the DIG-labeled 'ectA-ectB' DNA probe. A 11.2kb fragment containing the ectABC genes of the biosynthetic pathway of ectoine from the Halobacillus dabanensis D-8^T was obtained by twice Inverse PCR. The ORFs, of which we compared their deduced amino acid sequences to sequences available in databases, were designated orf1, orf2, ectA, ectB ,ectC orf3, orf4. They were predicted to encode proteins of 398,300,184,428,129,163, 361 amino acids with deduced molecular masses of 43508, 33956, 20921,46969,14956,18369,42186 Da, respectively. Subsequently, the entire ectABC cluster was cloned. It revealed that the putative promoter region of the ectABC operon was similar to to σ ⁷⁰ dependent promoters of E. coli. The intergenic regions of the ectABC genes from H. dabanensis D-8^T is more tightly spaced than that from Chromohalobacter salexigens, Halomonas elongata, Marinococcus halophiluss and Salibacillus. pasteurii. The amino acid sequence deduced from ectABC was highly homologous that from Virgibacillus. pantethenticus (EctA, 52%, EctB, 60%, EctC, 67%, respectively). The ectABC genes were cloned in the expression plasmid pMXBlO resulting in pMXB10ectABC. The ectoine produced by ectABC genes from cell extract in E.coli ER2566 containing pMXB10ecMBC was detected using ¹³C nuclear magnetic resonance spectroscopy.A defined medium(SSDM) containing all the inorganic components required for growth was developed. A number of compounds as potential sole carbon sources for H. dabanensis D-8^T were tested. Of the compounds examined, glycerol and glucose supported the most rapid growth rate, sourse, Betaine glutamate, proline, sorbitol and glutamine were able to support the growth. The whole-cell extracts prepared from H, dabanensis D-8^T grown in total sea salt 10% salinity SSDM, 15% salinity SSDM, M63 and G10. A representative set of ¹³C NMR spectra is shown signals corresponding to alanine glutamate, N_δ-acetylornithine and further unidentified signals could be identified. Interestingly, the ectoine signals is little observed.To study the glutamine transport system in H. dabanensis D-8^T, we cloned and sequenced its genes (glnABC) by two steps of Inverse PCR. The glnA encode the glutamine ABC transporter glutamine-binding piotem of 265 amino acids with a deduced molecular mass of 29382 Da, the glnB gene encodes the glutamine ABC transporter pennease of 218 amino acids, with a calculated molecular mass of 24474 Da, and glnC gene corresponds to the glutamine ABC transporter ATP-binding protein and codes for a 240-residue (26435 Da). The amino acid sequences of GlnA, GlnB and GlnC were individually compared with the protein sequences of B. clausii, O.iheyensis and E.coli k-12. In general, the GlnABC proteins of H. dabanensis D-8^T have somewhat greater sequence identity to those of the gram-positive bacteria B. clausii and O.iheyensis than to that from the gram-negative E. coli k-12. Hydrophobicity plot analysis showed that N-terminal portions of GlnA and GlnC have one and four predicted transmembrane domains, respectively.