Characteristics Of A Moderately Halophilic Bacterium BYS-1 (Halomonas Sp.) And Cloning Of Its Salt-Tolerance Related Gene

A moderately halophilic bacteria strain BYS-1 was isolated from the activated sludge of the wastewater-treating system of a phenyl acetic acid producing plant. BYS-1 could mineralize phenyl acetic acid. It was primarily identified as Halomonas.sp according to its physiological & biochemical characteristics and the homology analysis of its 16S rDNA sequence (GenBank Accession No. AY0662217).The optimal growth of BYS-1 was obtained with pH, temperature and NaCl concentration being 7.2,30℃and 1.0M respectively. BYS-1 could use anline, phenol and salicylic acid as substrate to support growth , but could not use catechol and quinol, it could use benzoic acid poorly. BYS-1 could tolerate many metal ions such as Zn2+, Cu2+, Ba2+,Mn2+ ,Co2+and Cr3+; but Cd2+and Pb2+ inhibited its growth.When BYS-1 grew in the media with different concentrations of NaCl, there was no obvious change in its intracellular Na+ contents, it accumulated K+,glutamic acid and betaine as osmoprotectants. Its intracellular contents of K+, glutamic acid and betaine increased by 1.9 times ,2.8 times and 6.7 times respectively when the concentration of NaCl increased from 0.1M to 2.0M. Betaine was its main osmoprotectant when the concentration of NaCl was above the level for its optimal growth .BYS-1could use choline as precursor to synthesis betaine de novo , there was a positive correlation between its synthesis and the concentration of NaCl. One pair of primers was designed according to the sequence of betB of Halomonas elongata registered in Genbank, betB of BYS-1 was amplified from its genome . There was a high homology (98%)between the betB sequence of BYS-1 and that of Halomonas elongata . The betB of BYS-1 was cloned to pET32a and expressed in E.coli ,the expressing product was 31.5% of the total cell protein, its enzyme activity was 38.5 U/mg.protein.A salt-sensitive mutant (BYS-1M) was obtained by nitriguanidine mutagenesis on Gibbons medium without NaCl, the mutant could grow in Gibbons medium with low concentrations of NaCl, while unable to grow in media containing NaCl above 10%. It was found that the salt–sensitive characteristics of BYS-1M was not due to destroy of betaine pathway , maybe due to the mutation of a regulon of the genes.Method to extract high quality total DNA of BYS-1 was established and the gene library of BYS-1 was constructed in E.coliDH5αwith pBBR1MCS-2 as the vector by the method of shotgun cloning. 90% of the recombinant plasmids (pBBR-X) had inserted fragments , the average lengh of them was 7.5Kb.Triparental conjucation were conducted with BYS-1M as recipient , the library of BYS-1...