Effects Of Heterologous Expression Of Dual-copies ButA Gene From Tetragenococcus Halophilus On Salt Tolerance Of Escherichia Coli

Tetragenococcus halophilus CICC10469, isolated from soy sauce broth, was a gram positive lactic acid bacterium(LAB) strain which had an optimum Na Cl concentration of approximately one molar and an upper limit of nearly 3.5 molar, and it was applied extensively in high salt food fermentation. Accumulating glycine betaine from an exogenous supply was a survival strategy employed by T. halophilus. But A was a secondary glycine betaine transport system which solely transported glycine betaine in T. halophilus,but its halophilus mechanism needed to be further illuminated. However,resistance of T. halophilus to electroporation or other transformation procedures made it difficult to study its halophilus mechanism in vivo. Thus, we turned to in vitro study. We tried to figure out expression of but A gene under different salinties by quantitative reverse transcription polymerase chain reaction(q RT-PCR) and demonstrated changes of membrane permeability over elevated salt concentrations. At the same time, we constructed multi-copy but A complete expression box(es), and transformed it to Escherichia coli MKH13(Δ(pro P)2 Δ(pro U)608 [Spcr]),which was unable to transport glycine betaine, in order to study its influence on salt tolerance of E.coli. We also used High Performance Liquid Chromatography(HPLC) to detect intracellular and extracellular contents of glycine bataine. In this study, we firstly confirmed that but A expression was up-regulated under salt stress condition(q RT-PCR) and also demonstrated that membrane permeability was generally increased over elevated salt concentrations. Subsequently, we discovered that recombinant E. coli MKH13 strains with single- and double-copy but A complete expression box(es) showed typical growth curves under high salt condition,while differed in salt tolerance ability. Meanwhile, results of HPLC experiment were consistent with growth curves under different salinity. In summary, our results indicated that regulation of but A expression was salt induced and double-copy but A cassettes confered higher salt tolerance to E. coli MKH13, which implied the potential of muti-copies of but A gene to be applied to genetic modification of T. halophilus for improvement of salt tolerance, and laid a solid foundation for further research and industrial application of T. halophilus.