Construction Of Sulfate Activating Complex And Pyrophosphatase Insertion Mutants Of Rhodobacter Sphaeroides

Rhodobacter sphaeroides, one widely spread photosynthetic bacteria, can perform photosynthesis without oxygen production, grows aerobically in absence of light or anaerobically in presence of light. Photosynthetic bacteria live in the environment of temperatures and pH in the range of28-36℃and6-9, respectively. It is one resource microorganism with versatile metabolism. The results of this study indicated that the best culture conditions were at30℃and pH7, and it could survive in presence of Co2+up to8mM.Sulfate activating complex(SAC) from R. sphaeroides is a bifunctional enzyme, composed of ATP sulfurylase (ATPS) catalyzing synthesis of adenosine5’-phosphosulfate (APS) with substrates ATP and sulfate, and adenosine5’-phosphosulfate kinase (APSK) catalyzing the phosphorylation of APS to form3’-phospho-adenosine5’-phosphosulfate(PAPS). In this study, the plasmid conjugation method for R. sphaeroides was optimized, and SAC and PPase insertion mutants were created, which would facilitate for the further analysis on functions of SAC and PPase for bacterium growth and stress tolerance.Inorganic pyrophosphatase (PPase) hydrolyzes pyrophosphate (PPi) generated from biosynthesis of macromolecules, such as protein, DNA and fatty acids, and provides the thermodynamic pull facilitating their biosynthesis. PPase from R. sphaeroides (RsPPase) is one of II soluble PPase, and divalent cation Co2+is essential for its activity. Regulation of RsPPase activity and effects of its expression on bacterium growth had not been reported yet. In this study, RsPPase was prokaryotically expressed and purified. It was found that fusion glutathione sulfotransferase tagged GST-PPase was non-cleavable by PreScission Protease in presence of Co2+, and had low catalytic efficiency (Km/kcat=2.0x104M-1·s-1). Little effect was found for R. sphaeroides growth by expressing PPase from E. coli (Km/kcat=5.5x107M-1·s-1), which indicated that intracellular RsPPase was enough for it growth. Based on the modeled structure, we speculated that in presence of Co2+with the closed conformation, GST tag would restrict the movement of C terminal domain; therefore decrease the activity by affect the substrate binding and product release. In the absence of Co2+with the open conformation, GST tag was more flexible, which made the protease recognition site available for protease to yield the non-tagged RsPPase. More work such as monomerization of RsPPase and extending the linker between GST tag and RsPPase, needs to be done to confirm this hypothesis.