Effects Of Sulfur On The Synthesis Of Phytotoxins And Sulfotransferase Raxst Of PXO99~A

Sulfur is the fourth important nutrient following nitrogen, phosphorus and potassium. Sulfur metabolism, a significant metabolic pathway in PXO99A, plays a major role in the synthesis process of pathogenic toxins and toxic factor. Researches on applying the antitoxin screening system to cultivate resistant plants and using the non-toxic factor as a drug target are two cardinal directions in the prevention and treatment of bacterial blight.In this study, the PXO99A pathogenic toxin is extracted by ethyl acetate under a treatment of sulfur gradient, followed by biological toxicity test. The results show that in a certain concentration range, the higher the concentration of sulfur, the better PXO99A pathogenic toxin synthesis, and the stronger its virulence. Rice CX6221B shows higher resistance to PXO99A toxin than D62B. The effect of toxin on growth inhibition of the rice radicle is significantly stronger than that of germ, and the toxic effect is strengthened with increasing concentration of toxin.1 mg/mL crude toxin can completely inhibit the germ and radicle.We constructed pSKB4-RaxST-ECFP plasmid vector, and obtained the recombinant strains PXO99A-RaxST-ECFP using electroporation method, which can be identified by fluorescence detection. Experimental results show that the synthesis quantity of RaxST varies in different bacterial growth periods, where the most RaxST expression is in the logarithmic growth phase. Within a certain range of concentrations, RaxST synthesis quantity shows positive correlation with the concentration of sulfur, and a low concentration of sulfur reduces the synthesis of RaxST.On the other hand, through cloning, expression and purification, we obtained four kinds of fusion proteins, fRaxST-MBP (full length RaxST), tnRaxST-MBP (N-terminal truncated RaxST), tcRaxST-MBP (C-terminal truncated RaxST), and tcnRaxST-MBP (N,C-terminal truncated RaxST). Using four synthetic peptides (10 peptides,15 peptides,17 peptides and 21 peptides) as substrates to analyze RaxST enzymatic activity. The results show that kinetic constants of RaxST vary in different sources of RaxST and substrates. fRaxST-MBP catalyzes sulfation of 10 peptides with kcat, Km and Ki of 6.13 s-1,1.36 mmol/L and 0.01 mmol/L, that of 15 peptides with kcat, Km and Ki; of 0.51 s-1,0.47 mmol/L and 0.49 mmol/L, that of 17 peptides with kcat, Km and Ki of 0.12 s-1,0.03 mmol/L and 1.72 mmol/L, that of 21 peptides with kcat, Km and Ki of 0.04 s-1,0.01 mmol/L and 0.01 mmol/L, respectively. tcRaxST-MBP catalyzes sulfation of 10 peptides with kcat, Km and Ki of 0.37 s-1,0.13 mmol/L and 1.94 mmol/L, that of 15 peptides with kcat, Km and Ki of 1.25 s-1,0.26 mmol/L and 0.10 mmol/L, that of 17 peptides with kcat, Km and Ki of 0.97 s-1,0.14 mmol/L and 0.07 mmol/L, that of 21 peptides with kcat, Km and Ki of 0.75 s-1,0.03 mmol/L and 0.02 mmol/L, respectively. tnRaxST-MBP catalyzes sulfation of 10 peptides with kcat, Km and Ki of 0.14 s-1,0.16 mmol/L and 0.92 mmol/L, that of 15 peptides with kcat, Km and Ki of 6.23 s-1,4.89 mmol/L and 0.02 mmol/L, that of 17 peptides with kcat, Km and Ki of 0.45 s-1,0.04 mmol/L and 0.21 mmol/L, that of 21 peptides with kcat, Km and Ki of 0.18 s-1,0.01 mmol/L and 0.14 mmol/L, respectively. tcnRaxST-MBP catalyzes sulfation of 10 peptides with kcat, Km and Ki of 0.25 s-1,0.11 mmol/L and 1.05 mmol/L, that of 15 peptides with kcat, Km and Ki of 65.3 s-1,4.20 mmol/L and 0.02 mmol/L, that of 17 peptides with kcat, Km and Ki of 0.15 s-1,0.02 mmol/L and 0.31 mmol/L, that of 21 peptides with kcat, Km and Ki of 5.75 s-1,0.22 mmol/L and 0.003 mmol/L, respectively. The studies on RaxST improved our understanding of the immune interactions between prokaryotes and their hosts, and the communication between prokaryote cells.