Purification And Partial Characterization Of A Hemolysin From Wild-type Strain Of Synechocystis Sp. PCC 6803

Synechocystis sp. PCC 6803 is a wide-used model organism for genetic and photosynthetic research. The entire genome of the cyanobacterium has already been sequenced. DNA homologic analysis suggests that there are 17 genes related with hemolysin of which 10 genes encode hemolysin or hemolysin-like proteins. Up to now, none of the products of these genes has been purified and characterized except for that of Sll1951, which showes no hemolytic activity against sheep erythrocytes. So the potential harmful impact of Synechocystis sp. PCC 6803 on environment has not been fully comfirmed and considered. In the support of the National Natural Science Fund of China and Shandong Provincial Natural Science Fundation, the extracellular product (ECP) of the wild-type strain of Synechocystis sp. PCC 6803 was examined by hemolysis assay. It was found that the cyanobacterium had a capability of secreting a hemolytic substance into the culture. Then the hemolysin were purified and partially characterized from both the cell-free culture and the cells. At last, we detected the effect of the composition changes in BG-11 medium on hemolysin secretion. The toxin we purified is the first proteinaceous hemolysin with apparent hemolytic activity, found in cyanobacteria up to now.The hemolytic activity could not be detected until 6 days after inoculation and remained quite slight up to the late exponential phase (6¹2 days). Then it increased rapidly, maximized at the stationary phase, showed a highest activity of 48.2%, and gradually declined at the death phase. The appearance and accumulation of an about 80 kDa protein band correlated with the increasing hemolytic activity, as shown by the gradually thicker staining of the protein band in SDS-PAGE. Further research suggested that the hemolysin was heat-labile since the activity was inhibited after heating at 100℃for more than 5 min.Ion exchange chromatography of the cell-free culture supernatants on a DEAE- Sepharose Fast Flow column produced two protein fractions (P1 and P2). Since P1 showed a potent hemolytic activity about 43.6%, P1 fraction was applied to gel filtration on a Sephacryl S-300 High Resolution column, and P3, P4, P5 were obtained. Of these fractions, P4 had a hemolytic activity of approximately 12.0% against rabbit erythrocytes while the others showed no apparent hemolytic activity. The SDS-PAGE analysis of P4 fraction revealed that it was a single band with a molecular mass about 81 kDa. Thus the P4 protein was considered to be the purified hemolysin secreted by the wild-type strain of Synechocystis sp. PCC 6803.SDS-PAGE showed that there was also hemolysin protein band in the cells of Synechocystis sp. PCC 6803. After broken up the cells by ultrasonic, the 81 kDa protein could be precipitated by ammonium sulfate at 25% saturation and be purified after Sephacryl S-300 High Resolution chromatography and Hydroxyapatite chromatography. But no distinct hemolytic activity of the intracellular hemolysin was observed. This could be interpreted by the presumption that the hemolysin protein was synthesized as a low-activity modality in the cells and need some unknown conformational changes to aquire hemolytic potency after being secreted into culture.The secretion of hemolysin by Synechocystis sp. PCC 6803 could be effected apparently when the composition of BG-11 medium was changed. The growth of cells in BG-11 mediums containing different concentration of Ca²⁺ showed no significant changes. However, hemolytic activity of the extraction from calcium-free medium declined drastically compared to control (0.24mM Ca²⁺). Doubling calcium concentration to 0.48 mM increased the hemolytic activity significantly. The result of SDS-PAGE further confirmed that very few hemolysin accumulated in calcium-free culture. These results suggested that the secretion of hemolysin by Synechocystis sp. PCC 6803 was calcium-dependent. Both the cell growth and the hemolytic activity declined obviously in BG-11 medium with omission of N and P element or sterilized air. The decline of hemolytic activity may due to the lower growth rate of cells. More over, the hemolytic activity declined drastically when adding 10μM Mn²⁺ or Fe³⁺into BG-11 medium, while the cell growth showed no significant changes. When adding 1.0¹00μM chloramphenicol (protein synthesis inhibitor) or sodium azide (energy metabolic inhibitor) into the medium, not only the hemolytic activity but also the growth of cells were inhibited drastically, suggesting that the secreted hemolysin from the wild type strain of Synechocystis sp. PCC 6803 was de novo synthesized in cells.