Effect of phosphate on two-compopent signal transduction system in the cyanobacterium, synechocystis sp. PCC 6803

Abstract

The two-component signal transduction, which typically consists of a histidine kinase and response regulator, is used by bacterial cells to sense changes in their environment. The histidine kinase S110337 and its cognate response regulator Slr0081, together with the negative regulator PhoU, are involved in the response to phosphate-limiting conditions in Synechocystis sp. PCC 6803. In this research, mutations have been introduced into Thr-214 in the conserved motif of the transmitter domain of S110337 and Asp-88 in the receiver domain of Slr0081 to examine the function of these specific amino acids. In addition, a PhoU deletion strain was constructed to determine the effect of the absence of this regulator when combined with Thr-214 histidine kinase and Asp-88 response regulator mutants. The measurement of alkaline phosphatase activity was carried out to monitor the effect of the mutations. The results showed that mutations of Thr-214 in the S110337, except T214R mutant, results in up-regulation of alkaline phosphatase activity in both phosphate-sufficient and phosphate-limiting conditions. However, removal of PhoU still retained the up-regulated of alkaline phosphatase activity in these Thr-214 mutants except T214N and T214R. The abolished alkaline phosphatase activity caused by Asp-88 mutation suggested that Asp-88 in the receiver domain of Slr0081 is the site of phosphorylation. Some response regulator proteins have been shown to be phosporylated by low molecular weight phophorylated compounds in the absence of the cognate histidine kinase. Therefore, phophorylation of Slr0081 by endogenous acetyl phosphate was also investigated. It was found that in the absence of s110337, the alkaline phosphatase activity could not be induced even when an endogenous acetyl phosphate was available. The results suggested that acetyl phosphate could not phosphorylate Slr0081. In addition, the exogenous acetyl phosphate can provide a source of phosphate to produce the energy for the cells even though acetate kinase and phosphotransacetylase, which are involved in acetyl-CoA production, are absent. Reverse transcription PCR demonstrated that the mRNA expression of acetate kinase and phosphotransacetylase genes of Synechocystis sp. PCC 6803 is up-regulated in response to phosphate limitation. It is suggested that these enzymes are important for energy metabolism allowing the cells to survive under phosphate stress. Acetate kinase was partially purified by 30% ammonium sulfate and phenyl-sepharose column chromatography. A partially purified enzyme was obtained with a specific activity of 2.46 μmol/min/mg protein. The subunit molecular weight determined by SDS-polyacrylamine gel electrophoresis was 48 kDa. The enzyme showed optimum activity at 40℃, with pH 6.5-8.5. The apparent K[subscript m] values for acetate and ATP were 20 mM, respectively. The enzyme showed high specificity toward the substrates acetate and ATP.