Studies On The Proteases Of Phycobiliproteins Degradation From Synechocystis Sp. PCC 6803

Nutrient deficiency causes a dramatic change of cyanobacteria in cell color from normal blue-green to yellow-green, which is known as bleaching or chlorosis. This bleaching is mainly due to the degradation of phycobilisome (PBS). NblA appeared to play the key role in PBS degradation process. It is poorly understood that how NblA leads to PBS degradation and what proteases play roles in this process. It is assumed that a protease should be found to degrade phycobiliproteins and further proteins or cofactors may be necessary for PBS degradation. Deg proteases (HhoA, HhoB and HtrA) and CtpA are important proteases in cyanobacteria. Little is known about functional relationships between these proteases and PBS degradation mechanism. In the paper, roles of Deg and CtpA proteases in the degradation of phycobiliproteins were studied.The cyanobacterial proteases HhoA, HtrA, HhoB and CtpA in Synechocystis PCC 6803 were first cloned and expressed in this work. After deletion of the predicted signal peptide or transmembrane segment, mature HhoA, HhoB, HtrA and CtpA protease were expressed as soluble recombinant His-tagged fusion protein in Escherichia coli. The purification of HhoAAN36, HhoBAN31, HtrAAN75 and CtpAAN32 constructs was achieved by nickel affinity chromatography using His-tag at the N-terminus.The proteolytic activity of Deg and CtpA proteases was tested in vitro using casein, BSA, five recombinant chromoproteins in E. coli and native CPC isolated from Synechocystis PCC 6803. The results showed that HhoA and HtrA had proteolytic activity on casein, five recombinant chromoproteins and native CPC in vitro.Therefore HhoA and HtrA were proved to be the proteases involved in the degradation of PBS in the study. No proteolytic activity of HhoB was obtained using all substrates in vitro, indicating functional difference among Deg proteases in Synechocystis PCC 6803. However, in the fluorescence quantitative PCR assay, the transcript level of hhoB increased more highly than that of hhoA and htrA after nitrogen step down. Therefore the role of HhoB in PBS degradation could not be completely ruled out and it was assumed that its proteolytic activity in vitro might still need stimulation of some unknown co-factors. CtpA had very low proteolytic activity on casein and several recombinant chromoproteins and no proteolytic activity on native CPC. At the same time, the transcript level of ctpA did not change significantly after nitrogen step down. Therefore it was impossible that CtpA was involved in the degradation of PBS.In the assay of co-factors effects on the proteolytic activities of HhoA, HhoB, HtrA and CtpA, two nblA genes from Synechocystis PCC 6803 were expressed as fusion protein in E. coli. NblB from Anabaena sp. PCC 7120 was also expressed in E. coli. In contrast to an increase, the adding of NblA and NblB triggered heavy inhibition on the proteolytic activity of HhoA and HtrA. Little effect of Mg2+ and Ca2+ on the HhoA activity was observed. The inhibition effect indicated that NblA and NblB were not co-factors stimulating proteolytic activity of HhoA and HtrA.In the binding assay in vitro, it was found that HhoA, HhoB and HtrA could not form complex with CpcA, NblA and NblB. The results demonstrated that the inhibition of proteolytic activity was due to the complex between NblA and phycobiliproteins, not the complex between NblA and proteases. No complex formation between HhoA and phycobiliproteins from Synechocystis PCC 6803 after nitrogen step down was found. Therefore it was assumed that there might be some unkown co-factors during phycobiliproteins degradation by HhoA and HtrA, which was not NblA or NblB.The RNA transcript of protease genes after nitrogen step down were analyzed by fluorescence quantitative PCR. According to the results, it was assumed that pepP had tight relation with the early start of PBS degradation, and it was possible that hhoB, htrA, ftsH, sppA and hhoA had relations with PBS degradation mechanism and play roles in the intermediate and later phase of PBS degradation. These seven genes clpP, ape2, sll2008, ctpA, ymxG, gcP, clpB were assumed to be not involved in the PBS degradation. This paper also includes two other study contents. One study is the reconstitution in vitro of deletion mutants of phycobilisome core-membrane linker protein ApcE in Anabaena sp. PCC 7120. Genes of N-terminus deletion mutants of ApcE were constructed by PCR. All deletion mutants were overexpressed in E. coli. The activities of expressed proteins were studied through in vitro reconstitution tests. The results showed that ApcE(25-240) could bind covalently PCB in an autocatalytic reaction, and other six expressed proteins could not bind covalently PCB, in the presence of 4 mol/L urea necessary to solubilize the proteins.The other study is in vivo biosynthesis of subunit of phycocyanin and allophycocyanin from Synechocystis sp. PCC 6803. In the in vivo reconstitution tests, genes of subunit (apcA, apcB, cpcA, cpcB) were cloned and expressed in E. coli, where PCB biosynthesizing gene(hol and pcyA) and lyase gene (cpeS, cpeT or cpcE/F) were transformed into E. coli and co-expressed. The results showed that reconstitution chromoprotein could bind covalently PCB in E. coli and had absorption and fluorescence properties. Therefore, in vivo biosynthesis of subunit of phycocyanin and allophycocyanin from Synechocystis sp. PCC 6803 was practicable.