Study On The Photochemistry Of Cyanobacteriochrome And The Function Of Slr0351

Phytochrome is the functional pigment protein photosensitizing light in plants and bacteria. It plays an important role in the formation of photomorphogenesis in plant and phototaxis movement in bacteria. The phytochrome in cyanobacteria is called cyanobacteriochrome (CBCRs).Nostoc.sp. PCC7120is one of the model organisms in the study of cyanobacteriochrome. The relatively large genome of Nostoc sp. PCC7120contains more than50ORFs coding for GAF domains in putative CBCRs. Phylogenetic analyses indicated a photoreceptor branch that contain the DXCF motif clearly distinct from other GAFs domains. While10GAFs locate in the branch of DXCF CBCRs, they are A11688, A112239, A113691GAF2, A111280GAF2, Alr1966GAF2, Alr3356, Alr2279, Alr3120GAF1, Alr3120GAF2and Alr5272GAF1. The GAF-coding domains were cloned individually into E. coli BL21, and then coexpressed with genes coding for PCB-generating enzymes. Eight GAFs could be chromophorylated and among them seven chromoproteins had various reversible photochemistries, in which the originally formed15Z states absorbed blue light around410-430nm and the15E states formed absorbing at wavelengths between490-600nm. These chromoproteins are putative photochromic blue-light photoreceptors. The bound chromophores were identified after removing non-covalent interactions with the protein by denaturation in acidic urea solutions. According the chromophores in DXCF CBCRs, We divided these eight DXCF CBCRs into three types. One type was DXCF CBCRs with PCB/TPcR, containing A111688, Alr3120GAF1, A112239and Alr3356. Another type was A113691GAF2with PVB/TPvR. The third type was DXCF CBCRs with PCB/TpcR and PVB/TPvR mixture, concluding A113691GAF2, A111280GAF2and Alr1966GAF2. By directed mutagenesis, single Cys site variant or both Cc and Cx variants were constructed.we tested the functions of the two cysteines in DXCF CBCRs in Nostoc sp, PCC7120. A conserved cysteine, Cc, is common to all CBCRs even to phytochromes:it binds to C-3of the chromophores. Replacement of this cysteine resulted in Alr2279, A112239, and A113691GAF2in the loss of chromophore, and in Alr1966GAF2and All1280GAF2in a strongly reduced absorption. This verifies that Cc is the conservative bilin-binding cysteine. In addition, DXCF CBCRs have an "extra" cysteine, Cx, that is responsible for a second bond to the chromophore, by thioether bond formation to C-10or C-5of PCB or PVB. Formation of this thioether bond interrupts the conjugation plane leading to the blue-light absorbing rubins, both TPcR and TPvR. Replacement of Cx resulted in a loss of the-420nm absorption and a concomitantly increased absorption in the560-650nm region. Rubin formation is inhibited, thus also confirming that Cx in these systems is the one forming the bond to C-10. Fluorescence spectra showed that six DXCF CBCRs with absorption reversibility all had fluorescence emission peaks in the range of630-660nm, also showing fluorescence reversibility between15E and15Z state. Among these six DXCF CBCRs, only A111280GAF2had particularly evident reversiblity fluorescence change, with red fluorescent/non-fluorescent reversibility. Most Cx mutants had fluorescence emission peak in the range of640-660nm with a slight red shift compared with their wild types. Due to high fluorescence activities and molar extinction coefficient, A111280GAF2(C53A) and Alr1966GAF2(C56A) were detected persistently red fluorescence using fluorescence microscopy. However, we could only detect the faint red fluorescent change of A111280GAF2. In order to obtain higher fluorescent variants, we mutated some key amino acid sites in A111280GAF2by site-directed mutagenesis. The resulting mutants all had not strong fluorescence activity.In the study of the cyanobacteriochrome A114261coexpressed with PCB in E. coli and its photochemical activities, we found that A114261was insoluble in water. In order to clarify the phtochemistry of A114261, we studied the photochemistry of A114261GAF1, A114261GAF2, A114261GAF3and A114261GAF4, respectively. The photochemical properties of these GAFs showed that only A114261GAF2combined with PCB covalently, while A114261GAF1, A114261GAF3and A114261GAF4failed to combine PCB. A114261GAF2has absorption peak at590nm and fluorescence emission peak at645nm. The Cys variants A114261GAF2(C81L) and A114261GAF2(C113A) both have high absorptive and fluorescent activites, while A114261GAF2(C124L) has no characteristic absorption and fluorescence peaks in visible range. Therefore Cys124is covalently binding site of PCB in A114261GAF2.In addition, we also studied the function of Slr0351from Synechocystis sp. PCC6803. Using homologous recombination technology,△slr0351was constructed when slr0351was knocked out by replacing slr0351with kanamycin gene in Synechocystis sp. PCC6803. Comparing the growth rates of△slr0351with those of wild type (WT) under different culture conditions, we found that in the absence of the N or S element in BG11media the growth rates of△slr0351were much slower than those of WT in the late stage of culture. We can conclude that△slr0351is very sensitive to the the absence of the N or S element stress conditions, which indicated slr0351may be involved in the regulation of S and N elements biochemical metabolism process. Meanwhile, we also found that the deletion of slr△351made△slr0351growing slower than WT in the low light condition, however, no effect on the growth of△slr0351under high light condition. It is suggested that slr0351may be involved in the regulation of light capture under low light condition.At the same time, we excised the transmembrane in the N-terminal of Slr0351and obtained the truncated Slr0351for further research on the function and property of Slr0351.