Functional Studies Of Photobiology Related Genes Sycrp2 And Piaf In Synechocystis Sp.PCC 6803

Cyanobacteria is Gram-negative bacteria and the first photosynthetic oxygen-evolving organism on earth,it has strong adaptability to changes in the external environment.Cyanobacteria can sense the intensity of external light signals and adjust their position by phototaxis or photophobotaxis,so that they are growing in a relatively favorable environment.How cyanobacteria sense the light signal and adjust its position through motion needs further study.Meanwhile,cyanobacteria can use light energy for photoautotrophic growth,its photosynthesis mechanism is similar to eukaryotic photosynthetic oxygen-evolving organisms,and the structural basis of photosynthesis,for example,the two major photosystem complexes are highly conserved from cyanobacteria to higher plant chloroplasts,therefore,the study of the cyanobacterial photosystem complexes assembly mechanism can be used to explain the assembly process of photosystem complexes involved in all photosynthetic oxygen-evolving organisms.In this study,a model cyanobacteria,Synechocystis sp.PCC 6803,was used as experimental material.We targeted knockout of genes that may be involved in phototaxis and photosystem assembly in Synechocystis sp.PCC 6803,through pre-screening and follow-up functional studies,found that Sycrp2 was essential for phototaxis and in Synechocystis sp.PCC 6803,while Piaf was involved in assembly process of photosystem ? in Synechocystis sp.PCC 6803.The specific experimental results are as follows:By selecting the genes encoding unknown functional protein containing GAF or PAS domain that may bind to the chromophore for photosensitive in Synechocystis sp.PCC 6803,and the homologous genes involved in the regulation of phototaxis in Synechocystis sp.PCC 6803,we knock out these genes and compare the phototaxis abilities of wild-type strains and mutants under different light qualities.We found that the sycrp2::C.K2 mutant loss its motility ability,it provides a preliminary basis for further study of the function of Sycrp2 in regulating phototaxis of Synechocystis sp.PCC 6803.To further investigate the function of Sycrp2,we use the slr0168 platform to reintroduce the sycrp2 gene into the genome of sycrp2 disrupted mutants,to obtain the sycrp2-com strain.The sycrp2-com strain restores phototaxis,indicating that the disruption of sycrp2 does affect the phototactic movement of Synechocystis sp.PCC 6803.Different strains were observed by transmission electron microscopy using negative stained method.Electron microscopy showed the sycrp2 disrupted mutant lost the pilus apparatus on the cell surface,while the surface pilus apparatus of wild-type and sycrp2-com strains are clearly visible,indicating the disappearance of pilus apparatus is the direct cause of loss of motility of sycrp2::C.K2 mutant.The transcript levels of genes involved in motility were examined using real time PCR assay,the pilA9-pilA11(encoding minor pilins)operon was markedly decreased in sycrp2 disrupted mutants compared with the wild-type strain.Moreover,yeast two-hybrid analysis and pull-down assay demonstrated that Sycrp2 interacted with Sycrpl(a known protein which is involved in motility by regulating the formation of pili)to form a heterodimer,and that Sycrpl and Sycrp2 interacted with themselves to form homodimers.Gel mobility shift assays revealed that Sycrpl specifically binds to the upstream region of pilA9,instead of Sycrp2.Together,these findings indicate that Sycrp2 regulates the transcription of pilA9-pilA11 operon and then the formation of pili and cell motility by interacting with Sycrpl in Synechocystis sp.PCC 6803.By selecting 12 unknown functions of hypothetical chloroplast open reading frames(ycfs)in Synechocystis sp.PCC 6803,we tried to knockout these genes and found that we can't obtain the complete isolation and purification mutants of y1,y2,y6,y9,y12 mutants.Growth curve measurement of the wild-type strain and mutants under normal light culture conditions indicated y2 mutant,y9 mutant and y10 mutant grew slower compared with wild type strain,while other mutant strains have similar growth rate compared with the wild type strain.The results of photosynthetic activity assay showed that the photosystem ? activity of y6 mutant strain after dark adaptation and light adaptation was lower than that of wild type strain,indicating that Y6 may play a role in the formation of photosystem ?.Meanwhile,the photosystem ? activity of y10 mutant was lower than that of wild-type strain only under light adaptation conditions,but there was no difference between wild-type strain and y10 mutant of photosystem ? activity after dark adaptation,indicating that the disruption of y10 affect the electron transfer rate after photosystem ?.Subsequent experiments focused on the function of y10 gene(y10 mutant is a complete isolation and purification mutant)in the photosynthesis in Synechocystis.We defined Y10 as photosystem ? assembly factor,Piaf.To further investigate the function of Piaf,we use the slr0168 platform to reintroduce the piaf gene into the genome of piaf disrupted mutants,to obtain the piaf-com strain.We also knockout the piaf downstream gene,piaf-down,to obtain the piaf-down-KO strain.The results of photoautotrophy growth under normal light conditions showed that the growth rate of piaf-KO mutant is much slower than the wild type strain,while the growth rate of other strains is similar to the wild type strain,demonstrated that disruption of piaf does significantly affect the photoautotrophy growth.Transmission electron microscopy showed that the distance between thylakoid membrane layers was wider in the piaf-KO mutant compared with the wild type strain.Subcellular localization experiments,photosynthetic physiology measurement and biochemical analysis results showed that Piaf is localized to the thylakoid membrane,loss of Piaf significantly affects the formation of photosystem ?.The RT-PCR and western blot experiment showed that Piaf disruption had no influence on the transcription of photosynthetic complex components,while specifically reduced PSI subunit(PsaA-D)accumulation in thylakoid membrane Combined with in vivo/vitro pull-down,LC-MS/MS and yeast two-hybrid analyses,Piaf was validated to be involved in PSI assembly via cooperating with PSI assembly factor Ycf3 in Synechocystis.Amino acid sequence analysis demonstrates Piaf is conserved in cyanobacteria and glaucophytes,but lost in other photosynthetic eukaryotic organisms,suggesting that Piaf is an ancient photosystem ? assembly factor present in cyanobacteria and glaucophytes.