Identification And Photochemistry Characterization Of Cyanobacteriochromes From Nostoc Flagelliforme

Cyanobacteriochromes?CBCRs?are important photoreceptors in cyanobacteria that respond to light signals.CBCRs in aquatic unicellular cyanobacterium Synechocystis sp.PCC 6803,multicellular filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp.PCC 7120 have been extensively studied while CBCRs in terrestrial drought-resistant cyanobacteria are still poorly investigated.In this study,on the basis of its complete genome sequencing,we screened and investigated the photoreceptor in the terrestrial drought-resistant cyanobacterium Nostoc flagelliforme to provide the basis support of light dependent signal transduction pathway.Through bioinformatics analysis,we found 72 GAF-containing genes,most of them including histidine kinase and REC domain,indicating that they may participate in light related two-component signal transduction pathway.Sequence analysis showed that some of genes in the present study are highly homologous with some known CBCRs,which may be potential photoreceptors.Then,the GAF and PAS domains were chosen to construct recombinant plasmids,and transformed into E.coli LMG194 strain which could induce BV,PCB and PEB chromophores.Through zinc stain and CBB stain after SDS-PAGE,we screened out 10 bilin-binding proteins.Among them,Nsfl₅654 bind BV,PCB and PEB,Nsfl₄3 bind BV and PCB,Nsfl₁0030 bind BV,other proteins are only binding PCB.Scanning spectrometer showed that these holoproteins respond to a wide wavelength ranging from near ultraviolet to far red.Meanwhile,the switch rate of each of CBCRs from one absorption station to another absorption station are different,indicating that the light sensitivity among different CBCRs varies greatly,and suggesting that they may have different function in N.flagelliforme.Various CBCRs in N.flagelliforme may be related to various light environments.The results of urea denaturation experiments showed that in addition to Nsfl₅097gl binding PVB,Nsfl₅654 and Nsfl₁0030g3/g4 binding BV,other proteins are bounding PCB.With the exception of Nsfl₁0030g3 binding trans-BV,the other CBCRs binding chromophores have conformation and anti-conformation changes.Furthermore,the amino acid sequences of Nsfl₄3 and Nsfl₁0181 are highly similar.Their GAF domain only have 21 different amino acids residues,but Nsfl₄3 can bind two chromophores and exhibit photochemical activities,while Nsfl₁0181 can not bind chromophore.Amino acid point mutation and chromophore binding experiments showed that Nsfl₄3 bind PCB through the conservative 87th and 115th cysteine,and 115th cysteine plays a key role in this process.Meanwhile,six mutants of Nsfl 10181 were constructed,and it found that the 132th threonine in Nsfl₁0181 can bind to PCB after mutation to proline,but when the same site in Nsfl₄3 of proline mutated to threonine,it can not bind PCB.These results indicated that the 132th proline is very important,but its function is still unknown.