Study On The Mechanism Of NfU In Iron-sulfur Cluster Biosynthesis Of Cyanobacteria

Iron-sulfur clusters are the oldest and most versatile inorganic cofactors that present in all living organisms and are required to sustain fundamental life processes.At present,three complete Fe-S cluster biosynthetic pathways have been identified.The nitrogen fixation（NIF）system,is responsible for the maturation of nitrogenase in nitrogen-fixing bacteria.The iron-sulfur cluster（ISC）system is proposed to play a housekeeping role in Fe-S cluster biosynthesis and is distributed widely in prokaryotes and the mitochondria of eukaryotes.The sulfur formation（SUF）system appears to be the major Fe-S biosynthetic pathway in cyanobacteria and chloroplasts in higher plants.Cyanobacteria is a model prokaryotic organism for the study of photosynthesis.Since the SUF system as the main Fe-S cluster biosynthesis pathway in cyanobacteria,but the genome also exist a gene which sequence homology with the C-terminal domain of nifU in Azotobacter vinelandii and named as nfU.The NfU protein can be used as a scaffolding protein for Fe-S cluster biosynthesis and nfU gene cannot be completely knocked out in cyanobacteria.To investigate the relationship between NfU protein and SUF system,Synechocystis sp.PCC 6803 was used as the experimental material,Ppet E-nfU strain was constructed to reveal the effects of knockdown the nfU gene on respiration,photosynthesis,pigmentation and electron transportation in cyanobacteria.1.Construction of the Ppet E-nfU mutant.A pair of homologous arms,nfU-up and nfU-down,were amplified upstream and downstream about 25 bp before the start codon ATG of nfU gene（ssl2667）.The Omega and Cu²⁺induced pet E promoter fragment were placed in the middle of the homologous arms by molecular biological methods to construct the Ppet E-nfU recombinant plastid.Then the plasmid was transformed into Synechocystis sp.PCC 6803 to produce an Ppet E-nfU strain,and we named this mutant as“NfU Knockdown”.The PCR results showed that the NfU Knockdown mutant was completely purified and meets the requirements.2.Physiological and biochemical responses of the NfU Knockdown mutant and wide type under photoautotrophic（Pa）and heterotrophic（Ht）conditions.Both strains were cultured in the BG-11 medium with 12 n M Cu²⁺concentration.The results showed that the NfU Knockdown mutant had a lower growth rate than WT under Pa condition.The F_v/F_m,NPQ,F_v’/F_m’of NfU Knockdown mutant were significantly lower than WT.And the Chl a content,Car content,α,E_k,r ETR_max were consistent with F_v/F_m.Net photosynthetic rate and dark respiration rate of NfU Knockdown mutant were significantly lower than WT,and the decline in net photosynthetic rate was even greater.However,the growth efficiency,respiration and photosynthesis of NfU Knockdown mutant were not significantly different from WT under Ht condition.So the study showed that limited amount of the NfU had a significant negative impact on growth,photosynthesis,respiration,pigment synthesis and electron transportation,and most inhibited photosynthesis.So we presumed that cyanobacteria reduced the intensity of photosynthesis and ensured respiration under unfavourable conditions.3.The relationship of NfU protein with SUF system.We used yeast two-hybrid system（Y2H）in this research.The results showed that the scaffolding protein NfU interacted weakly with cysteine desulfhydrase Suf S and Suf D protein which is a component of Suf BC₂D complex.It is suggested that Suf D and Suf S protein were required for the involvement of NfU protein in Fe-S cluster biosynthesis system.