| Iron-sulfur(Fe-S)cluster as the oldest,multifunctional and varied inorganic cofactor,has caused great concern since 1960s when it was found.[2Fe-2S],[4Fe-4S]and[3Fe-4S]are the most common forms of Fe-S clusters in living organisms.Fe-S cluster can be used as electron carrier,enzyme catalysis and sensor-regulated gene expression,and participate in a series of metabolic activities such as photosynthesis,nitrogen fixation and respiration.Although the composition and structures of Fe-S clusters are simple,their biosynthetic pathways are highly complex and closely controlled.At present,there are at least three Fe-S cluster biosynthetic pathways identified in the bacteria:the NIF system,which is the Fe-S cluster specifically needed for the synthesis of nitrogenase;the ISC system,which is the housekeeping pathway of the Fe-S cluster assembly,and present in almost all organisms;SUF system for the synthesis of Fe-S clusters under conditions of oxidative stress or iron confinement.Evolution of Fe-S cluster biosynthetic pathway from prokaryotes to eukaryotes is conservative.Compared with non-photosynthetic organisms,photosynthetic organisms Fe-S cluster assembly mechanism is limited.Cyanobacteria as the earliest photosynthetic oxygen organisms,have changed the early environment of the earth,and also are known as the ancestors of algae and higher plants.Unlike the E.coli dependent ISC system,the Fe-S cluster assembly mechanism in cyanobacteria is mainly SUF system,which core genes knockout to lethal,and the study of Fe-S cluster synthesis for cyanobacteria is very limited.Synechocystis PCC 6803 is a model of cyanobacteria,natural competent cells,and its genetic transformation is very mature.In this paper,Synechocystis PCC 6803 is used as a material to carry out the following work on SUF Fe-S cluster biosynthetic pathway:1.Study on transcriptional regulation of suf operon in Synechocystis PCC 6803.The transcriptional pattern of the suf operon in Synechocystis PCC 6803 is similar to that of E.coli:sufBCDS are co-transcriptional except that its transcriptional regulation is dependent on SufR.The growth of ?sufR was the same as that of WT,and the content of Chlorophyll a and Fv/Fm were not different from that of WT.The growth of sufR-OE was slow,Chl a content and Fv/Fm were lower than WT.SufBCD protein contents in WT and ? sufR were no difference,but were lowest in the in the sufR-OE strain,confirming that SufR was a transcription inhibitor of suf operon.Under normal condition knockout sufR has no effect on the growth and photosynthesis of Synechocystis PCC 6803.The expression of suf operon in MV and iron deficiency treatment indicates that SUF system can effectively deal with oxidative stress and iron limitation,which may be the reason why cyanobacteria choose SUF system as the main Fe-S cluster synthesis pathway.The results of the distribution of SUF system in organisms with various photosynthetic types were that all the photosynthetic oxygen organisms have SUF system.2.Effects of SUF System on photoautotrophic and heterotrophic growth in Synechocystis sp.PCC 6803.SUF system is very important for the photosynthesis and respiration in Synechocystis PCC 6803,but the cells usually choose to sacrifice photosynthesis to meet the needs of respiration Fe-S cluster.Attempts to knock out the suf gene were unsuccessful.For this reason,the petE promoter platform of Cu2+-induced expression,was introduced to construct the PpetE-sufB strain,so that the expression of the suf operon was controlled by the petE promoter.PpetE-sufB strains were gradually killed in BG11 medium without Cu2-,demonstrating that suf operon is very important for Synechocystis PCC 6803.When the Cu2+ content in the BG11 medium was 12 nM,the growth of the PpetE-sufB strain was about half that of WT and the content of SufBCD protein was reduced,named as SUF Knockdown.Under the photoautotrophic conditions,compared with WT,SUF Knockdown strain showed slower growth rate,lower Chl a contents,PSI/PSII and Fv/Fm,less photosynthetic oxygenation and lower respiratory oxygen consumption.However,the differences between these data in the heterotrophic condition disappeared.P700+ reduction kinetic data showed that the proportion of circulating electron transport in SUF Knockdown was higher than that in WT,and the difference was not obvious under heterotrophic conditions.The transcription of the suf operon was upregulated when its conditions changes to heterotopic from photoautotrophic conditions,but the upregulation of the SUF Knockdown strain was limited.The transcription of other genes on photosynthesis and electron transport chains under two conditions showed that the Hox-related genes in SUF Knockdown were up-regulated in photoautotrophic condition and down-regulated in heterotrophic condition.3.Effects of SUF system on iron uptake in Synechocystis PCC 6803.In order to study the relationship between SUF iron-sulfur cluster assembly system and iron uptake,the transcriptional changes of intracellular genes in SUF knockdown were analyzed by transcriptome sequencing technique.At the same time,mutant strains were used to compare the changes of iron homeostasis gene and the protein content of SUF system.Sequencing of transcriptions after SUF knockdown showed that the synthesis of Fe-S cluster reduced,intracellular pigment synthesis,photosynthesis,lipid metabolism,amino acid synthesis and carbon metabolism were inhibited,many factors involved in the transcription of binary signal transduction system down-regulated,the regulation of the gene changes,and the entire cell life metabolism decreased.SUF-OE grew well but the photosynthesis was consistent with WT,indicating that the increase in SUF system promoted cell growth but had no effect on photosynthesis.A series of iron-related genes of SUF-KD strains were down-regulated,and the transcripts of outer menbrane receptor proteins encoding genes in SUF-OE were up-regulated.Intracellular iron content was consistent with SUF system expression levels.The suf operon under the normal culture conditions of the exbB-exbD double mutant which encoding the iron-absorbing plasma membrane transporter is downregulated and the SufBCD protein content is lower.Therefore,the SUF system is directly involved in the regulation of iron absorption,and as its expression diminishes its demand for iron decreases,whereas the cells increase their uptake of extracellular iron.4.Study on the mechanism of SUF system in Synechocystis PCC 6803.The interactions among SUF system proteins in Synechocystis PCC 6803 were similar to that of E.coli.SufB and SufD interacted with SufC to form complexes as Fe-S cluster assembly scaffolds.SufS and SufE complexes were Fe Clusters are provided with sulfur;SufE interacts with SufrB in order to transfer sulfur to SxufB.SufB and NfLU,as the scaffold protein can be assembled Fe-S cluster and transferred to the apoprotein,can provide Fe-S cluster for Fd,SdhB and PsaC.In addition,SufB interacts with some transcriptional regulators.The yeast two-hybrid experiments and in vitro pull-down of SufD showedthat iron donors may have only a weak interaction with the SUF protein in Fe-S cluster biosynthesis. |
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