| Cyanobacteria are important global primary producers,accounting for about 25%of global photosynthetic carbon fixation.In the marine environments where cyanobacteria are widely distributed,there are many growth limiting factors of cyanobacteria.About one third of the primary productivity of oceans is limited by insufficient iron of seawater.In response to environmental iron limitations,cyanobacteria have evolved many adaptation strategies,that including:(1)synthesis and secretion some ferric iron chelates to assist iron uptake;(2)Upregulate expression the genes which related to iron response to increase iron uptake of cyanobacteria;(3)Induced the expression of IsiA(Iron stress-induced protein A),a photoprotective protein;(4)Using non-ferritin instead of ferritin to reduce cell iron demand;(5)Iron is stored in iron sufficient condition and released in iron limit condition by ferritin.However,it is not clear that how cyanobacteria sense iron limit signals and how to respond to this condition.In this study,three homologous iron limited-induced transcriptional regulators were found in Synechocystis sp.PCC 6803,there are S111205,S111408 and Slr1489.Its coding genes are,in the genome,distributed near to the coding genes of outer membrane iron transporter(TBDT)of TonB-dependent iron uptake system(Outer membrane iron uptake system).They may play important role in iron limit signal transduction in cyanobacteria,so these three transcription factors are classified as a class of transcription factors and named IutR(Iron uptake transcription regulator).Then a series of studies have been carried out and the main results are follows:1.Construction of single mutants,combination mutant and complementary strains of IutR transcription factors and their physiological phenotypes.Bioinformatics analysis showed that Sll1205,S111408 and Slr1489 had typical transcription factor domains.And their locations in genomic are located near of the TonB-dependent outer membrane iron uptake transporter(TBDT)genes,respectively.Their expression was induced by iron limit condition,so we named them IutR1,IutR2 and IutR3,respectively.Firstly,we single knockout of these three transcription factors,but the results showed that the physiological phenotype of mutant was no significantly different from wild type either in iron-sufficient or iron-limit conditions.So we further knocked out all the three transcription factors and obtained combination mutant of three iutR.And the results showed that there was no significant difference between the growth of iutR mutant and wild type in iron-sufficient condition.However,under iron-limit condition,the growth of three iutR combination mutants was significantly slower than that of wild type,indicating that the combination mutants were very sensitive to iron-limit condition.To eliminate the second point mutation and polarity effect in the knockout process,we constructed the complementary mutant that single iutR transcription factor complementary three iutR combination mutant strain.And the results showed that single iutR factor could partially complement the iron-limit sensitive phenotype of the combination mutants.Further,by yeast two-hybrid experiment we found there was no interaction between the three IutR,which indicated that they did not function in the way of complexes,and there might be functional redundancy among the three IutR.2.The mechanism of IutR transcription factors in regulate the adaptation of cyanobacteria in iron limit condition.The results of Quantitative fluorescence-PCR showed that the expression of iron uptake related genes such as TonB system in the iutR single mutants were reduced by iron-limit induction.But in three iutR combination mutants,iron deficiency induced expression of iron absorption related genes such as TonB system,FeoB system(ferrous iron transporter in inner membrance)and Fut system(ferric iron transporter in inner membrance)was significantly inhibited,and TBDT gene expression was the most significantly inhibited.To verify whether IutR transcription factor can bind the promoter of TBDT genes,we use yeast one-hybrid experiment and the EMSA assay,and found that IutR can bind the promoter of TBDT genes.And the results are as follows,by yeast one-hybrid experiment we found that IutR1 can bind the promoter of sll1206(tbtd1)and slr1490(tbtd4),IutR2 can bind the promoter of sll1206(tbtdl)and sll1409(tbtd3),and IutR3 can bind the promoter of slr1490(tbtd4).By EMSA assay we found that IutRl can bind the promoter of four tbtd(sll1206,sll1406,sll1409,slr1490),IutR2 can bind the promoter of sll1206(tbtd1),sll1406(tbtd3)and slr1490(tbtd4),and IutR3 can bind the promoter of sll1406(tbtd2)and slr1490(tbtd4).These results suggest that IutR can regulate the expression of iron uptake related genes such as tbdt genes.3.Target genes of IutR transcription factor identified by Chip-seq analysis.By Chip-seq analysis,we identified a series of target genes directly bound by IutRl transcription factors in Synechocystis sp.PCC 6803 under iron-limit conditions.These genes are involved in intracellular amino acid synthesis,carbon metabolism and iron uptake in cyanobacteria.Among them,iron uptake genes mainly include:TonB-dependent coding genes of TBDT1,TBDT2 and TonB in outer membrane iron uptake system,and those of FutAl,FutA2,FutB and FutC in plasma membrane ferric iron uptake system,as well as those of FecB,FecD and FecE in Fec iron uptake system(citrate-dependent ferric iron transport system).It is basically consistent with the previous experimental data.Therefore,we conclude that IutR is likely to directly regulate the genes of TBDT and TonB.And IutR may participates in the regulation of Fut and Fec systems,which is participate in Ferric iron uptake. |
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