The Role Of SHlyBD-sTolC Efflux System In Iron Homeostasis Of Synechocystis Sp.PCC 6803

Cyanobacteria are the oldest oxygen-producing organisms on the Earth and major contributors to marine primary productivity.In oligotrophic ocean,iron is a key factor limiting primary productivity.Iron is an essential micronutrient for all organisms and participates in key biological metabolic processes such as respiration,photosynthesis and nitrogen fixation.Iron deficiency affects the normal life activities of cyanobacterial cells,and excessive iron causes the Fenton reaction in the cells to produce a large number of hydroxyl radicals,which are toxic to cells.Therefore,the available iron concentration in cells must be strictly regulated.When facing iron-limiting environments,the expression of the iron absorption system in cyanobacteria is highly induced.While when iron is excessive,the cells will reduce iron toxicity through efflux system.The iron uptake mechanisms in cyanobacteria have been studied extensively,but it is still unclear how cyanobacteria use the efflux system to maintain cellular iron homeostasis.In this paper,the model cyanobacterium-Synechocystis sp.PCC 6803 was used to investigate the effects of cyanobacterial efflux system on maintaining cell iron homeostasis by bioinformatics,physiology and molecular biology.Through a large number of gene knockouts and physiological phenotypic analysis of mutants,a series of related genes involved in metal efflux were identified.In addition,an interesting phenomenon was discovered:the knockout of an efflux system component resulted in enhanced ability of the mutant strain to adapt to the high-iron environment and was sensitive to low-iron environments.The role of the main components of the efflux system,Sll1181(sHlyD)and Slr1270(sTo1C),in maintaining cellular iron homeostasis was further studied,and the three possible hypotheses were verified.The first possibility was ruled out:the inactivation of the sHlyBD-sTolC system led to the disappearance of substrate recognition and selectivity of extracellular metal ions,resulting in uncontrolled leakage of intracellular iron.Another possibility is that Synechocystis PCC 6803 may secrete related substances beneficial to iron uptake through the efflux system,but studies have shown that the cyanobacteria do not secrete Siderophore.Therefore,we analyzed the role of the main extracellular protein Sll1951(S-layer protein)secreted by the system in low-iron adaptation,and found that direct knockout of Sll1951 had no effect on the adaptation of cells to low-iron,thus eliminating the second possibility.Third,our results confirm that the sHlyBD-sTolC system mutant strain has decreased adherence ability,and the cells are more likely to settle.It is speculated that the loss of the system may lead to the decrease of extracellular polysaccharide(EPS)secretion,which in turn affects the adsorption and Absorption of iron by cells.Resulting in mutant strains sensitive to low iron environments.The main results are as follows:1.Screening and identification of genes related to efflux system of Synechocystis sp.PCC 6803.24 efflux related genes were knocked out and the physiological phenotype of the mutant was analyzed.It was found that under high-iron conditions,the slr0369::C.K2 and sll 1181::C.K2 mutants showed diametrically opposite physiological phenotypes,of which Slr0369 is a typical metal efflux system channel protein,and its mutant showed high iron sensitivity.Sll1181 is a periplasmic space-regulating protein in the efflux system,which is responsible for the recognition of the substrate,and its mutant exhibits tolerance in high iron condition and sensitivity in low iron condition.2.Function of the efflux system that Sll1181 involves.Overexpression of sll1181 resulted in cells adapting to a low iron environment,further confirming that the protein is involved in low iron adaptation,but Western blot results showed that the expression of Sll1181 was not induced by iron.Bioinformatics analysis indicated that Sll1181 may form a type 1 secretion system together with Sll1180 and Slr1270.The prototype is derived from the HlyBD-TolC secretion system of Escherichia coli.We named them sHlyD,sHlyB and sTolC.Yeast two-hybrid experiments demonstrated that sHlyD and sHlyB have protein interactions.Further knockout of sTolC revealed that the sTolC mutant(slr1270::C.K2)has a similar low-iron-sensitive phenotype with slr1181::C.K2,and the intracellular iron content is significantly lower than that of the wild type,indicating that these genes may be involved in maintenance.Intracellular iron homeostasis.3.The possible mechanism of sHlyBD-sTolC efflux system to participate in low-iron adaptation.No significant differences were found between the wild type and the sill1181::C.K2 mutant strains in the physiological experiments of high Cu condition and Mg deficiency condition,excluding the possibility of uncontrolled leakage of metal,indicating low iron sensitivity of mutants Specific.Further mutation analysis of the main extracellular protein Sll1951(S layer protein)secreted by the system revealed that the mutant strain did not have a corresponding low-iron-sensitive phenotype,thereby eliminating the possibility that the system promoted iron uptake by secreting S-layer protein.Finally,a preliminary analysis of the mutant strain and the extracellular polysaccharide of the mutant strain of the system showed that the adherence ability of the mutant strain decreased,and it was easier to settle than the wild type.It is speculated that the mutation of sHlyBD-sTolC may lead to the decrease of extracellular polysaccharide.Affecting the adsorption and uptake of iron by cells,the specific mechanism of exopolysaccharide secretion mechanism to promote iron uptake is still under further study.