The Regulatory Mechanism Of A Novel TCS MacRS In Streptomyces Coelicolor

Streptomyces are soil-dwelling bacteria that produce abundant natural products which play an important role in medical treatment,health care and biological control.The model organism Streptomyces coelicolor has uncovered to synthesize several secondary metabolites such as actinorhodin(ACT),undecylprodigiosin(RED),calcium dependent antibiotics(CDA),a zincophore called coelibactin and a yellow pigmented type I polyketide(yCPK).Streptomyces have a complex developmental life cycle that is exemplified by three different cell types:spores,vegetative mycelium,and aerial mycelium.A spore germinates to form a germ tube,which then grows to form hypha and followed by branching and development into vegetative hypha.The vegetative hypha grows into the air to form the aerial hypha,which then matures into spores.Spore-formation is the main mode of reproduction for Streptomyces to live in the soil with abundant resources but discontinuous distribution.Cell-division is the fundamental process for organism growth and proliferation.In most bacteria and archaea,the tubulin-like GTPase FtsZ is essential for this process,and it can polymerize into a Z-ring close to the cytoplasmic membrane at division site,recruiting additional cell division elements to form macromolecular machine called the divisome.The divisome drives cell membranes inward until they closure,as well as cell wall synthesis.In Streptomyyces,there are at least two kinds of cell-division.One of them is sporulation septation,in which protein SsgB recruits FtsZ to septum site dependent on the action of SsgA,leading to cell-cell separation and the release of equally sized,unigenomic spores.In the process of mycelium division,mycelium does not break by the septa,and the sparse Cross-wall separates the filamentous cells into a multi-nuclear compartment.FtsZ is also necessary for the formation of Cross-wall,but its dependence on FtsZ is different from that of spore division.When the alanine at position 249 of FtsZ protein is mutated to threonine,Cross-wall can form normally,but spores cannot form.Knockout of ftsZ results in the presence of a large number of membrane vesicle aggregation structures in the mycelium,which is known as Cross-membrane.This structure is also existed in the early stage of growth,and the presence of FtsZ and cell-wall components in some Cross-membrane structures may be related to the formation of Cross-wall.The studies on the morphogenesis and the process of cell division of Streptomyces will help us understand the role of cell division in the evolution of multicellular bacteria,as well as the development of anti-division drugs and the mechanism of drug resistance.The biosynthesis of secondary metabolites and development of Streptomyces are regulated by two-component signal transduction systems(TCSs).A typical TCS includes a histidine kinase and a response regulatory protein.Histidine kinase undergoes autophosphorylation on histidine after sensing external signals,and then activates the response regulatory protein which recognizes and activates or inhibits the transcription of target genes.In our study,we focused on the regulation mechanism of a new pair of TCS-MacRS in S.coelicolor M145 strain,and the functions of its target genes.Previous studies have shown that the mutation of regulatory gene sco2120 affected the synthesis of secondary metabolites.In order to determine the function of sco2120,the genes upstream or downstream of sco2120 were knockout,and based on the phenotypic observations of mutant strains,it is confirmed that sco2121 encodes a kinase which may active the function of SCO2120,but sco2118,sco2119 and sco2122 may be not connected with sco2120,indicating that SCO2120/2121 is a typical TCS.The knockout of SCO2120/2 121 resulted in a significant decrease in the production of ACT,RED,and CDA,while the aerial hyphae of the strain was produced earlier,and the phenotype of the complemented strain was close to that of the wild-type strain.RNA-Seq analysis indicated that SCO2120/2121 in S.coelicolor positively regulates the transcription of ACT,RED and CDA synthetic gene clusters,target-genes of Zur and promotes the metabolism of Zn2+.SCO2120/2121 was also found to positively regulate the target genes of SoxR and ?U,facilitate the expression of multiple membrane proteins and lipoproteins.Thus,SCO2120/2121 was identified to be a signal transduction system with global regulatory functions and named MacRS because of its transcriptional regulation of Membrane protein and ACT and CDA synthesisIn order to analyze the regulatory mechanism of MacR,we constructed the complemented strain C-?macR-Flag with the expression of MacR-Flag protein.The results of phenotypic observations showed that the phenotype of C-?macR-Flag was closed to wild type strain,indicating that MacR was expressed and functioned correctly;The results of Western blot showed the expression of MacR-Flag was properly.ChIP-Seq and ChIP-qPCR assays were performed with anti-flag M2 Affinity gel and the results showed that MacR could bind to the promoters of sco1700,sco4011,sco4225,sco4924,sco6728 and sco7613 genes encoding membrane proteins,sco0607 and sco 7460 genes encoding lipoproteins,and sco2101 encoding carotenoid dehydrogenase.The results of DNase I Footprinting assay confirmed that MacR can bind to the 16 bp palindromic sequence TGAGTACNNGTACTCA on the promoter;EMSA assays determined that MacR specifically bind to the promoters of target genes in vitro,and the palindromic sequence is conserved and specific;Real-time PCR results further confirmed that the transcription of target genes except sco2101 was regulated by MacR.Mining of genome sequences by Repredict software and EMSA experiments confirmed that there was a sequence similar to the MacR conserved binding sequence both in the promoters of act?-orf4 and cdaR,but the DNA probe containing the sequence did not bind to MacR in vitro,indicated that the effects of MacR on the syntheses of secondary metabolites are not achieved by directly regulating the expression of specific regulatory genes.MacR binds to the promoters of sco7681,sco7682 and sco0476 which are related to Zn2+ metabolism in vitro.According to the conserved binding sites of Zur,we speculated that MacR and Zur compete to regulate the expression of the genes associated with Zn2+ metabolism and jointly regulate the metabolism of Zn2+.However,no target sequences of MacR were found on the promoters of soxR,?u,and target genes of SoxR and ?u.Therefore,we speculated that the regulation of MacR on target genes of SoxR was achieved through the effect of ACT yield,while the regulation of target genes of ?U was caused by the decrease of Zn2+concentration.In conclusion,MacR indirectly regulates the synthesis of secondary metabolites and the transcription of SoxR and ?U target genes and indirectly or directly regulate the metabolism of Zn2+.In the study on the functions of target genes of MacRS,we found that there are at least three genes related to cell division.which are mmpA(sco6728).mmpB(sco4924)and mmpC(sco4011),and MmpA and MmpC mainly affect the formation of mycelial membranes,and MmpB plays an important role in the mycelial division and spore division.In the early growth stage of M145,FtsZ protein,Cross-membrane and cell wall components locate in the same position in the mycelium,involved in the formation of mycelial membrane.The localization of MmpA was determined by fluorescence and it is found that MmpA is mainly present in the inner membrane structure of the mycelium,including Cross-membrane.In the mmpA mutant strain,the density of FtsZ protein at the mycelial division point was decreased,and the vesicle did not aggregate around the Z-ring at the cell division point.The cell wall spanning the mycelium was formed under the guidance of FtsZ,but many cell wall synthetic components disorderly aggregated in the mycelium.The mycelial membrane was retained in the Cross-membrane state,and the Cross-wall was formed later than the wild-type strain.indicating that MmpA mainly guides vesicles aggregating around Z-ring and be related to the transportation and localization of cell wall synthetic components:MmpC also plays an important role in the assembly of divisome and it is mainly located in the Cross-wall and promotes the formation of mycelial membrane.The deletion of mmpC gene led to a decrease in the density of FtsZ protein at the mycelial division point,a delay in the formation of the cell wall,and an obstacle in the recruitment of the vesicle to the Z-ring.MmpB may anchor SsgB-FtsZ to the membrane structure during cell division,as DynAB and SepG during sporulation.In the mmpB mutant strain,the Z-rings in the mycelium and the reproductive hypha could not be assembled normally,resulting in a spherical or varying length of spores.Bacterial two-hybrid assays showed that MmpA,MmpB and MmpC interact with each other and interact with the FtsZ recruiting protein SsgB.In addition.MmpA interacts with the FtsZ partition protein CrgA.The results showed that the membrane components in the Cross-wall are likely to be fused by the Cross-membrane structure.The presence of the Z-ring and the cell wall accelerates the process,which explains the existence of the pores in the Cross-wall.MmpA and MmpC can directly participate in the formation of mycelial membrane.After the formation of Z ring by FtsZ,the cell membrane and cell wall forming materials are transported to the vicinity of the Z-ring during the formation of the membrane.MmpB can directly affect the formation and stability of the Z-ring which has a great influence on the formation of mycelium membrane and the normal division of spores.In summary,MacRS has global regulatory roles,including:(1)MacR directly regulates the transcription of membrane protein genes such as mmpA,mmpB and mmpC,and affects the formation of mycelium and spore septum of Streptomyces.(2)MacR regulates the transcription of the synthetic gene clusters of secondary metabolites,such as ACT,RED and CDA,and affects the syntheses of secondary metabolites;(3)since the conserved binding sites of MacR and Zur proteins are similar,the two may compete to regulate the genes related to the metabolism of Zn2+ and affect the Zn2+ metabolism.(4)The deletion of macR gene resulted in a significant decrease in the concentration of Zn2+ in the strain,which indirectly caused changes in the transcription level of ?U target genes.(5)MacR indirectly affects the transcription of SoxR target genes by regulating ACT synthesis.