Study On A Pair Of Two-component System Related To Nitrogen Metabolism Regulation In Micromonospora Carbonacea

Two-component system is a common signaling mechanism in microorganisms,which controls microbial growth and synthesis of secondary metabolites through phosphorylation of key effector proteins by protein kinases.Microbial nitrogen metabolism is regulated precisely.Microorganisms regulate their own physiology and synthesis of secondary metabolites by sensing changes in external nitrogen content.The research group isolated a Micromonospora carbonacea in the early stage,and found that the product with antibacterial ability in its fermentation liquid,and the level of nitrogen source have significant effects on the growth of M.carbonacea and the synthesis of secondary metabolites.Proteomic analysis revealed differences in the expression of nitrogen metabolism regulation proteins.Genome analysis screened a pair of proteins MCNtrB/MCNtrC homologous to the two-component NtrB/NtrC system of nitrogen metabolism regulation in gram-negative bacteria,but the specific mechanism of action is still unclear and needs further study.In this paper,M.carbonacea JXNU-1 was the research object.Firstly,the basic properties of MCNtrB/MCNtrC were analyzed by bioinformatics technology,and the target proteins were obtained by heterologous expression of prokaryotic vector.The function of protein was analyzed by phosphorylation experiment,and the interaction of MCNtrB/MCNtrC was verified by yeast two-hybrid.Finally,MCNtrB/MCNtrC was overexpressed in the target bacteria to verify its effect.It lays a foundation for the regulation mechanism of nitrogen metabolism of M.carbonacea.The specific content and result are as follow:1.Bioinformatics method was used to research two-component system proteins related to nitrogen metabolism,ProtParam and other related software were used to study the basic properties and structural interactions of proteins.The results showed that the two-component system protein MCNtrB,a two-component system protein involved in the regulation of nitrogen metabolism,was a hydrophobic protein with amino acid residue 477 and molecular weight 51.806 kDa,and MCNtrC is a hydrophilic protein with amino acid residue 246 and molecular weight 27.554 kDa.And MCNtrB and MCNtrC interact structurally.And MCNtrB interacts with MCNtrC structurally.2.The recombinant expression plasmid pET29a(+)-MCNtrB and pET29a(+)-MCNtrC were constructed by gene synthesis method and transformed into Escherichia coli.After IPTG induction,protein purification and SDS-PAGE,MCNtrB and MCNtrC proteins were obtained.At the same time,protein self-phosphorylation and phosphoric acid transfer were used to verify its TCSs function,and then LC-MS/MS was used to discover the active sites of protein phosphorylation.Using plasmids pET29a(+)-MCNtrB and pET29a(+)-MCNtrC as templates,the eukaryotic expression plasmids pGBT9-MCNtrB and pGAD10-MCNtrC were constructed,and the interaction between MCNtrB and MCNtrC was demonstrated by yeast two-hybrid method.3.The MCNtrB and MCNtrC genes of M.carbonacea were cloned into the Streptomyces overexpression plasmid pSET152 AKEx,and then the recombinant plasmid was introduced into M.carbonacea to obtain the strains that overexpressed MCNtrB and MCNtrC.M.carbonacea and overexpressed strains were cultured by lownitrogen fermentation.The Oxford Cup method was used to detect the antimicrobial activity of M.carbonacea.The results showed that the antimicrobial activity of M.carbonacea was significantly different from that of the overexpressed strains,which confirmed that the overexpression of MCNtrB and MCNtrC proteins had an effect on the synthesis of secondary metabolites of M.carbonacea.The above results laid the foundation for increasing the regulatory mechanism of nitrogen metabolism of M.carbonacea,and provided a basis for further elaborating the biosynthesis mechanism of M.carbonacea and its secondary metabolites.