Preliminary Study On Nitrogen Metabolism Regulation Of Micromonospora Carbonacea JXNU-1

The nitrogen metabolism of microorganisms is precise regulated.It is a hot topic in microbiology that microorganism regulate their physiology and synthesis of secondary metabolites by sensing the changes of nitrogen in real time.Micromonospora carbonacea JXNU-1 is a micromonospora actinomycetes with broad-spectrum antibacterial activity,and its antibacterial product is a new nucleoside antibiotic JX with important application prospects.According to the previous studies,nitrogen sources has significant effect on the growth and ferment production of antibiotic JX of Micromonospora carbonacea JXNU-1,but its specific effect and mechanism are still unclear.In this paper,we study the effect of nitrogen source on its growth and metabolism,then screen the differentially expression genes of nitrogen metabolism by i TRAQ quantitative proteomics,find and obtain nitrogen metabolism regulatory proteins by bioinformatic and heterologous expression method,which laid the experimental foundation for the complete analysis of nitrogen metabolism regulation system of Micromonospora carbonacea JXNU-1.The specific content and result are as follow:1.The mycelium morphology is studied by Gram staining observation method,the growth curve is drawn by wet weight method,and the antibacterial activity is detected by Oxford cup method.These results show that the cell morphology,growth curve and antibacterial activity of Micromonospora carbonacea JXNU-1 has obviously different characteristics in fermentation culture under different nitrogen source and different concentrations of the same nitrogen source.It is confirmed that nitrogen source has significant effect on the physiology and secondary metabolite synthesis of Micromonospora carbonacea JXNU-1.2.Collect the Micromonospora carbonacea JXNU-1 bacteria cultured by different nitrogen source,use i TRAQ quantitative proteomics technology to screen the nitrogen metabolism regulating protein of Micromonospora carbonacea JXNU-1.Obtain 1354 differentially expression proteins,of which 661 proteins are up-regulated and 693down-regulated.It is find that the expression of gln A,gln B and gln D genes related to nitrogen metabolism is up-regulated in the two-component metabolic pathway,find gd H2 gene related to nitrogen metabolism is down-regulated in the arginine synthesis pathway,and verify its expression trend by q RT-PCR.3.Comparing the i TRAQ quantitative proteomics results of Micromonospora carbonacea JXNU-1 with the currently widely studied Ntr two-component nitrogen metabolism regulation system,it is speculated that Ntr B/C nitrogen metabolism regulation system exists in Micromonospora carbonacea JXNU-1,but the ntr B/C gene is not found in this bacteria,then through bioinformatic analysis to find the 5 pairs of ntr B/C genes with the high similarity,Two pairs of ntr B/C homologous genes with the highest similarity are selected,and the recombinant expression plasmid of p ET29a(+)-ntr B/C homologous gene is constructed by gene synthesis and molecular biology methods,and then transformed into E.coli expression strain.Finally,two pairs of Ntr B/C homologous proteins are obtained by IPTG induction,histidine affinity chromatography purification,dialysis and SDS-PAGE identification.At the same time,preliminary verification its function by protein autophosphorylation and phosphate transfer.The above results lay the foundation for the follow-up in-depth study of the composition and regulation mechanism of the nitrogen metabolism regulation system of Micromonospora carbonacea JXNU-1,as well as,study the regulation mechanism of nitrogen sources on the synthesis of secondary metabolites on this basis.