Regulation Of NirA And Differential Proteomic Study Of Microcystis Aeruginosa Cultured On Different Nitrogen Nutrients And Different 2-OG Concentrations

In our research,we analyzed the structure of nitrate assimilation operon named nirA,cloned and expressed the nirA gene.Then,we detected the amount of NirA by western blotting and researched differential proteins by proteomic techniques of Microcystis aeruginosa 7806 cultured under different nutrients and differentα-ketoglutaric acid(2-OG) concentrations.The potential function of these different proteins was discussed.All these parameters gave us a deep insight of the nitrogen metabolism mechanisms:Firstly,we analyzed the nirA operon and found that gene nirA and narB are far away from nrtABCD structure.The reserved gene nirA encode for nitrite reductase was cloned into pGEX-6p-1 vector and then expressed in E.coli BL21(DE3) plysS, the purified product GST-NirA was used to immunize mice to prepare antiserum.Secondly,the impact of different nitrogen nutrition and 2-OG concentration on Microcystis aeruginosa 7806 was analyzed.The cyanobacteria were cultured on nitrogen-free medium previously and then grown on four different nitrogen enviroment(no nitrogen,10mM nitrite,15mM nitrate,3.75mM Ammonium sulfate) from 24h to 72h.During the next step of our experiment,the cyanobacteria were cultured on four different 2-OG concentrations(0,0.5,1,10mM) for 72h.We analyzed the fluctuations of NirA of the induced cyanobacteria.The results show that nitrate has a positive effect on the NirA expression,while nitrite and Ammonium sulfate have negative effect,the NirA content decrease sharply when cultured in the media without nitrogen;2-OG has a concentration dependent positive effect on NirA expression,the positive effect reduces when the concentration is over 10mM,and it's regulation extent is smaller than nitrate.We also put forward the scheme for the regulation of the nitrate assimilation operon by 2-OG in M.aeruginosa 7806 cells and discussed the feasibility of alage blooming controlling by adding 2-OG.Lastly,the protein profile of Microcystis aeruginosa 7806 treated in the same way was analyzed.Compared to the normal controlled condition(15mM nitrate),28 differential protein spots were found when the cyanobacteria were cultured on different nitrogen environment.On the other hand,14 protein spots are affected by adding 1mM 2-OG to the culture media.We discussed further effect of 10 protein spots on the regulation of NirA and nitrogen metabolism:glutamine synthetase, aspartate aminotransferase and aminotransferase are related to the transport and utilization of Ammonium;ABC transporter protein can transfer compounds between membranes;PtsN regulates the transcription of nitrogen metabolism genes by sigma factorσ⁵⁴;transcriptional regulator of LysR family is related to amino acid metabolism;histidine kinase senses signals on the outside of the cell and convert it into a response which can involve a change in the synthesis of proteins;elongation factor Tu charges of the elongation of peptides,and it may also acts as a chaperonin; peroxiredoxin is related to cell differentiation,apoptosis and signal truansduction.