Study On The Mechanism Of Cyanobacterial Nm1 In Nitrate Uptake

As one of the most important elements,nitrogen is important for lower organisms,such as bacteria,algae,and also for higher organisms.The elements made of nitrogen such as protein and nucleic acids are vital to maintain the normal life activities of living organisms.For cyanobacteria,a model organism,the nitrogen source utilization is very widely,nitrate,nitrite,ammonia salts,organic compounds such as urea and amino acids.Also the cyanobacteria with hetetocyst can also use nitrogen.Because of the widest range of nitrate distribution in many kinds of nitrogen sources,some achievements have been gotten in the study of the absorption of nitrate in cyanobacteria.In recent years,human beings find that the nitrate is transported into the cell via the NRT channel protein,and then nitrate reductase and nitrite reductase make nitrate to be reduced to ammonium,and then transported the GS-GOGAT pathway to be a basic amino acids as original of other N molecules.Some proteins involving in nitrate uptake have been found,such as protein NtcA and protein P?.But in the mutant of regulatory factors regulating nitrate absorption,the nitrate uptake is only partially affected,and the mutants can still survive.It is clear that there are still some other regulatory proteins unidentified involving in nitrate uptake.Under this background,this paper used Synechocystis 6803 transposons mutant library to select some genes related with nitrate uptake and identified a gene involved in nitrate uptake named gene nm1.Relevant achievements in nitrate uptake have been achieved and main results are as follows:1)In the low nitrate medium,we screened the transposon mutant library,and we screened two same mutant strains that could not grow normally under low nitrate,which are the same gene nm1.Further experimental evidences showed that nm1 mutant strain had less protein NRTA in low nitrate culture in some degree,compared with the wild type.It suggests that nm1 may be involved in nitrate uptake.2)The interaction between protein Nm1 and protein P? has been proved by threeprotein-protein interactions experiments such as GST pull-down,Yeast two hybrid and Co-IP.In the context of low nitrate and 2% carbon dioxide,the phosphorylation level of P? in the nm1 mutant is reduced,which changes the conformation of protein P? to regulate the absorption of nitrate.So we consider that Nm1 regulating nitrate uptake may functions through P? regulation..3)Interactions,between protein Nm1 and protein P?,change the conformation of P?,causing the competition between Pip X-NtcA protein complex and Pip X-P? protein complex to change.In WT,Pip X-NtcA protein complex is more than Pip X-P? protein complex,so the gene related with nitrate uptake can be expresses to absorb the nitrate.While in the nm1 mutant,Pip X-P? complex is more,so that the free NtcA with no binding protein Pip X cannot activate the nitrate gene including nrt gene,resulting in the nm1 mutant death in low nitrate.In conclusion,we successfully identified a novel gene involving in nitrate uptake.nm1 as a upstream gene of gln B(encoding P?),changes phosphorylation level of P? by an interaction with P?.Then phosphorylation level of P? made interaction between Pip X-NtcA and Pip X-P? changed,so that the state of NtcA with or without Pip X,makes a different nitrate uptake.