| Biological nitrogen fixation is a high energy-consuming process,and each molecule of N2 reduced consumes 16 molecules of ATP.Therefore,the expression of nitrogen-fixing genes is strictly regulated in the cell,and energy synthesis is an important factor affecting the expression of nitrogen-fixing genes Central carbon metabolism is the main source of energy required by an organism,and ?-ketoglutarate is a signaling molecule for intracellular carbon status.Studies have shown that GlnK,a nitrogen metabolism-regulating protein in microorganisms,can bind to ?-ketoglutarate to sense carbon signals in vivo and regulate the expression of nitrogen metabolism genes,and mediate the coupling of carbon and nitrogen metabolism through direct molecular dialogue.Also,the study found that in certain nitrogen-fixing bacteria,the nitrogen-fixing-specific regulatory protein NifA also can sense and bind?-ketoglutarate,indicating that the expression of nitrogen-fixing genes can directly respond to intracellular carbon signal levels.Pseudomonas stutzeri A1501 is a rice rhizosphere combined nitrogen-fixing bacterium.The bacterium has the ability of nitrogen-fixing under low nitrogen and microaerobic conditions.The mechanism of nitrogen-fixing gene expression regulation of this bacterium is relatively in-depth,but the induction transmission mode and regulation mechanism of the nitrogen metabolism regulation system to carbon signal is still unclear.In this study,the protein sequence characteristics of the nitrogen metabolism-regulating proteins GlnK and NifA in A1501 were analyzed,and GlnK and NifA in A1501 were determined by homology modeling,site mutation,protein expression,and protein-small molecule interaction.The molecular interaction between the protein and?-ketoglutarate compared the effects of NifA protein and mutant protein on nitrogen-fixing gene expression.The research results provided theoretical support for the in-depth analysis of the carbon-nitrogen metabolism coupling mechanism of A1501.The main results obtained are as follows1.Sequence comparison and phylogenetic tree analysis showed that the GlnK and NifA proteins of A1501 were closely related to their homologous proteins in nitrogen-fixing bacteria.Both the GlnK protein of A1501 and the homologous proteins of Azotobacter vinelandii and Klebsiella pneumoniae contain the same reported amino acid position G89(glycine)that binds ?-ketoglutarate.The NifA protein has the same amino acid position F119(phenylalanine)as ?-ketoglutarate bound by Azotobacter vinelandii.Domain homology modeling indicates that the 119th position of the NifA protein of A1501 bacteria is located on a random coil.The protein disorder after mutation to S(serine)significantly changed,suggesting that this site may have a significant impact on protein conformation and corresponding regulatory functions2.Expression vectors of GlnK wild-type and 89th amino acid point mutant protein(GlnK-G89A,from glycine to alanine)were constructed respectively.Those proteins were induced to expression and purification in Escherichia coli;microscale thermophoresis experiment(microscale thermophoresis,MST)measured the binding of GlnK and GlnK-G89A protein to ?-ketoglutarate in vitro,the results showed that GlnK and ?-ketoglutarate can bind with each other.The dissociation constant,Kd,was 9.75±4.72 ?M,and the result of GlnK-G89A protein bind with ?-ketoglutarate was not detected.The binding of ?-ketoglutarate in vitro indicated that the glycine residue at position 89 of GlnK may play an important role in the binding of GlnK to ?-ketoglutarate3.Expression vectors for NifA of wild-type and NifA with amino acid mutation at position 119(NifA-F119S,from phenylalanine to serine)were constructed respectively.The proteins were induced to expression and purification in Escherichia coli;The binding of NifA and NifA-F119S protein to?-ketoglutarate were determined by the MST experiment in vitro.The results have shown that NifA can bind to ?-ketoglutarate with a dissociation constant of 3.42±3.12 ?M and NifA-F119S protein has no binding ability to ?-ketoglutarate which indicating that the phenylalanine residue at position 119 may play an important role in the binding of GlnK to ?-ketoglutarate.In summary,the GlnK and NifA proteins of A1501 can bind to ?-ketoglutarate,thereby sensing carbon status signals and then participating in the regulation of the nitrogen-fixing network.The G89 site of GlnK and the F119 site of NifA protein may play an important role in the nitrogen fixation of A15014.The wild-type nifA gene and the mutant gene which expressing the NifA-F119S protein were used to supplement the nifA mutant strain.Nitrogenase activity measurement showed that both genes could supplement the nitrogen-fixing activity of the nifA mutant strain.The nitrogenase activity of complementary strain with wild-type nifA gene was 155%of A1501,and the complementary strain with mutation nifA gene was only about 30%of the wild-type.The result of quantitative real-time RT-PCR showed that the nifA gene was highly expressed in both complementary strains.The expression of nifH in complementary strain with a mutant nifA gene is reduced by 35%compared to complementary strain with wild-type nifA gene;Western Blot result showed that the expression of NifH in complementary strain with mutant nifA gene was decreased significantly compared to wild-type.The phenylalanine residue at position 119 plays an important role in the transcriptional regulation of the nifH gene activated by NifAIn summary,the GlnK and NifA proteins of A1501 both can binding with ?-ketoglutarate in vitro The G89 site of GlnK and the F119 site of NifA protein may be important active sites in the A1501 strain that sense intracellular carbon signal and initiate the expression of downstream genes. |
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