Regulation Of The Pseudomonas Stutzeri Nitrogen-fixing Island By The Escherichia Coli Nitrogen Regulatory Protein GlnG

The nitrogen-fixing island of Pseudomonas stutzeri A1501has been transferred into Escherichiacoli DH10B successfully in our laboratory. The nitrogenase activity of the EN-01(recombinant E. coliharboring the exogenous nitrogen fixation island) is far less than that of the donor strain A1501.Biological nitrogen fixation is a complex regulatory network involving a series of signal transductionand effector proteins. In Pseudomonas and Enterobacteria, nitrogen regulatory protein NR_I(encoded byntrC in P. stutzeri and by glnG in E.coli) is the master nitrogen regulator, which is required to activatethe expression of a number of genes involved in nitrogen uptake and metabolism, and also required fornitrogen fixation in Pseudomonas stutzeri. In this article, we have tried to identificate the regulatoryfunction of EN-01GlnG to the expression of nitrogen-fixing island genes from A1501. The main resultsare as following:1. Amino acid sequence alignment between DH10B GlnG and A1501NtrC showed the identity is65.35%. And the comparisons of secondary structures and advanced structures from two proteins foundthat they are very similar. They also have similar physical and chemical properties.2. Complementary experiments showed the DH10B GlnG can partially complement the A1501NtrC deletion mutant’s nitrogen-fixation ability in nitrogen-free medium K and the growth ability inmedium K when KNO3or urea was used as a sole nitrogen source. And the A1501NtrC can partiallycomplement the DH10B GlnG deletion mutant’s growth ability in medium M9containing arginine as asole nitrogen source. These results indicated that both DH10B GlnG and A1501NtrC could relativelycomplement the regulatory functions of each other in the process of general nitrogen metabolism andnitrogen fixation. But we also found the regulatory function of GlnG was below than NtrC innitrogen-fixing process, and this may be one of the reasons why the nitrogenase activity of the EN-01was lower.3. Because GlnG must be phosphorylated (GlnG-P) to catalyze ATP hydrolysis and activatetranscription in vitro, the purification of GlnG-P is the precondition to study the interaction betweenGlnG and target genes. We constructed a derivative of glnG encoding the substitutions D54E and S160Fby fusion PCR, which could give rise to a form of the activator that does not require phosphorylation byNR_IIto activate transcription. Next, to prove that the GlnG-P can activate the genes’ transcription fromA1501nitrogen-fixing island, the GlnG-P protein was expressed and purified. Finally, the experimentalresults of Gel retardation showed that GlnG-P protein could be combined with nifA promotor region. Sowe draw the conclusion that under the condition of nitrogen fixation, EN-01GlnG could activatetranscription of the related genes in nitrogen-fixing island from A1501.