Involvement of GlnK, a PII protein, in control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1501.

The nitrogen-fixing, root-associated strain Pseudomonas stutzeri A1501 carries a single gene encoding a protein from the PII family, designated glnK. The glnK gene is co-transcribed with two distantly related copies of amtB genes encoding putative ammonium channels. Transcription of glnK was decreased in the presence of ammonia and was partly dependent on NtrC and RpoN under nitrogen-limiting conditions. Inactivation of glnK led to a mutant strain devoid of nitrogenase activity, auxotrophic for glutamine and unable to deadenylylate glutamine synthetase, while inactivation of amtB1 led to a prototrophic and Nif+ mutant strain. RT-PCR analysis showed that nifA transcription was abolished in the glnK mutant, while glnA remained transcribed. Using the yeast two-hybrid system, an interaction between GlnK and the C-terminal domain of NifL was observed, suggesting GlnK-dependent control of NifA activity by NifL. Introduction of a plasmid that expressed nifA from a constitutive promoter restored nitrogen fixation to the glnK mutant, and nitrogenase activity was observed even in the presence of ammonia. GlnK signalling appears to be a key regulatory element in control of ammonia assimilation, of nifA expression and in modulation of NifA activity by NifL.

He S ，Chen M ，Xie Z ，Yan Y ，Li H ，Fan Y ，Ping S ，Lin M ，Elmerich C ... -  《ARCHIVES OF MICROBIOLOGY》

Regulation of nitrogen fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, transducing two environmental signals to the nif transcriptional activator NifA.

The enzymatic reduction of molecular nitrogen to ammonia requires high amounts of energy, and the presence of oxygen causes the catalyzing nitrogenase complex to be irreversible inactivated. Thus nitrogen-fixing microorganisms tightly control both the synthesis and activity of nitrogenase to avoid the unnecessary consumption of energy. In the free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii, products of the nitrogen fixation nifLA operon regulate transcription of the other nifoperons. NifA activates transcription of nif genes by the alternative form of RNA-polymerase, sigma54-holoenzyme; NifL modulates the activity of the transcriptional activator NifA in response to the presence of combined nitrogen and molecular oxygen. The translationally-coupled synthesis of the two regulatory proteins, in addition to evidence from studies of NifL/NifA complex formation, imply that the inhibition of NifA activity by NifL occurs via direct protein-protein interaction in vivo. The inhibitory function of the negative regulator NifL appears to lie in the C-terminal domain, whereas the N-terminal domain binds FAD as a redox-sensitive cofactor, which is required for signal transduction of the internal oxygen status. Recently it was shown, that NifL acts as a redox-sensitive regulatory protein, which modulates NifA activity in response to the redox-state of its FAD cofactor, and allows NifA activity only in the absence of oxygen. In K. pneumoniae, the primary oxygen sensor appears to be Fnr (fumarate nitrate reduction regulator), which is presumed to transduce the signal of anaerobiosis towards NifL by activating the transcription of gene(s) whose product(s) function to relieve NifL inhibition through reduction of the FAD cofactor. In contrast, the reduction of A. vinelandii-NifL appears to occur unspecifically in response to the availability of reducing equivalents in the cell. Nitrogen status of the cells is transduced towards the NifL/NifA regulatory system by the GlnK protein, a paralogue PII-protein, which appears to interact with the NifL/NifA regulatory system via direct protein-protein interaction. It is not currently known whether GlnK interacts with NifL alone or affects the NifL/NifA-complex; moreover the effects appear to be the opposite in K. pneumoniae and A. vinelandii. In addition to these environmental signals, adenine nucleotides also affect the inhibitory function of NifL; in the presence of ATP or ADP the inhibitory effect on NifA activity in vitro is increased. The NifL proteins from the two organisms differ, however, in that stimulation of K. pneumoniae-NifL occurs only when synthesized under nitrogen excess, and is correlated with the ability to hydrolyze ATP. In general, transduction of environmental signals to the nif regulatory system appears to involve a conformational change of NifL or the NifL/NifA complex. However, experimental data suggest that K. pneumoniae and A. vinelandii employ significantly different species-specific mechanisms of signal transduction.

Schmitz RA ，Klopprogge K ，Grabbe R 《JOURNAL OF MOLECULAR MICROBIOLOGY AND BIOTECHNOLOGY》

Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501.

The nitrogen-fixing Pseudomonas stutzeri strain A1501 contains two ammonium transporter genes, amtB1 and amtB2, linked to glnK. Growth of an amtB1-amtB2 double deletion mutant strain was not impaired compared to that of the wild type under any conditions tested, and it was still capable of taking up ammonium ions at nearly wild-type rates. Nitrogenase activity was repressed in wild-type strain A1501 in response to the addition of ammonium, but nitrogenase activity was only partially impaired in the amtB1 and amtB2 double mutant, suggesting that the two AmtB proteins are involved in regulating expression of nitrogenase or its activity in response to ammonium. An interaction between GlnK and AmtB1 or AmtB2 was observed in a yeast two-hybrid assay. Ammonium was excreted by the amtB double mutant strain under nitrogen fixation conditions, particularly when nifA was expressed constitutively. This suggests that AmtB proteins play a role in controlling the internal pool of ammonia within the cell.

Zhang T ，Yan Y ，He S ，Ping S ，Alam KM ，Han Y ，Liu X ，Lu W ，Zhang W ，Chen M ，Xiang W ，Wang X ，Lin M ... -  《-》

Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae.

In the diazotroph Klebsiella pneumoniae, the nitrogen sensory protein GlnK mediates the cellular nitrogen status towards the NifL/NifA system that regulates transcription of the nitrogen fixation genes in response to ammonium and molecular oxygen. To identify amino acids of GlnK essential for this signal transduction by protein-protein interaction, we performed random point mutagenesis by PCR amplification under conditions of reduced Taq polymerase fidelity. Three thousand two hundred mutated glnK genes were screened to identify those that would no longer complement a K. pneumoniaeDeltaglnK strain for growth under nitrogen fixing conditions. Twenty-four candidates resulting in a Nif(-) phenotype were identified, carrying 1-11 amino acid changes in GlnK. Based on these findings, as well as structural data, several single mutations were introduced into glnK by site-directed mutagenesis, and the Nif phenotype and the respective effects on NifA-mediated nif gene induction was monitored in K. pneumoniae using a chromosomal nifK'-'lacZ fusion. Single amino acid changes resulting in significant nif gene inhibition under nitrogen limiting conditions were located within the highly conserved T-loop (A43G, A49T and N54D), the body of the protein (G87V and K79E) and in the C-terminal region (I100M, R103S, E106Q and D108G). Complex formation analyses between GlnK (wild-type or derivatives) and NifL or NifA in response to 2-oxoglutarate indicated that: (a) besides the T-loop, the C-terminal region of GlnK is essential for the interaction with NifL and NifA and (b) GlnK binds both proteins in the absence of 2-oxoglutarate, whereas, in the presence of 2-oxoglutarate, NifA is released but NifL remains bound to GlnK.

Glöer J ，Thummer R ，Ullrich H ，Schmitz RA ... -  《-》

Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501.

Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.

Xie Z ，Dou Y ，Ping S ，Chen M ，Wang G ，Elmerich C ，Lin M ... -  《MICROBIOLOGY-SGM》