Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus

Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnB-GlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.     

Quaternary structure changes in a second Per-Arnt-Sim domain mediate intramolecular redox signal relay in the NifL regulatory protein

Per-Arnt-Sim (PAS) domains play a critical role in signal transduction in multidomain proteins by sensing diverse environmental signals and regulating the activity of output domains. Multiple PAS domains are often found within a single protein. The NifL regulatory protein from Azotobacter vinelandii contains tandem PAS domains, the most N-terminal of which, PAS1, contains a FAD cofactor and is responsible for redox sensing, whereas the second PAS domain, PAS2, has no apparent cofactor and its function is unknown. Amino acid substitutions in PAS2 were identified that either lock NifL in a form that constitutively inhibits NifA or that fail to respond to the redox status, suggesting that PAS2 plays a pivotal role in transducing the redox signal from PAS1 to the C-terminal output domains. The isolated PAS2 domain is a homodimer in solution and the subunits are in rapid exchange. PAS2 dimerization is maintained in the redox signal transduction mutants, but is inhibited by substitutions in PAS2 that lock NifL in the inhibitory conformer. Our results support a model for signal transduction in NifL, whereby redox-dependent conformational changes in PAS1 are relayed to the C-terminal domains via changes in the quaternary structure of the PAS2 domain.     

Two residues in the T-loop of GlnK determine NifL-dependent nitrogen control of nif gene expression

X-ray crystallographic analysis of the Escherichia coli P(II) protein paralogues GlnB and GlnK has shown that they share a superimposable structural core but can differ in conformation of the T-loop, a region of the protein (residues 37-54) that has been shown to be important for interaction with other proteins. In Klebsiella pneumoniae GlnK has been shown to have a clearly defined function in regulating NifL-mediated inhibition of NifA activity in response to the nitrogen status, and GlnB, when expressed from the chromosome, does not substitute for GlnK. Because the T-loops of K. pneumoniae and E. coli GlnB and GlnK differ at just three residues, 43, 52, and 54, we have used a previously constructed heterologous system, in which K. pneumoniae nifLA is expressed in E. coli, to investigate the importance of GlnK residues 43, 52, and 54 for regulation of the NifLA interaction. By site-directed mutagenesis of glnB we have shown that residue 54 is the single most important amino acid in the T-loop in the context of the regulation of NifA activity. Furthermore, a combination of just two changes, in residues 54 and 43, allows GlnB to function as GlnK and completely relieve NifL inhibition of NifA activity.     

Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein

PII-like signal transduction proteins, which respond to the nitrogen status via covalent modification and signal the carbon status through the binding of 2-oxoglutarate, have been implicated in the regulation of nitrogen fixation in several diazotrophs. The NIFL-NIFA two-component regulatory system, which integrates metabolic signals to fine-tune regulation of nitrogenase synthesis in Azotobacter vinelandii, is a potential target for PII-mediated signal transduction. Here we demonstrate that the inhibitory activity of the A.vinelandii NIFL protein is stimulated by interaction with the non-uridylylated form of PII-like regulatory proteins. We also observe that the NIFL-NIFA system is directly responsive to 2-oxoglutarate. We propose that the PII protein signals the nitrogen status by interaction with the NIFL-NIFA system under conditions of nitrogen excess, and that the inhibitory activity of NIFL is relieved by elevated levels of 2-oxoglutarate when PII is uridylylated under conditions of nitrogen limitation. Our observations suggest a model for signal transduction to the NIFL-NIFA system in response to carbon and nitrogen status which is clearly distinct from that suggested from studies on other diazotrophs.     

Role of GlnK in NifL-Mediated Regulation of NifA Activity in Azotobacter vinelandii

In several diazotrophic species of Proteobacteria, P(II) signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH(4)(+) by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the P(II)-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP in A. vinelandii were studied to determine whether the Nif (-) phenotype of glnD strains was due to an inability to modify GlnK, an effort previously hampered because glnK is an essential gene in this organism. A glnKY51F mutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH(4)(+), suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation. glnKY51F strains were significantly impaired for diazotrophic growth and expression of a nifH-lacZ fusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041-6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacterium Klebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.     

Unique mechanistic features of post-translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability

PII-like signal transduction proteins are found in all three domains of life and have been shown to play key roles in the control of bacterial nitrogen assimilation. This communication reports the first target protein of an archaeal PII-like protein, representing a novel PII receptor. The GlnK(1) protein of the methanogenic archaeon Methanosarcina mazei strain Go1 interacts and forms stable complexes with glutamine synthetase (GlnA(1)). Complex formation with GlnK(1) in the absence of metabolites inhibits the activity of GlnA(1). On the other hand, the activity of this enzyme is directly stimulated by the effector molecule 2-oxoglutarate. Moreover, 2-oxoglutarate antagonized the inhibitory effects of GlnK(1) on GlnA(1) activity but did not prevent GlnK(1)/GlnA(1) complex formation. On the basis of these findings, we hypothesize that besides the dominant effector molecule 2-oxoglutarate, the nitrogen sensor GlnK(1) allows finetuning control of the glutamine synthetase activity under changing nitrogen availabilities and propose the following model. (i) Under nitrogen limitation, increasing concentrations of 2-oxoglutarate stimulate maximal GlnA(1) activity and transform GlnA(1) into an activated conformation, which prevents inhibition by GlnK(1). (ii) Upon a shift to nitrogen sufficiency after a period of nitrogen limitation, GlnA(1) activity is reduced by decreasing internal 2-oxoglutarate concentrations through diminished direct activation and by GlnK(1) inhibition.