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.     

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.     

The product of the nitrogen fixation regulatory gene nfrX of Azotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded by glnD in enteric bacteria.

We sequenced the nitrogen fixation regulatory gene nfrX from Azotobacter vinelandii, mutations in which cause a Nif- phenotype, and found that it encodes a 105-kDa protein (NfrX), the N terminus of which is highly homologous to that of the uridylyltransferase-uridylyl-removing enzyme encoded by glnD in Escherichia coli. In vivo complementation experiments demonstrate that the glnD and nfrX products are functionally interchangeable. A vinelandii nfrX thus appears to encode a uridylyltransferase-uridylyl-removing enzyme, and in this paper we report the first sequence of such a protein. The Nif- phenotype of nfrX mutants can be suppressed by a second mutation in a recently identified nifL-like gene immediately upstream of nifA in A. vinelandii. NifL mediates nif regulation in response to the N status in A. vinelandii, presumably by inhibiting NifA activator function as occurs in Klebsiella pneumoniae; thus, one role of NfrX is to modify, either directly or indirectly, the activity of the nifL product.