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Isolation of the NIFL-NIFA complex directly from lysed cells. BL21(DE3)(pLysS) cells carrying plasmid pPR38 were grown as described in Materials and Methods. (a) The cell paste was resuspended in lysis buffer and divided into four aliquots. These were lysed and chromatographed as described in Materials and Methods with the following additions: lane 1, no nucleotide; lane 2, with 1 mM magnesium acetate; lane 3, with 1 mM ADP; lane 4, with 1 mM MgADP. Lanes 1 to 4, fractions containing protein which eluted with 0.5 M imidazole. Lane M contains molecular weight markers. (b) Cell paste was resuspended in lysis buffer containing 1 mM MgADP and was divided into two aliquots. These were applied to the nickel chelating column and washed with equilibration buffer containing 1 mM MgADP as described in Materials and Methods. Lane 1, cell supernatant; lane 2, nonbound protein from cell supernatant; lanes 3 to 7, NIFL-NIFA-containing fractions eluted with 0.5 M imidazole in the presence of 1 mM MgADP; lanes 8 to 13, fractions eluted with 0.5 M imidazole after washing the column in equilibration buffer without nucleotide to dissociate NIFA. (c and d) Absorbance spectra of oxidized NIFL and isolated NIFL-NIFA complex in the presence of 500 M MgADP. The spectra were recorded with a Shimadzu MP2000 spectrophotometer with a 1-cm light path and 1-nm slit width.
Source publication
In Azotobacter vinelandii, activation of nif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition...
Context in source publication
Context 1
... 6. Transcriptional activity of NIFA dissociated from the NIFL-NIFA complex. A single-round transcription assay from the nifH promoter was carried out as described in Materials and Methods. NIFA was dissociated from the NIFL-NIFA complex as described in the legend to Fig. 5. ϩ , present; Ϫ , absent.
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A flavoprotein, named azotoflavin, was isolated from an extract of Azotobacter vinelandii cells, which linked the reducing power generated by illuminated spinach chloroplasts to the Azotobacter nitrogen-fixing enzyme complex. The photoreduction of the yellow azotoflavin by chloroplasts produced a stable, free-radical semiquinone, blue in color, wit...
Citations
... A. vinelandii NifL is a tetrameric protein [109] in which each monomer can be divided into three discrete functional domains: the N-terminal-sensing domain, a C-terminal kinase-like domain and glutamine-rich linker domain connecting the N-terminal and C-terminal domains (Figure 1). The N-terminal domain contains tandem PAS domains, and the first N-terminal PAS domain (PAS1) binds an FAD that is responsive to oxidative status, and thus is necessary for sensing redox stress [110], whereas the PAS2 domain is proposed to transduce the redox signal perceived by the PAS1 domain by changing the quaternary structure of the protein [111]. ...
... A. vinelandii NifL is a tetrameric protein [109] in which each monomer can be divided into three discrete functional domains: the N-terminal-sensing domain, a C-terminal kinaselike domain and glutamine-rich linker domain connecting the N-terminal and C-terminal domains (Figure 1). The N-terminal domain contains tandem PAS domains, and the first Nterminal PAS domain (PAS1) binds an FAD that is responsive to oxidative status, and thus is necessary for sensing redox stress [110], whereas the PAS2 domain is proposed to transduce the redox signal perceived by the PAS1 domain by changing the quaternary structure of the protein [111]. ...
Bacteria, like humans, face diverse kinds of stress during life. Oxidative stress, which is produced by cellular metabolism and environmental factors, can significantly damage cellular macromolecules, ultimately negatively affecting the normal growth of the cell. Therefore, bacteria have evolved a number of protective strategies to defend themselves and respond to imposed stress by changing the expression pattern of genes whose products are required to convert harmful oxidants into harmless products. Structural biology combined with biochemical studies has revealed the mechanisms by which various bacterial redox sensor proteins recognize the cellular redox state and transform chemical information into structural signals to regulate downstream signaling pathways.
... The isolated N-terminal domain did not, however, inhibit NifA activity. On the other hand, the C-terminal domain of NifL has been found to bind ADP (S€ oderb€ ack et al., 1998) and it was the C-terminal domain of NifL that interacted with the N-terminal domain of NifA (Money, Jones, Dixon, & Austin, 1999). A complex of purified NifA and NifL formed in presence of MgADP, when subjected to limited proteolysis, revealed protection of the N-terminal region of NifA close to the Q-linker (Money, Barrett, Dixon, & Austin, 2001). ...
Azotobacters have been used as biofertilizer since more than a century. Azotobacters fix nitrogen aerobically, elaborate plant hormones, solubilize phosphates and also suppress phytopathogens or reduce their deleterious effect. Application of wild type Azotobacters results in better yield of cereals like corn, wheat, oat, barley, rice, pearl millet and sorghum, of oil seeds like mustard and sunflower, of vegetable crops like tomato, eggplant, carrot, chillies, onion, potato, beans and sugar beet, of fruits like mango and sugar cane, of fiber crops like jute and cotton and of tree like oak. In addition to the structural genes of the enzyme nitrogenase and of other accessory proteins, A. vinelandii chromosomes contain the regulatory genes nifL and nifA. NifA must bind upstream of the promoters of all nif operons for enabling their expression. NifL on activation by oxygen or ammonium, interacts with NifA and neutralizes it. Nitrogen fixation has been enhanced by deletion of nifL and by bringing nifA under the control of a constitutive promoter, resulting in a strain that continues to fix nitrogen in presence of urea fertilizer. Additional copies of nifH (the gene for the Fe-protein of nitrogenase) have been introduced into A. vinelandii, thereby augmenting nitrogen fixation. The urease gene complex ureABC has been deleted, the ammonia transport gene amtB has been disrupted and the expression of the glutamine synthase gene has been regulated to enhance urea and ammonia excretion. Gluconic acid has been produced by introducing the glucose dehydrogenase gene, resulting in enhanced solubilization of phosphate.
... GlnK interaction with NifL is dependent on both MgATP and 2-OG (Little et al., 2000). NifL binds ATP and ADP, and both nucleotides stimulate NifL-NifA complex formation (Eydmann et al., 1995;Söderbäck et al., 1998;Money et al., 1999), whereas the N-terminal NifA GAF domain binds 2-OG causing a reduction in NifL/ NifA interaction (Little et al., 2000;Little & Dixon, 2003;Martinez-Argudo et al., 2004a). The interaction between GlnK and NifL restores NifA inhibition even when the 2-OG level is saturating, therefore the GlnK signal overrides the 2-OG signal (Little & Dixon, 2003). ...
The P(II) proteins are one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably, P(II) proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in recent years the extent of these interactions has been recognized to be much greater than heretofore described. Major advances have been made in structural studies of P(II) proteins, including the solution of the first structures of P(II) proteins complexed with their targets. We have also begun to gain insights into how the key effector molecules, 2-oxoglutarate and ATP/ADP, influence the activities of P(II) proteins. In this review, we have set out to summarize our current understanding of P(II) biology and to consider where future studies of these extraordinarily adaptable proteins might lead us.
... This finding strongly indicates that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae (55). In Azotobacter vinelandii, another diazotrophic bacterium regulating its nif gene expression by a nifLA operon, NifA activity is also regulated in response to ammonium and molecular oxygen by direct protein-protein interaction by its negative regulator NifL (4,23,39,40,47). However, in contrast to K. pneumoniae a change in the cellular localization of NifL has not been observed, and the electrons for FAD reduction are presumably derived from reduction equivalents present in the cytoplasm during anaerobiosis (7,30,34,35). ...
In Klebsiella pneumoniae nitrogen fixation is tightly controlled in response to ammonium and molecular oxygen by the NifL/NifA regulatory system.
Under repressing conditions, NifL inhibits the nif-specific transcriptional activator NifA by direct protein-protein interaction, whereas under anaerobic and nitrogen-limited
conditions sequestration of reduced NifL to the cytoplasmic membrane impairs inhibition of cytoplasmic NifA by NifL. We report
here on a genetic screen to identify amino acids of NifL essential for sequestration to the cytoplasmic membrane under nitrogen-fixing
conditions. Overall, 11,500 mutated nifL genes of three independently generated pools were screened for those conferring a Nif− phenotype. Based on the respective amino acid changes of nonfunctional derivatives obtained in the screen, and taking structural
data into account as well, several point mutations were introduced into nifL by site-directed mutagenesis. The majority of amino acid changes resulting in a significant nif gene inhibition were located in the N-terminal domain (N46D, Q57L, Q64R, N67S, N69S, R80C, and W87G) and the Q-linker (K271E).
Further analyses demonstrated that positions N69, R80, and W87 are essential for binding the FAD cofactor, whereas primarily
Q64 and N46, but also Q57 and N67, appear to be crucial for direct membrane contact of NifL under oxygen and nitrogen limitation.
Based on these findings, we propose that those four amino acids most likely located on the protein surface, as well as the
presence of the FAD cofactor, are crucial for the correct overall protein conformation and respective surface charge, allowing
NifL sequestration to the cytoplasmic membrane under derepressing conditions.
... As mentioned above, the GHKL domain of NifL binds adenosine nucleotides but does not hydrolyse ATP (Söderbäck et al., 1998). NifL is incompetent to bind NifA in the absence of nucleotide in vitro (Eydmann et al., 1995) and ADP binding has been shown to stabilise the NifL-NifA binary complex (Eydmann et al., 1995;Money et al., 1999). NifL has a higher affinity for ADP (K d = 16 μM) than for ATP (K d = 130 μM). ...
... It has previously been demonstrated that NifL requires nucleotide binding to the Cterminal GHKL domain in order to inhibit NifA activity. NifL is incompetent to bind NifA in vitro in the absence of nucleotide and the binding of ADP to the GHKL domain has been shown to stabilise the NifL-NifA binary complex (Eydmann et al., 1995;Money et al., 1999). Mutations in the GHKL domain that prevent binding of adenosine nucleotides result in the inability of NifL to inhibit NifA in vivo (Martinez-Argudo et al., 2004c;Perry et al., 2005). ...
This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise its copyright rests with the author and that no quotation from the thesis, nor any information derived therefrom, may be published without the author's prior written consent.
... It has previously been shown that NifL requires nucleotide binding to the C-terminal GHKL domain in order to inhibit NifA activity. NifL is incompetent to bind NifA in vitro in the absence of nucleotide and the binding of ADP to the GHKL domain has been shown to stabilize the NifL-NifA binary complex (Eydmann et al., 1995;Money et al., 1999). Mutations in the GHKL domain that prevent binding of adenosine nucleotides result in the inability of NifL to inhibit NifA in vivo (Martinez-Argudo et al., 2004b;Perry et al., 2005). ...
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.
... In certain nitrogen-fixing organisms of the gamma subgroup of the Proteobacteria, for example, expression of the nitrogen fixation or nif genes is under the control of the NifA activator, whose activity is, however, modulated by NifL in response to both oxygen and fixed nitrogen (31). Apparently, NifL negatively controls NifA activity via a stable protein-protein interaction (24,37) that is modulated by redox changes (which NifL can sense), ligand binding, and interactions with other proteins (31). Similarly, redox and blue light control of expression of photosynthesis genes in Rhodobacter sphaeroides is mediated by a repressor, PpsR, whose activity is modulated by the AppA antirepressor that responds to redox and blue light and forms a complex with PpsR (33). ...
nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [K(D)], 158.0 +/- 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative "noninteracting" mutant MexR protein, MexR(I104F), was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; K(D), 853.2 +/- 151.1 nM). Consistent with this, MexR(I104F) repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal alpha-helix of the protein that alone is sufficient for interaction with MexR.
... As in the case of bona fide HPKs, this domain binds adenosine nucleotides, although it exhibits neither ATPase nor transphosphorylation activities. The binding of nucleotide to the GHKL domain potentiates the inhibitory functions of NifL and its interaction with NifA (13)(14)(15). The GHKL domain is also the target for interaction with the signal transduction protein GlnK, which in its noncovalently modified form interacts with the GHKL domain to convey the fixed nitrogen status (16 -18). ...
... We have shown previously that the presence of adenosine nucleotides increases the stability of the NifL⅐NifA binary complex (14,15). Accordingly, co-retention of NifA with wild-type NifL was stimulated by the inclusion of 50 M ADP in the buffers (data not shown). ...
The NifL protein from Azotobacter vinelandii senses both the redox and fixed nitrogen status to regulate nitrogen fixation by controlling the activity of the transcriptional activator NifA. NifL has a domain architecture similar to that of the cytoplasmic histidine protein kinases. It contains two N-terminal PAS domains and a C-terminal transmitter region containing a conserved histidine residue (H domain) and a nucleotide binding GHKL domain corresponding to the catalytic core of the histidine kinases. Despite these similarities, NifL does not exhibit kinase activity and regulates its partner NifA by direct protein-protein interactions rather than phosphorylation. NifL senses the redox status via a FAD co-factor located within the PAS1 domain and responds to the nitrogen status by interaction with the signal transduction protein GlnK, which binds to the GHKL domain. The ability of NifL to inhibit NifA is antagonized by the binding of 2-oxoglutarate to the N-terminal GAF domain of NifA. In this study we have performed site-directed mutagenesis of the H domain of NifL to examine its role in signal transmission. Our results suggest that this domain plays a major role in transmission of signals perceived by the PAS1 and GHKL domains to ensure that NifL achieves the required conformation necessary to inhibit the 2-oxoglutarate-bound form of NifA. Some of the substitutions discriminate the redox and fixed nitrogen sensing functions of NifL implying that the conformational requirements and/or domain interactions necessary for NifA inhibition differ with respect to the signal input.
... NifL contains the conserved DHp and catalytic domains of histidine kinases and binds adenine nucleotides (40), but amino acid substitutions at the conserved phosphorylation site, H304, do not disable NifL (49). Rather than phosphorylating its partner protein, the transcriptional regulator NifA, NifL blocks the transcription of genes for nitrogen fixation under the appropriate conditions by sequestering NifA in a complex (28). In the second example, the hybrid histidine kinase RpfC of Xanthomonas campestris performs two distinct functions, namely, regulating virulence factor production via phosphoryl transfer to the cognate response regulator RpfG and regulating synthesis of the cell-cell communication signal DSF by a protein-protein interaction between the RpfC receiver domain and the enzyme RpfF (12). ...
The Caulobacter cell cycle is regulated by a network of two-component signal transduction proteins. Phosphorylation and stability of the
master transcriptional regulator CtrA are controlled by the CckA-ChpT phosphorelay, and CckA activity is modulated by another
response regulator, DivK. In a screen to identify suppressors of the cold-sensitive divK341 mutant, we found point mutations in the essential gene divL. DivL is similar to histidine kinases but has a tyrosine instead of a histidine at the conserved phosphorylation site (Y550).
Surprisingly, we found that the ATPase domain of DivL is not essential for Caulobacter viability. We show that DivL selectively affects CtrA phosphorylation but not CtrA proteolysis, indicating that DivL acts
in a pathway independent of the CckA-ChpT phosphorelay. divL can be deleted in a strain overproducing the phosphomimetic protein CtrAD51E, but unlike ΔctrA cells expressing CtrAD51E, this strain is profoundly impaired in the control of chromosome replication and cell division.
Thus, DivL performs a second function in addition to promoting CtrA phosphorylation. DivL is required for bipolar DivK localization
and positively regulates DivK phosphorylation. Our results show that DivL controls two key cell cycle regulators, CtrA and
DivK, and that phosphoryl transfer is not DivL's essential cellular activity.
... However, in addition to these regulatory constraints, interactions between NifL and NifA are further controlled by interactions with two other ligands, namely adenosine nucleotides and 2-oxoglutarate. Binding of adenosine nucleotides to NifL increases its affinity for NifA [7][8][9][10], whereas the binding of 2-oxoglutarate to NifA antagonizes this effect, enabling release of NifL from NifA under conditions appropriate for nitrogen fixation [11,12]. ...
... Although this C-terminal GHKL domain of NifL contains residues corresponding to the N, G1, F and G2 motifs that are highly conserved in this superfamily, including the histidine protein kinases, NifL does not exhibit either ATPase or transphosphorylation activity. However, the binding of ADP to this domain increases the affinity of NifL for NifA and stabilizes the NifL-NifA complex [7,8,10]. The GHKL domain is also involved in sensing the nitrogen status since it is the site of interaction for the GlnK signal transduction protein [6]. ...
The NifL regulatory protein is an anti-activator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA through the formation of a protein-protein complex. NifL modulates the activity of NifA in response to the redox, carbon and nitrogen status to ensure that nitrogen fixation occurs only under physiological conditions that are appropriate for nitrogenase activity. The domain architecture of NifL is similar to that of some histidine protein kinases, with two N-terminal PAS (PER, ARNT, SIM) domains, one of which contains an FAD cofactor that senses the redox status, and a C-terminal domain containing conserved residues that constitutes the nucleotide-binding domain of the GHKL (gyrase, Hsp90, histidine kinase, MutL) superfamily of ATPases. We have evidence that the central region of NifL, which is located between the PAS domains and the C-terminal GHKL nucleotide-binding domain, plays a crucial role in the propagation of signals perceived in response to the redox and fixed nitrogen status and that this region participates in conformational changes that switch NifL between active and inactive states. We have identified a critical arginine residue in the central region of NifL that participates in the conformational switch that activates NifL. Mutations in the central region of NifL that disable the redox-sensing function of NifL but leave the protein competent to respond to the nitrogen signal conveyed by the signal transduction protein GlnK have also been isolated. Our results suggest that the topological relationship between the central region and the GHKL domain may alter as a consequence of conformational changes induced in response to signal perception.
