Membrane association of Klebsiella pneumoniae NifL is affected by molecular oxygen and combined nitrogen

Klopprogge, Kai; Grabbe, Roman; Hoppert, Michael; Schmitz, Ruth A.

doi:10.1007/s00203-001-0379-x

Cited by 29 publications

(76 citation statements)

References 47 publications

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“…In a variety of diverse bacteria, AmtB interacts directly with unmodified GlnK to form a membrane-bound complex under NH z 4 -excess conditions Detsch & Stülke, 2003;Javelle et al, 2004;Klopprogge et al, 2002;Strösser et al, 2004). This interaction is reversible and is thought to block the NH 3 -transporting function of AmtB Javelle et al, 2004), and a docking model for the interaction of these two proteins from A. fulgidus has been proposed (Andrade et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

et al. 2006

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The AmtB protein transports uncharged NH 3 into the cell, but it also interacts with the nitrogen regulatory protein P II , which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P II homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to NH + 4 addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB 1 and amtB 2 , have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB 1 are defective in their response to both NH + 4 addition and darkness, while mutants lacking AmtB 2 show little effect on the regulation of nitrogenase activity. These responses to darkness and NH + 4 appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB 1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB 1 -P II regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P II is proposed. Other results demonstrate a degree of specificity in interaction of AmtB 1 with the different P II homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P II proteins regulate processes involved in nitrogen acquisition and utilization.

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Section: Introductionmentioning

confidence: 99%

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

et al. 2006

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“…Quantification of in vivo distribution of NifL or derivatives in membrane and cytoplasmic fractions of the K. pneumoniae ⌬nifLA strain carrying pRS315 or derivatives under nitrogen-fixing conditions was performed as described by Klopprogge et al (21).…”

Section: Methodsmentioning

confidence: 99%

“…NifL antagonizes cytoplasmic NifA in the presence of ammonium and/or molecular oxygen by direct protein-protein interaction (10,14,54). However, upon anaerobiosis and simultaneous absence of combined nitrogen, the flavoprotein NifL is sequestered to the cytoplasmic membrane (21,54). This membrane sequestration of the negative regulator NifL, allowing cytoplasmic NifA to induce nif gene expression by activating the alternative 54 RNA-polymerase (16,41), is the key mechanism for regulating nitrogen fixation in K. pneumoniae in response to the environmental signals (12,21).…”

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confidence: 99%

“…However, upon anaerobiosis and simultaneous absence of combined nitrogen, the flavoprotein NifL is sequestered to the cytoplasmic membrane (21,54). This membrane sequestration of the negative regulator NifL, allowing cytoplasmic NifA to induce nif gene expression by activating the alternative 54 RNA-polymerase (16,41), is the key mechanism for regulating nitrogen fixation in K. pneumoniae in response to the environmental signals (12,21). In previous studies we obtained evidence that membrane sequestration of NifL under nitrogen-fixing conditions is achieved by the reduction of the N-terminally bound FAD cofactor by electrons derived from the reduced menaquinone pool (11,12,55).…”

mentioning

confidence: 99%

“…Pulldown experiments revealed that K. pneumoniae GlnK effects stability of the inhibitory NifL/ NifA complexes in response to nitrogen (54). Under nitrogen sufficiency, GlnK simultaneously binds to NifL and NifA, whereas under nitrogen limitation -in the presence of higher internal 2-oxoglutarate concentrations-NifA is released, while NifL remains bound to GlnK, resulting in NifA/NifL complex dissociation followed by NifL sequestration to the cytoplasmic membrane in case oxygen is simultaneously limiting (9,21,54).…”

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Insights into Membrane Association of Klebsiella pneumoniae NifL under Nitrogen-Fixing Conditions from Mutational Analysis

et al. 2011

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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.

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Regulation of nif Gene Expression in Azotobacter vinelandii

Poza‐Carrión

Echávarri-Erasun

Rubio

2015

Biological Nitrogen Fixation

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Membrane association of Klebsiella pneumoniae NifL is affected by molecular oxygen and combined nitrogen

Cited by 29 publications

References 47 publications

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

Insights into Membrane Association of Klebsiella pneumoniae NifL under Nitrogen-Fixing Conditions from Mutational Analysis

Regulation of nif Gene Expression in Azotobacter vinelandii

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