Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic Bacterium Rhodobacter capsulatus (original) (raw)
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Journal of Bacteriology, 2008
Updated information and services can be found at: These include: REFERENCES http://jb.asm.org/content/190/5/1588#ref-list-1 at: This article cites 41 articles, 17 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on December 30, 2013 by guest http://jb.asm.org/ Downloaded from on December 30, 2013 by guest
Microbiology-sgm, 2003
In most bacteria, nitrogen metabolism is tightly regulated and P II proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK ) encoding P II -like proteins. The glnB gene forms part of a glnB-glnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both P II -like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnB-glnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnB-glnK double mutant. Furthermore, the P II -like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnB-glnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.
Microbiology-sgm, 2008
The activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL-NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein-protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations.
Journal of Biological Chemistry, 2006
P II proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the P IIhomologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other P II proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other P II -like proteins, Mg 2؉ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK-or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mM ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions.
Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense
Microbiological Research, 2015
a b s t r a c t PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ.
Journal of Bacteriology, 2007
Updated information and services can be found at: These include: REFERENCES http://jb.asm.org/content/189/16/5850#ref-list-1 at: This article cites 63 articles, 28 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on December 30, 2013 by guest http://jb.asm.org/ Downloaded from on December 30, 2013 by guest
MicrobiologyOpen, 2013
Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein-protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor r N (r 54 ) are not conserved in archaea suggesting a novel mechanism of transcriptional control.
Molecular Microbiology, 2004
P II -type signal transduction proteins play a central role in nitrogen regulation in many bacteria. In response to the intracellular nitrogen status, these proteins are rendered in their function and interaction with other proteins by modification/demodification events, e.g. by phosphorylation or uridylylation. In this study, we show that GlnK, the only P II -type protein in Corynebacterium glutamicum, is adenylylated in response to nitrogen starvation and deadenylylated when the nitrogen supply improves again. Both processes depend on the GlnD protein. As shown by mutant analyses, the modifying activity of this enzyme is located in the N-terminal part of the enzyme, while demodification depends on its C-terminal domain. Besides its modification status, the GlnK protein changes its intracellular localization in response to changes of the cellular nitrogen supply. While it is present in the cytoplasm during nitrogen starvation, the GlnK protein is sequestered to the cytoplasmic membrane in response to an ammonium pulse following a nitrogen starvation period. About 2-5% of the GlnK pool is located at the cytoplasmic membrane after ammonium addition. GlnK binding to the cytoplasmic membrane depends on the ammonium transporter AmtB, which is encoded in the same transcriptional unit as GlnK and GlnD, the amtB-glnK-glnD operon. In contrast, the structurally related methylammonium/ammonium permease AmtA does not bind GlnK. The membrane-bound GlnK protein is stable, most likely to inactivate AmtB-dependent ammonium transport in order to prevent a detrimental futile cycle under post-starvation ammonium-rich conditions, while the majority of GlnK is degraded within 2-4 min. Proteolysis in the transition period from nitrogen starvation to nitrogen-rich growth seems to be specific for GlnK; other proteins of the nitrogen metabolism, such as glutamine synthetase, or proteins unrelated to ammonium assimilation, such as enolase and ATP synthase subunit F 1 b b b b, are stable under these conditions. Our analyses of different mutant strains have shown that at least three different proteases influence the degradation of GlnK, namely FtsH, the ClpCP and the ClpXP protease complex.
Microbiology, 2012
The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energyexpensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme's activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the P II signal transduction proteins act as key players. Alphaproteobacteria Deltaproteobacteria Azospirillum brasilense Anaeromyxobacter sp. Fw109-5 Azospirillum lipoferum Anaeromyxobacter sp. K Azospirillum sp. B510 Desulfobacterium autotrophicum HRM2 Magnetospirillum magneticum AMB1 Desulfuromonas acetoxidans DSM 684 Magnetospirillum magnetotacticum MS-1 Geobacter bemidjiensis Bem Magnetospirillum gryphiswaldense MSR-1 Geobacter lovleyi SZ Rhodobacter capsulatus SB 1003 Geobacter metallireducens GS-15 Rhodobacter sphaeroides ATCC 17025 Geobacter sp. M21 Rhodopseudomonas palustris BisB5 Geobacter sp. FRC-32 Rhodopseudomonas palustris BisB18 Geobacter uraniireducens Rf4 Rhodopseudomonas palustris HaA2 Geobacter sulfurreducens PCA Rhodopseudomonas palustris CGA009 Pelobacter carbinolicus DSM 2380 Rhodopseudomonas palustris TIE-1 Pleobacter propionicus DSM 2379 Rhodopseudomonas palustris BisA53 Betaproteobacteria Rhodospirillum rubrum ATCC 11170 Accumulibacter phosphatis UW1 Gammaproteobacteria Azoarcus sp. BH72 Acidithiobacillus ferroxidans ATCC 23270 Dechloromonas aromatica RCB Acidithiobacillus ferroxidans ATCC 53993 Sideroxydans lithotrophicus ES-1 Allochromatium vinosum DSM 180 Rubrivivax benzoatilyticus JA2 Methylobacter tundripaludum SV96 Unclassified Proteobacteria Methylococcus capsulatus str. Bath Magnetococcus sp. MC-1 Methylomonas methanica MC09 Verrucomicrobia Teredinibacter turnerae T7901 Coraliomargarita akajimensis DSM 45221 Tolumonas auensis DSM 9187 Verrucomicrobiae bacterium DG1235 Deferribacteres Chrysiogenetes Calditerrivibrio nitroreducens DSM 19672 Desulfurispirillum indicum S5 Denitrovibrio acetiphilus DSM 12809