Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2203-2212 ◽  
Author(s):  
Thomas Drepper ◽  
Silke Groß ◽  
Alexander F. Yakunin ◽  
Patrick C. Hallenbeck ◽  
Bernd Masepohl ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Translational Control ◽  
Double Mutant ◽  
Rhodobacter Capsulatus ◽  
Mutational Analysis ◽  
Nitrogenase Activity ◽  
Constitutive Expression ◽  
Ammonium Transporter ◽  
Adp Ribosylation ◽  
Ammonium Regulation

In most bacteria, nitrogen metabolism is tightly regulated and PII proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK) encoding PII-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 PII-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 PII-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.

scholarly journals Ammonia-Induced Formation of an AmtB-GlnK Complex Is Not Sufficient for Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

2007 ◽  
Vol 190 (5) ◽  
pp. 1588-1594 ◽  
Author(s):  
Pier-Luc Tremblay ◽  
Patrick C. Hallenbeck
Keyword(s):  
Rhodobacter Capsulatus ◽  
Mutational Analysis ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
The Other ◽  
Amino Acid Residues ◽  
Adp Ribosylation ◽  
Nitrogenase Regulation ◽  
Ammonia Transport ◽  
First Time

ABSTRACT A series of Rhodobacter capsulatus AmtB variants were created and assessed for effects on ammonia transport, formation of AmtB-GlnK complexes, and regulation of nitrogenase activity and NifH ADP-ribosylation. Confirming previous reports, H193 and H342 were essential for ammonia transport and the replacement of aspartate 185 with glutamate reduced ammonia transport. Several amino acid residues, F131, D334, and D335, predicted to be critical for AmtB activity, are shown here for the first time by mutational analysis to be essential for transport. Alterations of the C-terminal tail reduced methylamine transport, prevented AmtB-GlnK complex formation, and abolished nitrogenase switch-off and NifH ADP-ribosylation. On the other hand, D185E, with a reduced level of transport, was capable of forming an ammonium-induced complex with GlnK and regulating nitrogenase. This reinforces the notions that ammonia transport is not sufficient for nitrogenase regulation and that formation of an AmtB-GlnK complex is necessary for these processes. However, some transport-incompetent AmtB variants, i.e., F131A, H193A, and H342A, form ammonium-induced complexes with GlnK but fail to properly regulate nitrogenase. These results show that formation of an AmtB-GlnK complex is insufficient in itself for nitrogenase regulation and suggest that partial ammonia transport or occupation of the pore by ammonia is essential for this function.

scholarly journals Distinct Roles of PII-Like Signal Transmitter Proteins and amtB in Regulation of nif Gene Expression, Nitrogenase Activity, and Posttranslational Modification of NifH in Azoarcus sp. Strain BH72

2002 ◽  
Vol 184 (8) ◽  
pp. 2251-2259 ◽  
Author(s):  
Dietmar E. Martin ◽  
Barbara Reinhold-Hurek
Keyword(s):  
Gene Transcription ◽  
Protein Interactions ◽  
Double Mutant ◽  
Nitrogenase Activity ◽  
Covalent Modification ◽  
Ammonium Transporter ◽  
Protein Protein Interactions ◽  
Oxygen Control ◽  
Functional Inactivation ◽  
Signal Transduction Proteins

ABSTRACT PII-like signal transmitter proteins, found in Bacteria, Archaea, and plants, are known to mediate control of carbon and nitrogen assimilation. They indirectly regulate the activity of key metabolic enzymes and transcription factors by protein-protein interactions with signal transduction proteins. Many Proteobacteria harbor two paralogous PII-like proteins, GlnB and GlnK, whereas a novel third PII paralogue (GlnY) was recently identified in Azoarcus sp. strain BH72, a diazotrophic endophyte of grasses. In the present study, evidence was obtained that the PII-like proteins have distinct roles in mediating nitrogen and oxygen control of nif gene transcription and nitrogenase activity. Full repression of nif gene transcription in the presence of a combined nitrogen source or high oxygen concentrations was observed in wild-type and glnB and glnK knockout mutants, revealing that GlnB and GlnK can complement each other in mediating the repression. In contrast, in a glnBK double mutant strain in the presence of only GlnY, nif gene transcription was still detectable, albeit at a lower level, on nitrate or 20% oxygen. As another level of control, nitrogenase activity was regulated by at least three types of mechanisms in strain BH72: covalent modification of dinitrogenase reductase (NifH), probably by ADP-ribosylation, and two other, unknown means. Functional inactivation upon ammonium addition (switch-off) required the putative high-affinity ammonium transporter AmtB and GlnK, but not GlnB or GlnY. Functional inactivation in response to anaerobiosis did not depend on AmtB, GlnK, or GlnB. In contrast, covalent modification of NifH required both GlnB and GlnK and AmtB as response to ammonium addition, whereas it required either GlnB or GlnK and not AmtB when cells were shifted to anaerobiosis. In a glnBK double mutant expressing only GlnY, NifH modification was completely abolished, further revealing functional differences between the three PII paralogues.

scholarly journals Nitrogenase Switch-Off by Ammonium Ions in Azospirillum brasilense Requires the GlnB Nitrogen Signal-Transducing Protein

2005 ◽  
Vol 71 (9) ◽  
pp. 5637-5641 ◽  
Author(s):  
Giseli Klassen ◽  
Emanuel M. Souza ◽  
M. Geoffrey Yates ◽  
Liu Un Rigo ◽  
Roberta M. Costa ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Azospirillum Brasilense ◽  
Double Mutant ◽  
Nitrogenase Activity ◽  
Ammonium Ions ◽  
Anaerobic Conditions ◽  
Adp Ribosylation ◽  
Fe Protein

ABSTRACT Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.

scholarly journals The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in Rhodobacter capsulatus Is Correlated with Cellular Nitrogen Status

1999 ◽  
Vol 181 (7) ◽  
pp. 1994-2000 ◽  
Author(s):  
Alexander F. Yakunin ◽  
Tatyana V. Laurinavichene ◽  
Anatoly A. Tsygankov ◽  
Patrick C. Hallenbeck
Keyword(s):  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
Growth Conditions ◽  
Covalent Modification ◽  
Nitrogen Status ◽  
Modification System ◽  
Adp Ribosylation ◽  
Fe Protein ◽  
Batch Growth

ABSTRACT The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4 +. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4 +, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4 +, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.

scholarly journals Urea Utilization in the Phototrophic BacteriumRhodobacter capsulatus Is Regulated by the Transcriptional Activator NtrC

2001 ◽  
Vol 183 (2) ◽  
pp. 637-643 ◽  
Author(s):  
Bernd Masepohl ◽  
Björn Kaiser ◽  
Nazila Isakovic ◽  
Cynthia L. Richard ◽  
Robert G. Kranz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nitrogen Source ◽  
Urease Activity ◽  
Rhodobacter Capsulatus ◽  
Mutational Analysis ◽  
Nitrogenase Activity ◽  
Sole Source ◽  
Purple Bacterium ◽  
Dependent Manner ◽  
Induced Mutations

ABSTRACT The phototrophic nonsulfur purple bacteriumRhodobacter capsulatus can use urea as a sole source of nitrogen. Three transposon Tn5-induced mutations (Xan-9, Xan-10, and Xan-19), which led to a Ure−phenotype, were mapped to the ureF and ureCgenes, whereas two other Tn5 insertions (Xan-20 and Xan-22) were located within the ntrC and ntrB genes, respectively. As in Klebsiella aerogenes and other bacteria, the genes encoding urease (ureABC) and the genes required for assembly of the nickel metallocenter (ureD andureEFG) are clustered in R. capsulatus(ureDABC-orf136-ureEFG). No homologues of Orf136 were found in the databases, and mutational analysis demonstrated that orf136 is not essential for urease activity or growth on urea. Analysis of aureDA-lacZ fusion showed that maximum expression of the ure genes occurred under nitrogen-limiting conditions (e.g., serine or urea as the sole nitrogen source), but ure gene expression was not substrate (urea) inducible. Expression of the ure genes was strictly dependent on NtrC, whereas ς54 was not essential for urease activity. Expression of the ure genes was lower (by a factor of 3.5) in the presence of ammonium than under nitrogen-limiting conditions, but significant transcription was also observed in the presence of ammonium, approximately 10-fold higher than in an ntrC mutant background. Thus, ure gene expression in the presence of ammonium also requires NtrC. Footprint analyses demonstrated binding of NtrC to tandem binding sites upstream of the ureD promoter. Phosphorylation of NtrC increased DNA binding by at least eightfold. Although urea is effectively used as a nitrogen source in an NtrC-dependent manner, nitrogenase activity was not repressed by urea.

scholarly journals AmtB Is Necessary for NH4+-Induced Nitrogenase Switch-Off and ADP-Ribosylation in Rhodobacter capsulatus

2002 ◽  
Vol 184 (15) ◽  
pp. 4081-4088 ◽  
Author(s):  
Alexander F. Yakunin ◽  
Patrick C. Hallenbeck
Keyword(s):  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Growth Defect ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Adp Ribosylation ◽  
Fe Protein ◽  
Knockout Mutants

ABSTRACT Rhodobacter capsulatus possesses two genes potentially coding for ammonia transporters, amtB and amtY. In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants. Strains mutated in either amtB or amtY did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an amtB mutant was no longer capable of transporting methylamine. The amtB strain but not the amtY strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH4 + addition. ADP-ribosylation in response to darkness was unaffected in amtB and amtBY strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity.

Regulation of nitrogenase in the photosynthetic bacteriumRhodobacter sphaeroidescontainingdraTGandnifHDKgenes fromRhodobacter capsulatus

2001 ◽  
Vol 47 (3) ◽  
pp. 206-212 ◽  
Author(s):  
Alexander F Yakunin ◽  
Alexander S Fedorov ◽  
Tatyana V Laurinavichene ◽  
Vadim M Glaser ◽  
Nikolay S Egorov ◽  
...  
Keyword(s):  
Photosynthetic Bacteria ◽  
Rhodospirillum Rubrum ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
Adp Ribosylation ◽  
Fe Protein ◽  
Dinitrogenase Reductase ◽  
Different Strains

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.Key words: nitrogenase regulation, nitrogenase modification, photosynthetic bacteria.

scholarly journals In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

2013 ◽  
Vol 57 (9) ◽  
pp. 4470-4480 ◽  
Author(s):  
Min Jung Kwun ◽  
Gabriela Novotna ◽  
Andrew R. Hesketh ◽  
Lionel Hill ◽  
Hee-Jeon Hong
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Streptomyces Coelicolor ◽  
Constitutive Expression ◽  
Transcriptional Response ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Expression Of Genes ◽  
Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

scholarly journals Mutational Analysis of Regulatory cis-acting Elements for the Transcriptional Activation of the dmsCBA Operon in Rhodobactersphaeroides f. sp. denitrificans

2001 ◽  
Vol 42 (7) ◽  
pp. 703-709 ◽  
Author(s):  
Isamu Yamamoto ◽  
Takeshi Ujiiye ◽  
Yoshinori Ohshima ◽  
Toshio Satoh
Keyword(s):  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Cis Acting

Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication

1996 ◽  
Vol 271 (6) ◽  
pp. L963-L971 ◽  
Author(s):  
M. A. Fiedler ◽  
K. Wernke-Dollries ◽  
J. M. Stark
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Viral Replication ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
A549 Cells ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Syncytial Virus ◽  
Time Point

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

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