Biomedical subjects

Karl Forchhammer

Publications and source records attributed to Karl Forchhammer.

At least 19 recordsLinked to original sources

PubMed2007

Glutamine signalling in bacteria.

Glutamine is a metabolite of central importance in bacterial physiology. In addition to its function as one of the 20 standard amino acids in protein synthesis, glutamine is required for the biosynthesis of a variety of nitrogen-containing compounds. Of particular importance is glutamine synthesis as primary reaction of ammonium assimilation. Because of this versatile role, glutamine metabolism is tightly controlled in response to the cellular nitrogen status in bacteria. Recent progress in elucidating the molecular basis of nitrogen signalling has shed light on the role of glutamine as a signalling molecule. Bacteria belonging to the phylogenetic domains of proteobacteria and low G+C gram-positives (firmicutes) have evolved different mechanisms to monitor glutamine as an indicator of the state of nitrogen metabolism, which then regulates nitrogen metabolism at the transcriptional and post-transcriptional levels. Using the conserved PII signal transduction system, major groups of prokaryotes, including the cyanobacteria, have evolved yet another strategy to monitor the cellular nitrogen status, which relies on 2-oxoglutarate instead of glutamine as the signalling molecule. In addition to monitoring the intracellular glutamine level, bacteria may respond to extracellular glutamine, which is used as a nutrient. This overview details our current knowledge of glutamine-regulated processes in bacteria.

Bacteria↗

PubMed2006

Interaction of the membrane-bound GlnK-AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis.

PII 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 PII-homologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other PII 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 PII-like proteins, Mg2+ 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.

Bacillus subtilis↗

PubMed2006

Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2-oxoglutarate dependent manner with PII and NtcA.

Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. The global nitrogen transcription factor NtcA and the signal transduction protein PII are both involved in 2-oxoglutarate sensing. PII proteins, probably the most conserved signal transduction proteins in nature, are remarkable for their ability to interact with very diverse protein targets in different systems. Despite widespread efforts to understand nitrogen signalling in cyanobacteria, the involvement of PII in the regulation of transcription activation by NtcA remains enigmatic. Here we show that PipX, a protein only present in cyanobacteria, interacts with both PII and NtcA and provides a mechanistic link between these two factors. A variety of in vivo and in vitro approaches were used to study PipX and its interactions with PII and NtcA. 2-Oxoglutarate favours complex formation between PipX and NtcA, but impairs binding to PII, suggesting that partner swapping between these nitrogen regulators is driven by the 2-oxoglutarate concentration. PipX is required for NtcA-dependent transcriptional activation in vivo, thus implying that PipX may function as a prokaryotic transcriptional coactivator.

Adenosine Triphosphate↗

PubMed2006

Regulatory links between carbon and nitrogen metabolism.

The metabolism of carbon- and nitrogen-containing compounds is fundamental to all forms of life. To cope with changing environmental conditions, bacteria have to sense the nutrient supply and adapt their metabolism accordingly. In addition to nutrient- and pathway-specific responses, they integrate information from the different branches of metabolism to coordinate the control of the expression of many metabolic genes. Two major players interconnecting carbon and nitrogen regulation are the PII proteins and the phosphotransferase system. Moreover, several DNA-binding transcription regulators sense signals are derived from both carbon and nitrogen metabolism. The regulatory networks enable the bacteria to make the appropriate metabolic responses to changing nutrient availabilities in the environment.

Antigens, Bacterial↗

PubMed2006

Alanine dehydrogenase activity is required for adequate progression of phycobilisome degradation during nitrogen starvation in Synechococcus elongatus PCC 7942.

Degradation of the cyanobacterial light-harvesting antenna, the phycobilisome, is a general acclimation response that is observed under various stress conditions. In this study we identified a novel mutant of Synechococcus elongatus PCC 7942 that exhibits impaired phycobilisome degradation specifically during nitrogen starvation, unlike previously described mutants, which exhibit aberrant degradation under nitrogen, sulfur, and phosphorus starvation conditions. The phenotype of the new mutant, AldOmega, results from inactivation of ald (encoding alanine dehydrogenase). AldOmega is deficient in transcription induction of a number of genes during nitrogen starvation. These genes include the "general nutrient stress-related" genes, nblA and nblC, the products of which are essential for phycobilisome degradation. Furthermore, transcripts of several specific nitrogen-responsive genes accumulate at lower levels in AldOmega than in the wild-type strain. In contrast, ald inactivation did not decrease the accumulation of transcripts during sulfur starvation. Transcription of ald is induced upon nitrogen starvation, which is consistent with the ability of wild-type cells to maintain a low cellular content of alanine under these conditions. Unlike wild-type cells, AldOmega accumulates alanine upon nitrogen starvation. Our analyses suggest that alanine dehydrogenase activity is necessary for an adequate cellular response to nitrogen starvation. Decomposition of alanine may be required to provide a sufficient amount of ammonia. Furthermore, the accumulated alanine, or a related metabolite, may interfere with the cues that modulate acclimation during nitrogen starvation. Taken together, our results provide novel information regarding cellular responses to nitrogen starvation and suggest that mechanisms related to nitrogen-specific responses are involved in modulation of a general acclimation process.

Alanine Dehydrogenase↗

PubMed2006

PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp. strain PCC 6803.

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein PII.

Arginine↗

PubMed2005

Analysis of a non-canonical NtcA-dependent promoter in Synechococcus elongatus and its regulation by NtcA and PII.

In this communication, we present a genetic analysis of the glnN promoter region of Synechococcus elongatus PCC 7942. luxAB reporter fusions were used to characterize the glnN promoter by deletion and site-directed mutational analysis. Reporter gene expression analysis was performed in S. elongatus wild-type and mutant strains to reveal the role of the global nitrogen responsive transcription factor NtcA and of the P(II) signalling protein on regulation of glnN gene expression. A non-canonical NtcA-binding motif is responsible for NtcA-dependent, nitrogen-responsive regulation of glnN. The P(II) signalling protein has opposing effects on NtcA-dependent glnN expression. Under conditions of nitrate-growth, it depresses expression, whereas under conditions of combined nitrogen starvation, it is required for full induction. Furthermore, sequences upstream of the NtcA-binding site have repressive effect on glnN promoter activity.

Bacterial Proteins↗

PubMed2005

Protein phosphatase PphA from Synechocystis sp. PCC 6803: the physiological framework of PII-P dephosphorylation.

The phosphorylated signal transduction protein P(II) (P(II)-P) in the cyanobacterium Synechocystis sp. strain PCC 6803 is dephosphorylated by PphA, a protein phosphatase of the 2C family (PP2C). In this study, the physiological conditions of P(II)-P dephosphorylation were investigated with respect to the in vivo specificity of P(II)-P towards PphA and the cellular abundance of PphA in cells growing under different nitrogen regimes. Furthermore, the consequences of impaired P(II)-P dephosphorylation with respect to short-term inhibition of glutamine synthetase (GS) were studied. With a contribution of approximately 15 % of total Mn(2+)-dependent p-nitrophenyl phosphate hydrolysis activity, PphA has only a minor impact on the total PP2C activity in Synechocystis extracts. Nevertheless, residual P(II)-P dephosphorylation in PphA-deficient cells could only be observed after prolonged incubation in the presence of ammonium. The abundance of PphA correlates with the phosphorylation state of P(II) under nitrogen-replete conditions and is specifically enhanced by nitrite. Regulation of pphA expression operates at the post-transcriptional level. In the presence of nitrate/nitrite, PphA is present in molar excess over P(II)-P, enabling the cells to rapidly dephosphorylate P(II)-P in response to changing environmental conditions. A PphA-deficient mutant is not impaired in short-term inhibition of GS activity following ammonium treatment. Down-regulation of GS occurs by induction of gif genes (encoding GS inactivating factors 7 and 17), which is controlled by NtcA-mediated gene repression. Thus, impaired P(II)-P dephosphorylation does not affect ammonium-prompted inactivation of NtcA.

Bacterial Proteins↗

PubMed2005

Acclimation of unicellular cyanobacteria to macronutrient deficiency: emergence of a complex network of cellular responses.

Cyanobacteria are equipped with numerous mechanisms that allow them to survive under conditions of nutrient starvation, some of which are unique to these organisms. This review surveys the molecular mechanisms underlying acclimation responses to nitrogen and phosphorus deprivation, with an emphasis on non-diazotrophic freshwater cyanobacteria. As documented for other micro-organisms, nutrient limitation of cyanobacteria elicits both general and specific responses. The general responses occur under any starvation condition and are the result of the stresses imposed by arrested anabolism. In contrast, the specific responses are acclimation processes that occur as a result of limitation for a particular nutrient; they lead to modification of metabolic and physiological routes to compensate for the restriction. First, the general acclimation processes are discussed, with an emphasis on modifications of the photosynthetic apparatus. The molecular mechanisms underlying specific responses to phosphorus and nitrogen-limitation are then outlined, and finally the cross-talk between pathways modulating specific and general responses is described.

Acclimatization↗

PubMed2005

Unique mechanistic features of post-translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability.

PII-like signal transduction proteins are found in all three domains of life and have been shown to play key roles in the control of bacterial nitrogen assimilation. This communication reports the first target protein of an archaeal PII-like protein, representing a novel PII receptor. The GlnK(1) protein of the methanogenic archaeon Methanosarcina mazei strain Go1 interacts and forms stable complexes with glutamine synthetase (GlnA(1)). Complex formation with GlnK(1) in the absence of metabolites inhibits the activity of GlnA(1). On the other hand, the activity of this enzyme is directly stimulated by the effector molecule 2-oxoglutarate. Moreover, 2-oxoglutarate antagonized the inhibitory effects of GlnK(1) on GlnA(1) activity but did not prevent GlnK(1)/GlnA(1) complex formation. On the basis of these findings, we hypothesize that besides the dominant effector molecule 2-oxoglutarate, the nitrogen sensor GlnK(1) allows finetuning control of the glutamine synthetase activity under changing nitrogen availabilities and propose the following model. (i) Under nitrogen limitation, increasing concentrations of 2-oxoglutarate stimulate maximal GlnA(1) activity and transform GlnA(1) into an activated conformation, which prevents inhibition by GlnK(1). (ii) Upon a shift to nitrogen sufficiency after a period of nitrogen limitation, GlnA(1) activity is reduced by decreasing internal 2-oxoglutarate concentrations through diminished direct activation and by GlnK(1) inhibition.

Adaptation, Physiological↗

PubMed2005

Signal transduction protein PII phosphatase PphA is required for light-dependent control of nitrate utilization in synechocystis sp. strain PCC 6803.

Signal transduction protein P(II) is dephosphorylated in Synechocystis sp. strain PCC 6803 by protein phosphatase PphA. To determine the impact of PphA-mediated P(II) dephosphorylation on physiology, the phenotype of a PphA-deficient mutant was analyzed. Mutants lacking either PphA or P(II) were impaired in efficient utilization of nitrate as the nitrogen source. Under conditions of limiting photosystem I (PSI)-reduced ferredoxin, excess reduction of nitrate along with impaired reduction of nitrite occurred in P(II) signaling mutants, resulting in excretion of nitrite to the medium. This effect could be reversed by increasing the level of PSI-reduced ferredoxin. We present evidence that nonphosphorylated P(II) controls the utilization of nitrate in response to low light intensity by tuning down nitrate uptake to meet the actual reduction capacity. This control mechanism can be bypassed by exposing cells to excess levels of nitrate. Uncontrolled nitrate uptake leads to light-dependent nitrite excretion even in wild-type cells, confirming that nitrate uptake controls nitrate utilization in response to limiting photon flux densities.

Light↗

PubMed2004

Complex formation and catalytic activation by the PII signaling protein of N-acetyl-L-glutamate kinase from Synechococcus elongatus strain PCC 7942.

The signal transduction protein P(II) from the cyanobacterium Synechococcus elongatus strain PCC 7942 forms a complex with the key enzyme of arginine biosynthesis, N-acetyl-l-glutamate kinase (NAGK). Here we report the effect of complex formation on the catalytic properties of NAGK. Although pH and ion dependence are not affected, the catalytic efficiency of NAGK is strongly enhanced by binding of P(II), with K(m) decreasing by a factor of 10 and V(max) increasing 4-fold. In addition, arginine feedback inhibition of NAGK is strongly decreased in the presence of P(II), resulting in a tight control of NAGK activity under physiological conditions by P(II). Analysis of the NAGK-P(II) complex suggests that one P(II) trimer binds to one NAGK hexamer with a K(d) of approximately 3 nm. Complex formation is strongly affected by ATP and ADP. ADP is a strong inhibitor of complex formation, whereas ATP inhibits complex formation only in the absence of divalent cations or in the presence of Mg(2+) ions, together with increased 2-oxoglutarate concentrations. Ca(2+) is able to antagonize the negative effect of ATP and 2-oxoglutarate. ADP and ATP exert their adverse effect on NAGK-P(II) complex formation through binding to the P(II) protein.

Adenosine Diphosphate↗

PubMed2004

Cell-type specific modification of PII is involved in the regulation of nitrogen metabolism in the cyanobacterium Anabaena PCC 7120.

In the heterocystous cyanobacterium Anabaena PCC 7120, the modification state of the signalling PII protein is regulated according to the nitrogen regime of the cells, as already observed in some unicellular cyanobacteria. However, during the adaptation to diazotrophic growth conditions, PII is phosphorylated in vegetative cells while unphosphorylated in heterocysts. Isolation of mutants affected on PII modification state and analysis of their phenotypes allow us to show the implication of PII in the regulation of molecular nitrogen assimilation and more specifically, the requirement of unmodified state of PII in the formation of polar nodules of cyanophycin in heterocysts.

Anabaena↗

PubMed2004

Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets.

PII signal transduction plays a pervasive role in microbial nitrogen control. Different phylogenetic lineages have developed various signal transduction schemes around the highly conserved core of the signalling system, which consists of the PII proteins. Among all various bacterial PII signalling systems, the one in cyanobacteria is so far unique: in unicellular strains, the mode of covalent modification is by serine phosphorylation and the interpretation of the cellular nitrogen status occurs by measuring the 2-oxoglutarate levels. Recent advances have been the identification of the phospho-PII phosphatase, the resolution of the crystal structure of PII proteins from Synechococcus and Synechocystis strains and the identification of novel functions of PII regulation in cyanobacteria, which highlight the central role of PII signalling for the acclimation to changing carbon-nitrogen regimes.

Bacterial Proteins↗

PubMed2004

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge.

This communication presents a short outline of the current knowledge on the molecular basis of P(II) signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P(II) signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P(II) signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P(II) phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P(II) (P(II)-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P(II)-P dephosphorylation, novel insights into the signals, to which P(II)-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P(II)-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P(II)-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P(II)-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.

Journal Article↗

PubMed2004

The Synechococcus elongatus P signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase.

This communication identifies, for the first time, a receptor protein for signal perception from the P(II) signal transduction protein in the cyanobacterium Synechococcus elongatus. P(II), a phosphoprotein that signals the carbon/nitrogen status of the cells, forms a tight complex with the key enzyme of the arginine biosynthetic pathway, N-acetylglutamate (NAG) kinase. In complex with P(II), the catalytic activity of NAG kinase is strongly enhanced. Complex formation does not require the effector molecules of P(II), 2-oxoglutarate and ATP, but it is highly susceptible to modifications at the phosphorylation site of P(II), Ser-49. Stable complexes were only formed with the non-phosphorylated form of P(II) but not with Ser-49 mutants. In accordance with these data, NAG kinase activity in S. elongatus extracts correlated with the phosphorylation state of P(II), with high NAG kinase activities corresponding to non-phosphorylated P(II) (nitrogen-excess conditions) and low activities to increased levels of P(II) phosphorylation (nitrogen-poor conditions), thus subjecting the key enzyme of arginine biosynthesis to global nitrogen control.

Adenosine Triphosphate↗

PubMed2003

The structures of the PII proteins from the cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803.

The PII proteins from the cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 have been crystallized and high-resolution structures have been obtained using X-ray crystallography. The core of these new structures is similar to that of the PII proteins from Escherichia coli, although the structures of the T- and C-loops differ. The T-loop of the Synechococcus protein is ordered, but appears to be stabilized by crystal contacts. The same loop in the Synechocystis protein is disordered. The C-terminus of the Synechocystis protein is stabilized by hydrogen bonding to the same region of a crystallographically related molecule. The same terminus in the Synechococcus protein is stabilized by coordination with a metal ion. These observations are consistent with the idea that both the T-loop and the C-terminus of PII proteins are flexible in solution and that this flexibility may be important for receptor recognition. Sequence comparisons are used to identify regions of the sequence unique to the cyanobacteria.

Amino Acid Sequence↗

PubMed2003

Carbon-source-dependent nitrogen regulation in Escherichia coli is mediated through glutamine-dependent GlnB signalling.

The P(II) signal transduction proteins GlnB and GlnK are uridylylated/deuridylylated in response to the intracellular glutamine level, the primary signal of the cellular nitrogen status. Furthermore, GlnB was shown to be allosterically regulated by 2-oxoglutarate, and thus GlnB was suggested to integrate signals of the cellular carbon and nitrogen status. Receptors of GlnB signal transduction in Escherichia coli are the NtrB/NtrC two-component system and GlnE, an enzyme which adenylylates/deadenylylates glutamine synthetase. In this study, the authors investigated the effect of different carbon sources on the expression of the NtrC-dependent genes glnA and glnK and on the uridylylation status of GlnB and GlnK. With glutamine as nitrogen source, high levels of glnA and glnK expression were obtained when glucose was used as carbon source, but expression was strongly decreased when the cells were grown with poor carbon sources or when cAMP was present. This response correlated with the uridylylation status of GlnB, suggesting that the carbon/cAMP effect was mediated through GlnB uridylylation, a conclusion that was confirmed by mutants of the P(II) signalling pathway. When glutamine was replaced by low concentrations of ammonium as nitrogen source, neither glnAglnK expression nor GlnB uridylylation responded to the carbon source or to cAMP. Furthermore, glutamine synthetase could be rapidly adenylylated in vivo by the external addition of glutamine; however, this occurred only when cells were grown in the presence of cAMP, not in its absence. Together, these results suggest that poor carbon sources, through cAMP signalling, favour glutamine uptake. The cellular glutamine signal is then transduced by uridylyltransferase and GlnB to modulate NtrC-dependent gene expression.

Bacterial Proteins↗