scholarly journals Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

PLoS Genetics ◽  
2021 ◽  
Vol 17 (1) ◽  
pp. e1009092
Author(s):  
Larissa Krüger ◽  
Christina Herzberg ◽  
Hermann Rath ◽  
Tiago Pedreira ◽  
Till Ischebeck ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cellular Response ◽  
Second Messengers ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Complex Medium ◽  
Glutamate Toxicity ◽  
Potassium Homeostasis ◽  
Suppressor Mutants ◽  
Free Strain

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

scholarly journals Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

2020 ◽  
Author(s):  
Larissa Krüger ◽  
Christina Herzberg ◽  
Hermann Rath ◽  
Tiago Pedreira ◽  
Anja Poehlein ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cellular Response ◽  
Second Messengers ◽  
Glutamate Transporter ◽  
Second Messenger ◽  
Complex Medium ◽  
Potassium Homeostasis ◽  
Suppressor Mutants ◽  
External Signals ◽  
Free Strain

ABSTRACTIn order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) suggesting the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal mediu. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.AUTHOR SUMMARYBacteria are exposed to constantly changing environmental conditions. In order to respond to these changes, they use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Among the repertoire of bacterial second messenger nucleotides, cyclic di-AMP (c-di-AMP) stands out as it is the only second messenger that is essential for the bacteria that produce it, including the Gram-positive model organism Bacillus subtilis. C-di-AMP plays a major role in the control of potassium homeostasis, and we found that glutamate is toxic to a B. subtilis strain lacking c-di-AMP to the same extent as potassium. These toxic conditions were the starting point for an extensive suppressor analysis, which led to the identification of a novel glutamate transporter (AimA). If the B. subtilis strain lacking c-di-AMP was cultivated on complex medium, the isolation of suppressor mutants was only possible under anaerobic conditions and if either of the two c-di-AMP-binding signal transduction proteins was absent. This suggests that these proteins are a major burden for the cell on complex medium in their c-di-AMP free state. Our result underline the complexity of c-di-AMP signaling and propose new directions for research.

Secondary Messenger Systems in PTSD

2016 ◽  
Author(s):  
Ulrike Schmidt
Keyword(s):  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Brain Regions ◽  
Guanosine Monophosphate ◽  
Intracellular Camp ◽  
Post Traumatic Stress ◽  
Secondary Messenger

Second messengers such as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), inositoltriphosphate, and diacylglycerol (DAG) are a prerequisite for the signal transduction of extracellular receptors. The latter are central for cellular function and thus are implicated in the pathobiology of a variety of disorders, such as schizophrenia, bipolar disorder, major depression, and post-traumatic stress disorder (PTSD). This chapter focuses on the involvement of second messenger molecules and their regulators as direct targets in human and animal PTSD and aims to stimulate the underdeveloped research in this field. The synthesis of literature reveals that second messengers clearly play a central role in PTSD-associated brain regions and processes. In particular, pituitary adenylate cyclase-activating polypeptide (PACAP), an important regulator of intracellular cAMP levels, as well as protein kinase c, the major target of DAG, belong to the hitherto most promising PTSD candidate molecules directly involved in second messenger signaling.

scholarly journals An Essential Poison: Synthesis and Degradation of Cyclic Di-AMP in Bacillus subtilis

2015 ◽  
Vol 197 (20) ◽  
pp. 3265-3274 ◽  
Author(s):  
Jan Gundlach ◽  
Felix M. P. Mehne ◽  
Christina Herzberg ◽  
Jan Kampf ◽  
Oliver Valerius ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Nitrogen Source ◽  
Second Messengers ◽  
Regulatory Protein ◽  
Control Cell ◽  
Model Organism ◽  
Second Messenger ◽  
Content Type ◽  
Gram Positive Bacteria

ABSTRACTGram-positive bacteria synthesize the second messenger cyclic di-AMP (c-di-AMP) to control cell wall and potassium homeostasis and to secure the integrity of their DNA. In the firmicutes, c-di-AMP is essential for growth. The model organismBacillus subtilisencodes three diadenylate cyclases and two potential phosphodiesterases to produce and degrade c-di-AMP, respectively. Among the three cyclases, CdaA is conserved in nearly all firmicutes, and this enzyme seems to be responsible for the c-di-AMP that is required for cell wall homeostasis. Here, we demonstrate that CdaA localizes to the membrane and forms a complex with the regulatory protein CdaR and the glucosamine-6-phosphate mutase GlmM. Interestingly,cdaA,cdaR, andglmMform a gene cluster that is conserved throughout the firmicutes. This conserved arrangement and the observed interaction between the three proteins suggest a functional relationship. Our data suggest that GlmM and GlmS are involved in the control of c-di-AMP synthesis. These enzymes convert glutamine and fructose-6-phosphate to glutamate and glucosamine-1-phosphate. c-di-AMP synthesis is enhanced if the cells are grown in the presence of glutamate compared to that in glutamine-grown cells. Thus, the quality of the nitrogen source is an important signal for c-di-AMP production. In the analysis of c-di-AMP-degrading phosphodiesterases, we observed that both phosphodiesterases, GdpP and PgpH (previously known as YqfF), contribute to the degradation of the second messenger. Accumulation of c-di-AMP in agdpP pgpHdouble mutant is toxic for the cells, and the cells respond to this accumulation by inactivation of the diadenylate cyclase CdaA.IMPORTANCEBacteria use second messengers for signal transduction. Cyclic di-AMP (c-di-AMP) is the only second messenger known so far that is essential for a large group of bacteria. We have studied the regulation of c-di-AMP synthesis and the role of the phosphodiesterases that degrade this second messenger. c-di-AMP synthesis strongly depends on the nitrogen source: glutamate-grown cells produce more c-di-AMP than glutamine-grown cells. The accumulation of c-di-AMP in a strain lacking both phosphodiesterases is toxic and results in inactivation of the diadenylate cyclase CdaA. Our results suggest that CdaA is the critical diadenylate cyclase that produces the c-di-AMP that is both essential and toxic upon accumulation.

scholarly journals A rendezvous of two second messengers: The c-di-AMP receptor protein DarB controls (p)ppGpp synthesis in Bacillus subtilis

2020 ◽  
Author(s):  
Larissa Krüger ◽  
Christina Herzberg ◽  
Dennis Wicke ◽  
Heike Bähre ◽  
Jana L. Heidemann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Second Messengers ◽  
Stringent Response ◽  
Second Messenger ◽  
Receptor Protein ◽  
Rna Molecules ◽  
Regulatory Interactions ◽  
Cystathionine Beta Synthase ◽  
Interaction Partners ◽  
Osmotic Homeostasis

AbstractMany bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Most likely these proteins transduce their signal by regulatory interactions with other proteins. Here, we have investigated the function of the c-di-AMP-binding protein DarB in B. subtilis, a protein consisting of two CBS (cystathionine-beta synthase) domains. We have used an unbiased search for DarB interaction partners and identified the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. Indeed, c-di-AMP inhibits the binding of DarB to Rel. The interaction results in the Rel-dependent accumulation of pppGpp. Our results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.

scholarly journals A meet-up of two second messengers: the c-di-AMP receptor DarB controls (p)ppGpp synthesis in Bacillus subtilis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Larissa Krüger ◽  
Christina Herzberg ◽  
Dennis Wicke ◽  
Heike Bähre ◽  
Jana L. Heidemann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Second Messengers ◽  
Stringent Response ◽  
Second Messenger ◽  
Rna Molecules ◽  
Cystathionine Beta Synthase ◽  
Interaction Partners ◽  
Osmotic Homeostasis ◽  
Low Potassium ◽  
Cbs Domains

AbstractMany bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB–Rel interaction results in the Rel-dependent accumulation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.

scholarly journals Physiology of guanosine-based second messenger signaling in Bacillus subtilis

2020 ◽  
Vol 401 (12) ◽  
pp. 1307-1322
Author(s):  
Gert Bange ◽  
Patricia Bedrunka
Keyword(s):  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Second Messengers ◽  
Model Organism ◽  
Physiological Regulation ◽  
Open Questions ◽  
Key Players ◽  
Stress Signals ◽  
Second Messenger Signaling ◽  
Synthesis And Degradation

AbstractThe guanosine-based second messengers (p)ppGpp and c-di-GMP are key players of the physiological regulation of the Gram-positive model organism Bacillus subtilis. Their regulatory spectrum ranges from key metabolic processes over motility to biofilm formation. Here we review our mechanistic knowledge on their synthesis and degradation in response to environmental and stress signals as well as what is known on their cellular effectors and targets. Moreover, we discuss open questions and our gaps in knowledge on these two important second messengers.

Rapid kinetics of second messenger production in bitter taste

1996 ◽  
Vol 270 (3) ◽  
pp. C926-C931 ◽  
Author(s):  
A. I. Spielman ◽  
H. Nagai ◽  
G. Sunavala ◽  
M. Dasso ◽  
H. Breer ◽  
...  
Keyword(s):  
Bitter Taste ◽  
Second Messengers ◽  
Second Messenger ◽  
Mouse Strains ◽  
Protective Mechanism ◽  
Transient Nature ◽  
Sucrose Octaacetate ◽  
Rapid Kinetics ◽  
First Time

The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5-trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA-taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste.

scholarly journals Increased intracellular cyclic adenosine monophosphate inhibits T lymphocyte-mediated cytolysis by two distinct mechanisms.

1988 ◽  
Vol 167 (6) ◽  
pp. 1963-1968 ◽  
Author(s):  
L S Gray ◽  
J Gnarra ◽  
E L Hewlett ◽  
V H Engelhard
Keyword(s):  
Cholera Toxin ◽  
T Lymphocyte ◽  
Pertussis Toxin ◽  
Target Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Second Messenger ◽  
Adenosine Monophosphate ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Stimulation Of

Cholera toxin (CT), but not pertussis toxin (PT), treatment of cloned murine CTL inhibited target cell lysis in a dose-dependent fashion. The effects of CT were mimicked by forskolin and cyclic adenosine monophosphate (cAMP) analogues. Inhibition of cytotoxicity by CT and cAMP analogs was mediated in part by attenuation of conjugate formation. Additionally, both CT and cAMP analogs blocked the increase in intracellular Ca2+ induced by stimulation of the TCR complex by mAbs. These findings indicate that cAMP inhibits the activity of CTL by two distinct mechanisms and suggests a role for this second messenger in CTL-mediated cytolysis.

Stimulation of eating by the second messenger cAMP in the perifornical and lateral hypothalamus

1997 ◽  
Vol 273 (1) ◽  
pp. R107-R112 ◽  
Author(s):  
E. R. Gillard ◽  
A. M. Khan ◽  
A. ul-Haq ◽  
R. S. Grewal ◽  
B. Mouradi ◽  
...  
Keyword(s):  
Food Intake ◽  
Lateral Hypothalamus ◽  
Second Messengers ◽  
Second Messenger ◽  
Cyclic Monophosphate ◽  
Camp Analog ◽  
Complex Goal ◽  
Intracellular Action ◽  
Stimulation Of ◽  
Goal Oriented Behavior

Despite intense study of neurotransmitters mediating hypothalamic controls of food intake, little is known about which second messengers are critical for these mechanisms. To determine whether adenosine 3',5'-cyclic monophosphate (cAMP) might participate in these mechanisms, we injected the membrane-permeant cAMP analog 8-bromo-cAMP (8-BrcAMP) hypothalamically in satiated rats. Injection of 8-BrcAMP (10-100 nmol) into the perifornical (PFH) and lateral hypothalamus (LH) dose dependently stimulated food intake of up to 15.7 g in 2 h. Significantly smaller responses were obtained with thalamic injections. In contrast to the strong stimulatory effects of PFH and LH 8-BrcAMP, cAMP and 8-bromo-guanosine 3',5'-cyclic monophosphate (100 nmol) were ineffective, suggesting a chemically specific, intracellular action. Consistent with this, combined PFH injection of 7-deacetyl-7-O-(N-methylpiperazino)-tau-butyryl-forskolin dihydrochloride and 3-isobutyl-1-methylxanthine, agents that increase endogeneous cAMP, stimulated eating of up to 9.9 g in 2 h. These results demonstrate that increases in PFH/LH cAMP can elicit complex, goal-oriented behavior, suggesting an important role for cAMP in hypothalamic mechanisms stimulating food intake.

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