scholarly journals RelA Inhibits Bacillus subtilis Motility and Chaining

2014 ◽  
Vol 197 (1) ◽  
pp. 128-137 ◽  
Author(s):  
Qutaiba O. Ababneh ◽  
Jennifer K. Herman
Keyword(s):  
Bacillus Subtilis ◽  
Second Messengers ◽  
Cell Formation ◽  
Stringent Response ◽  
Alternative Sigma Factor ◽  
Content Type ◽  
Biofilm Production ◽  
Motile Cells ◽  
Persister Cell ◽  
Developmental Decision

The nucleotide second messengers pppGpp and ppGpp [(p)ppGpp] are responsible for the global downregulation of transcription, translation, DNA replication, and growth rate that occurs during the stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many nonstringent processes, including virulence, persister cell formation, and biofilm production. InBacillus subtilis, (p)ppGpp production is primarily determined by the net activity of RelA, a bifunctional (p)ppGpp synthetase/hydrolase, and two monofunctional (p)ppGpp synthetases, YwaC and YjbM. We observe that inB. subtilis, arelAmutant grows exclusively as unchained, motile cells, phenotypes regulated by the alternative sigma factor SigD. Our data indicate that therelAmutant is trapped in a SigD “on” state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility inB. subtilis. Our results also suggest that minor variations in basal (p)ppGpp levels can significantly skew developmental decision-making outcomes.

scholarly journals From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

2015 ◽  
Vol 197 (18) ◽  
pp. 2908-2919 ◽  
Author(s):  
Anthony O. Gaca ◽  
Pavel Kudrin ◽  
Cristina Colomer-Winter ◽  
Jelena Beljantseva ◽  
Kuanqing Liu ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Second Messengers ◽  
Stringent Response ◽  
Synthetase Activity ◽  
Protective Effects ◽  
Content Type ◽  
Regulatory Effects ◽  
Complex Target

ABSTRACTThe bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. InEnterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolaseE. faecalisRel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation inFirmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEfsynthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEfalso efficiently utilized GMP to form GMP 3′-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity ofE. faecalisenzymes involved in GTP biosynthesis and, to a lesser extent, transcription ofrrnBbyEscherichia coliRNA polymerase. Activation ofE. coliRelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEfwas activated only by ppGpp. Furthermore, enzymatic activity of RelQEfis insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of “long” RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp.IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ ofEnterococcus faecalis(RelQEf), we found that, in addition to (p)ppGpp, RelQEfis an efficient producer of pGpp (GMP 3′-diphosphate).In vitroanalysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEfand suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

scholarly journals ClpP is required for proteolytic regulation of type II toxin–antitoxin systems and persister cell formation in Streptococcus mutans

2019 ◽  
Vol 1 (8) ◽  
Author(s):  
Xiao-Lin Tian ◽  
Miao Li ◽  
Zachariah Scinocca ◽  
Heather Rutherford ◽  
Yung-Hua Li
Keyword(s):  
Streptococcus Mutans ◽  
Type Species ◽  
Critical Role ◽  
Cell Formation ◽  
Type Ii ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Viability Assay ◽  
Persister Cell

The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.

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 Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis

2011 ◽  
Vol 55 (9) ◽  
pp. 4326-4337 ◽  
Author(s):  
Anna-Barbara Hachmann ◽  
Elif Sevim ◽  
Ahmed Gaballa ◽  
David L. Popham ◽  
Haike Antelmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Stringent Response ◽  
Resistant Isolate ◽  
Cross Resistance ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Wild Type ◽  
Content Type

ABSTRACTDaptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2+-dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, DapR1, from the model bacteriumBacillus subtilis168. DapR1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. DapR1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of DapR1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator genemreB, the stringent response generelA, and the phosphatidylglycerol synthase genepgsA. Genetic reconstruction studies indicated that thepgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-typepgsArestored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca2+-DAP complex.

scholarly journals Single-Molecule Dynamics at a Bacterial Replication Fork after Nutritional Downshift or Chemically Induced Block in Replication

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Rogelio Hernández-Tamayo ◽  
Hannah Schmitz ◽  
Peter L. Graumann
Keyword(s):  
Dna Damage ◽  
Bacillus Subtilis ◽  
Single Molecule ◽  
Stringent Response ◽  
Stress Conditions ◽  
High Plasticity ◽  
Replication Forks ◽  
Content Type ◽  
Single Molecule Dynamics ◽  
Molecule Dynamics

ABSTRACT Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single-molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and lagging-strand polymerase DnaE in the model bacterium Bacillus subtilis, we show that proteins react differently to stress conditions in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after the arrest of polymerase, but binds less frequently after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone (p)ppGpp due to stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behavior of DnaG and DnaE suggests that both proteins are recruited independently to the forks rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension. IMPORTANCE All cells need to adjust DNA replication, which is achieved by a well-orchestrated multiprotein complex, in response to changes in physiological and environmental conditions. For replication forks, it is extremely challenging to meet with conditions where amino acids are rapidly depleted from cells, called the stringent response, to deal with the inhibition of one of the centrally involved proteins or with DNA modifications that arrest the progression of forks. By tracking helicase (DnaC), primase (DnaG), and polymerase (DnaE), central proteins of Bacillus subtilis replication forks, at a single molecule level in real time, we found that interactions of the three proteins with replication forks change in different manners under different stress conditions, revealing an intriguing plasticity of replication forks in dealing with replication obstacles. We have devised a new tool to determine rates of exchange between static movement (binding to a much larger complex) and free diffusion, showing that during stringent response, all proteins have highly increased exchange rates, slowing down overall replication, while inactivation of polymerase or replication roadblocks leaves forks largely intact, allowing rapid restart once obstacles are removed.

scholarly journals High persister cell formation by clinical Staphylococcus aureus strains belonging to clonal complex 30

Microbiology ◽  
2020 ◽  
Vol 166 (7) ◽  
pp. 654-658 ◽  
Author(s):  
Liping Liu ◽  
Ying Wang ◽  
Martin Saxtorph Bojer ◽  
Paal Skytt Andersen ◽  
Hanne Ingmer
Keyword(s):  
Staphylococcus Aureus ◽  
Membrane Potential ◽  
Type Species ◽  
Cell Formation ◽  
Susceptible Population ◽  
Content Type ◽  
Clinical Strains ◽  
Link Type ◽  
Persister Cell

Bacterial persisters form a subpopulation of cells that survive lethal concentrations of antibiotics without being genetically different from the susceptible population. They are generally considered to be phenotypic variants that spontaneously have entered a dormant state with low ATP levels or reduced membrane potential. In Staphylococcus aureus , a serious opportunistic human pathogen, persisters are believed to contribute to chronic infections that are a major global healthcare problem. While S. aureus persisters have mostly been studied in laboratory strains, we have here investigated the ability of clinical strains to form persisters. For 44 clinical strains belonging to the major clonal complexes CC5, CC8, CC30 or CC45, we examined persister cell formation in stationary phase when exposed to 100 times the MIC of ciprofloxacin, an antibiotic that targets DNA replication. We find that while all strains are able to form persisters, those belonging to CC30 displayed on average 100-fold higher persister cell frequencies when compared to strains of other CCs. Importantly, there was no correlation between persister formation and the cellular ATP content of the individual strains, but the group of CC30 strains displayed slightly lower membrane potential compared to the non-CC30 group. CC30 strains have previously been associated with chronic and reoccuring infections and we hypothesize that there could be a correlation between lineage-specific characteristics displayed via in vitro persister assays and the observed clinical spectrum of disease.

scholarly journals The Listeria monocytogenes σB Regulon and Its Virulence-Associated Functions Are Inhibited by a Small Molecule

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
M. Elizabeth Palmer ◽  
Soraya Chaturongakul ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Bacillus Subtilis ◽  
Listeria Monocytogenes ◽  
Small Molecule ◽  
Sigma Factor ◽  
Pathogen Transmission ◽  
Bile Salt Hydrolase ◽  
Alternative Sigma Factor ◽  
Bacterial Survival ◽  
Gram Positive ◽  
Content Type

ABSTRACTThe stress-responsive alternative sigma factor σBis conserved across diverse Gram-positive bacterial genera. InListeria monocytogenes, σBregulates transcription of >150 genes, including genes contributing to virulence and to bacterial survival under host-associated stress conditions, such as those encountered in the human gastrointestinal lumen. An inhibitor ofL. monocytogenesσBactivity was identified by screening ~57,000 natural and synthesized small molecules using a high-throughput cell-based assay. The compound fluoro-phenyl-styrene-sulfonamide (FPSS) (IC50= 3.5 µM) downregulated the majority of genes previously identified as members of the σBregulon inL. monocytogenes10403S, thus generating a transcriptional profile comparable to that of a 10403S ΔsigBstrain. Specifically, of the 208 genes downregulated by FPSS, 75% had been identified previously as positively regulated by σB. Downregulated genes included key virulence and stress response genes, such asinlA,inlB,bsh,hfq,opuC, andbilE. From a functional perspective, FPSS also inhibitedL. monocytogenesinvasion of human intestinal epithelial cells and bile salt hydrolase activity. The ability of FPSS to inhibit σBactivity in bothL. monocytogenesandBacillus subtilisindicates its utility as a specific inhibitor of σBacross multiple Gram-positive genera.IMPORTANCEThe σBtranscription factor regulates expression of genes responsible for bacterial survival under changing environmental conditions and for virulence; therefore, this alternative sigma factor is important for transmission ofL. monocytogenesand other Gram-positive bacteria. Regulation of σBactivity is complex and tightly controlled, reflecting the key role of this factor in bacterial metabolism. We present multiple lines of evidence indicating that fluoro-phenyl-styrene-sulfonamide (FPSS) specifically inhibits activity of σBacross Gram-positive bacterial genera, i.e., in bothListeria monocytogenesandBacillus subtilis. Therefore, FPSS is an important new tool that will enable novel approaches for exploring complex regulatory networks inL. monocytogenesand other Gram-positive pathogens and for investigating small-molecule applications for controlling pathogen transmission.

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 (p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Bret N. Peterson ◽  
Megan K. M. Young ◽  
Shukun Luo ◽  
Jeffrey Wang ◽  
Aaron T. Whiteley ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Binding Protein ◽  
Second Messengers ◽  
Stringent Response ◽  
Intracellular Pathogen ◽  
Microbial World ◽  
Content Type ◽  
Secondary Messenger ◽  
Biochemical Analyses ◽  
Protein B

ABSTRACT The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes. IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes. Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

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