The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5'-monophosphate-3'-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here, we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis gene expression upon amino acid starvation. We first identified PurR as a receptor of (p)ppGpp in Bacillus anthracis. A co-structure with Bacillus subtilis PurR reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of phosphoribosyltransferase enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant in B. subtilis fails to downregulate purine synthesis genes upon amino acid starvation. Our work establishes the precedent of (p)ppGpp as an effector of a classical transcription repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from soil bacteria to pathogens.

Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K+) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K+, leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K+ levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K+ fluxes perturb Mg2+ homeostasis: import of K+ upon osmotic upshift is correlated with Mg2+ efflux, and Mg2+ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg2+ and a decrease in protein translation. Conversely, the reimport of Mg2+ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg2+ and K+ levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. IMPORTANCE Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K+), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Here, using Bacillus subtilis as a model, we demonstrate that concomitant with K+ import there is a large reduction in intracellular magnesium (Mg2+) mediated by specific efflux pumps. Further, it is the reimport of Mg2+ that is rate-limiting for the resumption of growth. These coordinated fluxes of K+ and Mg2+ are orchestrated by cyclic-di-AMP, an essential second messenger in Firmicutes. These findings amend the conventional model for osmoadaptation and reveal that Mg2+ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.

Degradation of RNA polymers, an ubiquitous process in all cells, is catalyzed by specific subsets of endo- and exoribonucleases that together recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, 3-'5' exoribonucleases comprise up to eight distinct enzymes. Among them, Oligoribonuclease (Orn) is unique as its activity is required for clearing short RNA fragments, which is important for cellular fitness. However, the molecular basis of Orn's unique cellular function remained unclear. Here, we show that Orn exhibits exquisite substrate preference for diribonucleotides. Crystal structures of substrate-bound Orn reveal an active site optimized for diribonucleotides. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, Orn is the one and only diribonucleotidase that completes the terminal step of RNA degradation. Together, our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of a specific intermediate pool, diribonucleotides, that affects cellular physiology and viability.

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in Pseudomonas aeruginosa as the 3'-to-5' exoribonuclease oligoribonuclease (Orn). Mutants of orn accumulated pGpG, which inhibited the linearization of c-di-GMP. This product inhibition led to elevated c-di-GMP levels, resulting in increased aggregate and biofilm formation. Thus, the hydrolysis of pGpG is crucial to the maintenance of c-di-GMP homeostasis. How species that utilize c-di-GMP signaling but lack an orn ortholog hydrolyze pGpG remains unknown. Because Orn is an exoribonuclease, we asked whether pGpG hydrolysis can be carried out by genes that encode protein domains found in exoribonucleases. From a screen of these genes from Vibrio cholerae and Bacillus anthracis, we found that only enzymes known to cleave oligoribonucleotides (orn and nrnA) rescued the P. aeruginosa Δorn mutant phenotypes to the wild type. Thus, we tested additional RNases with demonstrated activity against short oligoribonucleotides. These experiments show that only exoribonucleases previously reported to degrade short RNAs (nrnA, nrnB, nrnC, and orn) can also hydrolyze pGpG. A B. subtilisnrnA nrnB mutant had elevated c-di-GMP, suggesting that these two genes serve as the primary enzymes to degrade pGpG. These results indicate that the requirement for pGpG hydrolysis to complete c-di-GMP signaling is conserved across species. The final steps of RNA turnover and c-di-GMP turnover appear to converge at a subset of RNases specific for short oligoribonucleotides.IMPORTANCE The bacterial bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling molecule regulates complex processes, such as biofilm formation. c-di-GMP is degraded in two-steps, linearization into pGpG and subsequent cleavage to two GMPs. The 3'-to-5' exonuclease oligoribonuclease (Orn) serves as the enzyme that degrades pGpG in Pseudomonas aeruginosa Many phyla contain species that utilize c-di-GMP signaling but lack an Orn homolog, and the protein that functions to degrade pGpG remains uncharacterized. Here, systematic screening of genes encoding proteins containing domains found in exoribonucleases revealed a subset of genes encoded within the genomes of Bacillus anthracis and Vibrio cholerae that degrade pGpG to GMP and are functionally analogous to Orn. Feedback inhibition by pGpG is a conserved process, as strains lacking these genes accumulate c-di-GMP.

Potassium is the most abundant metal ion in every living cell. This ion is essential due to its requirement for the activity of the ribosome and many enzymes but also because of its role in buffering the negative charge of nucleic acids. As the external concentrations of potassium are usually low, efficient uptake and intracellular enrichment of the ion is necessary. The Gram-positive bacterium Bacillus subtilis possesses three transporters for potassium, KtrAB, KtrCD, and the recently discovered KimA. In the absence of the high-affinity transporters KtrAB and KimA, the bacteria were unable to grow at low potassium concentrations. However, we observed the appearance of suppressor mutants that were able to overcome the potassium limitation. All these suppressor mutations affected amino acid metabolism, particularly arginine biosynthesis. In the mutants, the intracellular levels of ornithine, citrulline, and arginine were strongly increased, suggesting that these amino acids can partially substitute for potassium. This was confirmed by the observation that the supplementation with positively charged amino acids allows growth of B. subtilis even at the extreme potassium limitation that the bacteria experience if no potassium is added to the medium. In addition, a second class of suppressor mutations allowed growth at extreme potassium limitation. These mutations result in increased expression of KtrAB, the potassium transporter with the highest affinity and therefore allow the acquisition and accumulation of the smallest amounts of potassium ions from the environment.IMPORTANCE Potassium is essential for every living cell as it is required for the activity for many enzymes and for maintaining the intracellular pH by buffering the negative charge of the nucleic acids. We have studied the adaptation of the soil bacterium Bacillus subtilis to life at low potassium concentrations. If the major high-affinity transporters are missing, the bacteria are unable to grow unless they acquire mutations that result in the accumulation of positively charged amino acids such as ornithine, citrulline, and arginine. Supplementation of the medium with these amino acids rescued growth even in the absence of externally added potassium. Moreover, these growth conditions, which the bacteria experience as an extreme potassium limitation, can be overcome by the acquisition of mutations that result in increased expression of the high-affinity potassium transporter KtrAB. Our results indicate that positively charged amino acids can partially take over the function of potassium.

Healthcare-associated infections are major causes of complications that lead to extended hospital stays and significant medical costs. The use of medical devices, including catheters, increases the risk of bacterial colonization and infection through the presence of a foreign surface. Two outcomes are observed for catheterized patients: catheter-associated asymptomatic bacteriuria and catheter-associated urinary tract infection (CAUTI). However, the relationship between these two events remains unclear. To understand this relationship, we studied a murine model of Pseudomonas aeruginosa CAUTI. In this model, we also observe two outcomes in infected animals: acute symptoms that is associated with CAUTI and chronic colonization that is associated with asymptomatic bacteriuria. The timing of the acute outcome takes place in the first week of infection, whereas chronic colonization occurs in the second week of infection. We further showed that mutants lacking genes encoding type III secretion system (T3SS), T3SS effector proteins, T3SS injection pore, or T3SS transcriptional activation all fail to cause acute symptoms of CAUTI. Nonetheless, all mutants defective for T3SS colonized the catheter and bladders at levels similar to the parental strain. In contrast, through induction of the T3SS master regulator ExsA, all infected animals showed acute phenotypes with bacteremia. Our results demonstrated that the acute symptoms, which are analogous to CAUTI, and chronic colonization, which is analogous to asymptomatic bacteriuria, are independent events that require distinct bacterial virulence factors. Experimental delineation of asymptomatic bacteriuria and CAUTI informs different strategies for the treatment and intervention of device-associated infections.

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3'3'-cGAMP) but not c-di-GMP or 2'3'-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA.

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.

The Gram-positive bacterium Bacillus subtilis can utilize several proteinogenic and non-proteinogenic amino acids as sources of carbon, nitrogen, and energy. The utilization of the amino acids arginine, citrulline, and ornithine is catalyzed by enzymes encoded in the rocABC and rocDEF operons and by the rocG gene. The expression of these genes is controlled by the alternative sigma factor SigL. RNA polymerase associated with this sigma factor depends on ATP-hydrolyzing transcription activators to initiate transcription. The RocR protein acts as a transcription activator for the roc genes. However, the details of amino acid metabolism via this pathway are unknown. Here, we investigated the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline, and ornithine. We identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that RocB acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to form ornithine and carbamate. Moreover, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments, we demonstrate that ornithine is the molecular inducer of RocR activity. Taken together, our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector of RocR.

In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. In some bacteria, c-di-AMP also binds to the pyruvate carboxylase to inhibit its activity. We have discovered that in B. subtilis the c-di-AMP target protein DarB, rather than c-di-AMP itself, specifically binds to pyruvate carboxylase both in vivo and in vitro. This interaction stimulates the activity of the enzyme, as demonstrated by in vitro enzyme assays and in vivo metabolite determinations. Both the interaction and the activation of enzyme activity require apo-DarB and are inhibited by c-di-AMP. Under conditions of potassium starvation and corresponding low c-di-AMP levels, the demand for citric acid cycle intermediates is increased. Apo-DarB helps to replenish the cycle by activating both pyruvate carboxylase gene expression and enzymatic activity via triggering the stringent response as a result of its interaction with the (p)ppGpp synthetase Rel and by direct interaction with the enzyme, respectively. IMPORTANCE If bacteria experience a starvation for potassium, by far the most abundant metal ion in every living cell, they have to activate high-affinity potassium transporters, switch off growth activities such as translation and transcription of many genes or replication, and redirect the metabolism in a way that the most essential functions of potassium can be taken over by metabolites. Importantly, potassium starvation triggers a need for glutamate-derived amino acids. In many bacteria, the responses to changing potassium availability are orchestrated by a nucleotide second messenger, cyclic di-AMP. c-di-AMP binds to factors involved directly in potassium homeostasis and to dedicated signal transduction proteins. Here, we demonstrate that in the Gram-positive model organism Bacillus subtilis, the c-di-AMP receptor protein DarB can bind to and, thus, activate pyruvate carboxylase, the enzyme responsible for replenishing the citric acid cycle. This interaction takes place under conditions of potassium starvation if DarB is present in the apo form and the cells are in need of glutamate. Thus, DarB links potassium availability to the control of central metabolism.

Any signal transduction requires communication between a sensory component and an effector. Some enzymes engage in signal perception and transduction, as well as in catalysis, and these proteins are known as "trigger" enzymes. In this report, we detail the trigger properties of RocG, the glutamate dehydrogenase of Bacillus subtilis. RocG not only deaminates the key metabolite glutamate to form alpha-ketoglutarate but also interacts directly with GltC, a LysR-type transcription factor that regulates glutamate biosynthesis from alpha-ketoglutarate, thus linking the two metabolic pathways. We have isolated mutants of RocG that separate the two functions. Several mutations resulted in permanent inactivation of GltC as long as a source of glutamate was present. These RocG proteins have lost their ability to catabolize glutamate due to a strongly reduced affinity for glutamate. The second class of mutants is exemplified by the replacement of aspartate residue 122 by asparagine. This mutant protein has retained enzymatic activity but has lost the ability to control the activity of GltC. Crystal structures of glutamate dehydrogenases that permit a molecular explanation of the properties of the various mutants are presented. Specifically, we may propose that D122N replacement affects the surface of RocG. Our data provide evidence for a correlation between the enzymatic activity of RocG and its ability to inactivate GltC, and thus give insights into the mechanism that couples the enzymatic activity of a trigger enzyme to its regulatory function.

Many 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.