DarA-the central processing unit for the integration of osmotic with potassium and amino acid homeostasis in Bacillus subtilis.

Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes including osmolyte uptake, cell wall homeostasis, as well as antibiotic and heat resistance. This study investigates the role of the c-di-AMP receptor protein DarA in the osmotic stress response in Bacillus subtilis. Through a series of experiments, we demonstrate that DarA plays a central role in the cellular response to osmotic fluctuations. Our findings show that DarA becomes essential under extreme potassium limitation as well as upon salt stress, highlighting its significance in mediating osmotic stress adaptation. Suppressor screens with darA mutants reveal compensatory mechanisms involving the accumulation of osmoprotectants, particularly potassium and citrulline. Mutations affecting various metabolic pathways, including the citric acid cycle as well as glutamate and arginine biosynthesis, indicate a complex interplay between the osmotic stress response and metabolic regulation. In addition, the growth defects of the darA mutant during potassium starvation and salt stress in a strain lacking the high-affinity potassium uptake systems KimA and KtrAB can be rescued by increased affinity of the remaining potassium channel KtrCD or by increased expression of ktrD, thus resulting in increased potassium uptake. Finally, the darA mutant can respond to salt stress by the increased expression of MleN , which can export sodium ions.IMPORTANCEEnvironmental bacteria are exposed to rapidly changing osmotic conditions making an effective adaptation to these changes crucial for the survival of the cells. In Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in this adaptation by controlling (i) the influx of physiologically compatible organic osmolytes and (ii) the biosynthesis of such osmolytes. In several bacteria, cyclic di-adenosine monophosphate (c-di-AMP) can bind to a signal transduction protein, called DarA, in Bacillus subtilis. So far, no function for DarA has been discovered in any organism. We have identified osmotically challenging conditions that make DarA essential and have identified suppressor mutations that help the bacteria to adapt to those conditions. Our results indicate that DarA is a central component in the integration of osmotic stress with the synthesis of compatible amino acid osmolytes and with the homeostasis of potassium, the first response to osmotic stress.

Exploring Synergistic Effects of Levan and Levan-Metabolizing Bacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat.

Boosting plant immunity by priming agents can lower agrochemical dependency in plant production. Levan and levan-derived oligosaccharides (LOS) act as priming agents against biotic stress in several crops. Additionally, beneficial microbes can promote plant growth and protect against fungal diseases. This study assessed possible synergistic effects caused by levan, LOS and five levan- and LOS-metabolizing Bacillaceae (Bacillus and Priestia) strains in tomato and wheat. Leaf and seed defense priming assays were conducted in non-soil (semi-sterile substrate) and soil-based systems, focusing on tomato-Botrytis cinerea and wheat-Magnaporthe oryzae Triticum (MoT) pathosystems. In the non-soil system, seed defense priming with levan, the strains (especially Bacillus velezensis GA1), or their combination significantly promoted tomato growth and protection against B. cinerea. While no growth stimulatory effects were observed for wheat, disease protective effects were also observed in the wheat-MoT pathosystem. When grown in soil and subjected to leaf defense priming, tomato plants co-applied with levan and the bacterial strains showed increased resistance to B. cinerea compared with plants treated with levan or single strains, and these effects were synergistic in some cases. For seed defense priming in soil, more synergistic effects on disease tolerance were observed in a non-fertilized soil as compared to a fertilized soil, suggesting that potential prebiotic effects of levan are more prominent in poor soils. The potential of using combinations of Bacilliaceae and levan in sustainable agriculture is discussed.

c-di-AMP determines the hierarchical organization of bacterial RCK proteins.

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

Metabolic rewiring enables ammonium assimilation via a non-canonical fumarate-based pathway.

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle.

Control of three-carbon amino acid homeostasis by promiscuous importers and exporters in Bacillus subtilis: role of the "sleeping beauty" amino acid exporters.

The Gram-positive model bacterium Bacillus subtilis can acquire amino acids by import, de novo biosynthesis, or degradation of proteins and peptides. The accumulation of several amino acids inhibits the growth of B. subtilis, probably due to misincorporation into cellular macromolecules such as proteins or peptidoglycan or due to interference with other amino acid biosynthetic pathways. Here, we studied the adaptation of B. subtilis to toxic concentrations of the three-carbon amino acids L-alanine, ß-alanine, and 2,3-diaminopropionic acid, as well as the two-carbon amino acid glycine. Resistance to the non-proteinogenic amino acid ß-alanine, which is a precursor for coenzyme A biosynthesis, is achieved by mutations that either activate a cryptic amino acid exporter, AexA (previously YdeD), or inactivate the amino acid importers AimA, AimB (previously YbxG), and BcaP. The aexA gene is very poorly expressed under most conditions studied. However, mutations affecting the transcription factor AerA (previously YdeC) can result in strong constitutive aexA expression. AexA is the first characterized member of a group of amino acid exporters in B. subtilis, which are all very poorly expressed. Therefore, we suggest to call this group "sleeping beauty amino acid exporters." 2,3-Diaminopropionic acid can also be exported by AexA, and this amino acid also seems to be a natural substrate of AerA/AexA, as it can cause a slight but significant induction of aexA expression, and AexA also provides some natural resistance toward 2,3-diaminopropionic acid. Moreover, our work shows how low-specificity amino acid transporters contribute to amino acid homeostasis in B. subtilis.IMPORTANCEEven though Bacillus subtilis is one of the most-studied bacteria, amino acid homeostasis in this organism is not fully understood. We have identified import and export systems for the C2 and C3 amino acids. Our work demonstrates that the responsible amino acid permeases contribute in a rather promiscuitive way to amino acid uptake. In addition, we have discovered AexA, the first member of a group of very poorly expressed amino acid exporters in B. subtilis that we call "sleeping beauty amino acid exporters." The expression of these transporters is typically triggered by mutations in corresponding regulator genes that are acquired upon exposure to toxic amino acids. These exporters are ubiquitous in all domains of life. It is tempting to speculate that many of them are not expressed until the cells experience selective pressure by toxic compounds, and they protect the cells from rare but potentially dangerous encounters with such compounds.

Control of asparagine homeostasis in Bacillus subtilis: identification of promiscuous amino acid importers and exporters.

The Gram-positive model bacterium B. subtilis is able to import all proteinogenic amino acids from the environment as well as to synthesize them. However, the players involved in the acquisition of asparagine have not yet been identified for this bacterium. In this work, we used d-asparagine as a toxic analog of l-asparagine to identify asparagine transporters. This revealed that d- but not l-asparagine is taken up by the malate/lactate antiporter MleN. Specific strains that are sensitive to the presence of l-asparagine due to the lack of the second messenger cyclic di-AMP or due to the intracellular accumulation of this amino acid were used to isolate and characterize suppressor mutants that were resistant to the presence of otherwise growth-inhibiting concentrations of l-asparagine. These screens identified the broad-spectrum amino acid importers AimA and BcaP as responsible for the acquisition of l-asparagine. The amino acid exporter AzlCD allows detoxification of l-asparagine in addition to 4-azaleucine and histidine. This work supports the idea that amino acids are often transported by promiscuous importers and exporters. However, our work also shows that even stereo-enantiomeric amino acids do not necessarily use the same transport systems.IMPORTANCETransport of amino acid is a poorly studied function in many bacteria, including the model organism Bacillus subtilis. The identification of transporters is hampered by the redundancy of transport systems for most amino acids as well as by the poor specificity of the transporters. Here, we apply several strategies to use the growth-inhibitive effect of many amino acids under defined conditions to isolate suppressor mutants that exhibit either reduced uptake or enhanced export of asparagine, resulting in the identification of uptake and export systems for l-asparagine. The approaches used here may be useful for the identification of transporters for other amino acids both in B. subtilis and in other bacteria.