Katrin Gunka

Cyclic Di-AMP Homeostasis in Bacillus subtilis BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH

Background: Bacillus subtilis encodes three diadenylate cyclases. Results: Cyclic di-AMP is essential for the viability of B. subtilis; however, excess c-di-AMP also harms the cells. The activity of the cyclases is subject to regulation. Conclusion: The control of c-di-AMP homeostasis is crucial for B. subtilis. Significance: c-di-AMP is the first essential signaling nucleotide in bacteria. The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.

Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation.

Glutamate, the major amino group donor in anabolism, is synthesized by the combined action of the glutamine synthetase (GS) and the glutamate synthase (GOGAT) in Bacillus subtilis. The glutamate dehydrogenase (GDH) exclusively degrades glutamate. GS and GDH are both trigger enzymes, active in nitrogen metabolism and in controlling gene expression. Feedback‐inhibited GS (FBI‐GS) controls DNA‐binding activities of two transcription factors, the repressor GlnR and TnrA, the global regulator of nitrogen metabolism. FBI‐GS binds to and activates GlnR. This protein complex inhibits GS formation and thus glutamine synthesis. Moreover, FBI‐GS inhibits DNA‐binding activity of TnrA. Glutamate biosynthesis, the reaction linking carbon with nitrogen metabolism, is controlled by GDH. Together with glutamate GDH inhibits GltC, the transcription factor that activates expression of the GOGAT genes. Thus, GS and GDH control glutamine and glutamate synthesis, respectively, depending on the nitrogen status of the cell. B. subtilis lacking a functional GDH show a severe growth defect. Interestingly, the growth defect is suppressed by the rapid activation of an inactive GDH. Thus, maintenance of glutamate homeostasis is crucial for cellular vitality. This review covers the recent work on the complex control of glutamine and glutamate metabolism in the Gram‐positive model organism B. subtilis.

Glutamate Metabolism in Bacillus subtilis: Gene Expression and Enzyme Activities Evolved To Avoid Futile Cycles and To Allow Rapid Responses to Perturbations of the System

Glutamate is a central metabolite in all organisms since it provides the link between carbon and nitrogen metabolism. In Bacillus subtilis, glutamate is synthesized exclusively by the glutamate synthase, and it can be degraded by the glutamate dehydrogenase. In B. subtilis, the major glutamate dehydrogenase RocG is expressed only in the presence of arginine, and the bacteria are unable to utilize glutamate as the only carbon source. In addition to rocG, a second cryptic gene (gudB) encodes an inactive glutamate dehydrogenase. Mutations in rocG result in the rapid accumulation of gudB1 suppressor mutations that code for an active enzyme. In this work, we analyzed the physiological significance of this constellation of genes and enzymes involved in glutamate metabolism. We found that the weak expression of rocG in the absence of the inducer arginine is limiting for glutamate utilization. Moreover, we addressed the potential ability of the active glutamate dehydrogenases of B. subtilis to synthesize glutamate. Both RocG and GudB1 were unable to catalyze the anabolic reaction, most probably because of their very high K(m) values for ammonium. In contrast, the Escherichia coli glutamate dehydrogenase is able to produce glutamate even in the background of a B. subtilis cell. B. subtilis responds to any mutation that interferes with glutamate metabolism with the rapid accumulation of extragenic or intragenic suppressor mutations, bringing the glutamate supply into balance. Similarly, with the presence of a cryptic gene, the system can flexibly respond to changes in the external glutamate supply by the selection of mutations.

A Novel Factor Controlling Bistability in Bacillus subtilis: the YmdB Protein Affects Flagellin Expression and Biofilm Formation

Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the σ(D)-dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation.

The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis.

The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA.

Control of potassium homeostasis is an essential function of the second messenger cyclic di-AMP in Bacillus subtilis

The second messenger cyclic di-AMP enables bacteria to adapt to changes in environmental potassium concentrations. c-di-AMP controls potassium homeostasis in bacteria In Bacillus subtilis, the second messenger cyclic di-AMP (c-di-AMP) regulates the expression of many genes encoding potassium transporters by binding to a regulatory RNA structure called a riboswitch in a gene called ydaO, preventing transcription beyond the riboswitch. Gundlach et al. found that ydaO encoded a high-affinity potassium transporter and renamed it kimA (K+ importer A). Binding of c-di-AMP to the kimA riboswitch under high external concentrations of potassium and the resulting inhibition of kimA expression were essential to ensure bacterial viability under these conditions. KimA is a member of an evolutionarily conserved family of potassium transporters, suggesting that this regulatory mechanism for potassium homeostasis could be widespread among diverse bacterial taxa. The second messenger cyclic di–adenosine monophosphate (c-di-AMP) is essential in the Gram-positive model organism Bacillus subtilis and in related pathogenic bacteria. It controls the activity of the conserved ydaO riboswitch and of several proteins involved in potassium (K+) uptake. We found that the YdaO protein was conserved among several different bacteria and provide evidence that YdaO functions as a K+ transporter. Thus, we renamed the gene and protein KimA (K+ importer A). Reporter activity assays indicated that expression beyond the c-di-AMP–responsive riboswitch of the kimA upstream regulatory region occurred only in bacteria grown in medium containing low K+ concentrations. Furthermore, mass spectrometry analysis indicated that c-di-AMP accumulated in bacteria grown in the presence of high K+ concentrations but not in low concentrations. A bacterial strain lacking all genes encoding c-di-AMP–synthesizing enzymes was viable when grown in medium containing low K+ concentrations, but not at higher K+ concentrations unless it acquired suppressor mutations in the gene encoding the cation exporter NhaK. Thus, our results indicated that the control of potassium homeostasis is an essential function of c-di-AMP.

Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes

Background: Listeria monocytogenes CdaA is an essential diadenylate cyclase. Results: CdaA activity depends on manganese and cobalt ions. Conclusion: CdaA has an unusual requirement for metal cofactors. Significance: Characterization of essential enzymes is important for developing novel antibiotics. The recently identified second messenger cyclic di-AMP (c-di-AMP) is involved in several important cellular processes, such as cell wall metabolism, maintenance of DNA integrity, ion transport, transcription regulation, and allosteric regulation of enzyme function. Interestingly, c-di-AMP is essential for growth of the Gram-positive model bacterium Bacillus subtilis. Although the genome of B. subtilis encodes three c-di-AMP-producing diadenlyate cyclases that can functionally replace each other, the phylogenetically related human pathogens like Listeria monocytogenes and Staphylococcus aureus possess only one enzyme, the diadenlyate cyclase CdaA. Because CdaA is also essential for growth of these bacteria, the enzyme is a promising target for the development of novel antibiotics. Here we present the first crystal structure of the L. monocytogenes CdaA diadenylate cyclase domain that is conserved in many human pathogens. Moreover, biochemical characterization of the cyclase revealed an unusual metal cofactor requirement.

A High-Frequency Mutation in Bacillus subtilis: Requirements for the Decryptification of the gudB Glutamate Dehydrogenase Gene

Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10(-4). Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes.

SPABBATS: A pathway-discovery method based on Boolean satisfiability that facilitates the characterization of suppressor mutants

BackgroundSeveral computational methods exist to suggest rational genetic interventions that improve the productivity of industrial strains. Nonetheless, these methods are less effective to predict possible genetic responses of the strain after the intervention. This problem requires a better understanding of potential alternative metabolic and regulatory pathways able to counteract the targeted intervention.ResultsHere we present SPABBATS, an algorithm based on Boolean satisfiability (SAT) that computes alternative metabolic pathways between input and output species in a reconstructed network. The pathways can be constructed iteratively in order of increasing complexity. SPABBATS allows the accumulation of intermediates in the pathways, which permits discovering pathways missed by most traditional pathway analysis methods. In addition, we provide a proof of concept experiment for the validity of the algorithm. We deleted the genes for the glutamate dehydrogenases of the Gram-positive bacterium Bacillus subtilis and isolated suppressor mutant strains able to grow on glutamate as single carbon source. Our SAT approach proposed candidate alternative pathways which were decisive to pinpoint the exact mutation of the suppressor strain.ConclusionsSPABBATS is the first application of SAT techniques to metabolic problems. It is particularly useful for the characterization of metabolic suppressor mutants and can be used in a synthetic biology setting to design new pathways with specific input-output requirements.

Localization of Components of the RNA-Degrading Machine in Bacillus subtilis

In bacteria, the control of mRNA stability is crucial to allow rapid adaptation to changing conditions. In most bacteria, RNA degradation is catalyzed by the RNA degradosome, a protein complex composed of endo- and exoribonucleases, RNA helicases, and accessory proteins. In the Gram-positive model organism Bacillus subtilis, the existence of a RNA degradosome assembled around the membrane-bound endoribonuclease RNase Y has been proposed. Here, we have studied the intracellular localization of the protein that have been implicated in the potential B. subtilis RNA degradosome, i.e., polynucleotide phosphorylase, the exoribonucleases J1 and J2, the DEAD-box RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase. Our data suggests that the bulk of these enzymes is located in the cytoplasm. The RNases J1 and J2 as well as the RNA helicase CshA were mainly localized in the peripheral regions of the cell where also the bulk of messenger RNA is localized. We were able to demonstrate active exclusion of these proteins from the transcribing nucleoid. Taken together, our findings suggest that the interactions of the enzymes involved in RNA degradation in B. subtilis are rather transient.