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 B. 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

The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in <em>Bacillus subtilis</em>

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

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

BACKGROUND: Several 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. RESULTS: Here 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. CONCLUSIONS: SPABBATS 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

The ␥-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

b PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-DL-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the ␥-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHPresistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates

Allosteric regulation of glycogen breakdown by the second messenger cyclic di-GMP

Streptomyces are our principal source of antibiotics, which they generate concomitant with a complex developmental transition from vegetative hyphae to spores. c-di-GMP acts as a linchpin in this transition by binding and regulating the key developmental regulators, BldD and WhiG. Here we show that c-di-GMP also binds the glycogen-debranching-enzyme, GlgX, uncovering a direct link between c-di-GMP and glycogen metabolism in bacteria. Further, we show c-di-GMP binding is required for GlgX activity. We describe structures of apo and c-di-GMP-bound GlgX and, strikingly, their comparison shows c-di-GMP induces long-range conformational changes, reorganizing the catalytic pocket to an active state. Glycogen is an important glucose storage compound that enables animals to cope with starvation and stress. Our in vivo studies reveal the important biological role of GlgX in Streptomyces glucose availability control. Overall, we identify a function of c-di-GMP in controlling energy storage metabolism in bacteria, which is widespread in Actinobacteria

The impact of the glutamate dehydrogenases on the link between carbon and nitrogen metabolism in Bacillus subtilis

Glutamat ist der zentrale Aminogruppendonor für alle stickstoffhaltigen Verbindungen in der Zelle. In dem Gram-positiven Bodenbakterium Bacillus subtilis wird Glutamat ausschließlich in den gekoppelten Reaktionen der Glutaminsynthetase und der Glutamatsynthase synthetisiert. Da für den Aufbau von Glutamat 2 Oxoglutarat aus dem Citrat Zyklus benötigt wird, stellt die Glutamatsynthese einen wichtigen Knotenpunkt zwischen dem Kohlenstoff- und Stickstoffstoffwechsel dar. Deshalb ist die Expression der beteiligten Enzyme streng reguliert. Der Abbau von Glutamat wird von der Glutamatdehydrogenase RocG katalysiert. Durch die geringe Affinität zu Ammonium kann dieses Enzym nicht die Synthese von Glutamat katalysieren. Neben der enzymatischen Funktion reguliert RocG die Aktivität des Transkriptionsregulators GltC, der bedeutend für die Expression der Glutamatsynthase ist. In dieser Arbeit wurden mutierte RocG-Varianten isoliert und charakterisiert. Durch einen Aminosäureaustausch wurden die beiden Funktion von RocG voneinander getrennt. Eine Klasse von Varianten inaktiviert GltC sehr stark und ist in ihrer zeigt enzymatischen Aktivität schwer beeinträchtigt. Die zweite Klasse kann GltC nicht mehr inhibieren, zeigt aber enzymatische Aktivität. Diese Ergebnisse geben einen Einblick in den regulatorischen Mechanismus der RocG-GltC Interaktion. B. subtilis 168 kodiert für eine zweite Glutamatdehydrogenase, GudB. Im Laborstamm 168 ist das gudB-Gen durch eine direkte Wiederholung von neun Basenpaaren kryptisch. Beim Wachstum einer rocG-Mutante auf Komplexmedium wird das gudB-Allel schnell dekryptifiziert, wobei immer eine Hälfte der Sequenzwiederholung deletiert wird. In dieser Arbeit wurde gezeigt, dass die gudB-Mutation mit einer extrem hohen Rate von 10-4 auftritt. Außerdem wurde bewiesen, dass eine perfekte Basen-wiederholung für die hohe Mutationsrate notwendig ist. Darüber hinaus zeigte sich in einem artifiziellen Mutagenese-System, dass die Transkription für die hohe Mutationsrate des gudB-Gens notwendig ist. Das Mfd Protein, welches die Transkription mit der DNA Reparatur koppelt, spielt eine entscheidende Rolle bei der Dekryptifizierung des gudB Gens. Die Ergebnisse dieser Arbeit unterstreichen die herausragende Bedeutung der Glutamathomöostase in B. subtilis

The Highly Conserved Asp23 Family Protein YqhY Plays a Role in Lipid Biosynthesis in Bacillus subtilis

In most bacteria, fatty acid biosynthesis is an essential process that must be controlled by the availability of precursors and by the needs of cell division. So far, no mechanisms controlling synthesis of malonyl-coenzyme A (CoA), the committed step in fatty acid synthesis, have been identified in the Gram-positive model bacterium Bacillus subtilis. We have studied the localization and function of two highly expressed proteins of unknown function, YqhY and YloU. Both proteins are members of the conserved and widespread Asp23 family. While the deletion of yloU had no effect, loss of the yqhY gene induced the rapid acquisition of suppressor mutations. The vast majority of these mutations affect subunits of the acetyl-CoA carboxylase (ACCase) complex, the enzyme that catalyzes the formation of malonyl-CoA. Moreover, lack of yqhY is accompanied by the formation of lipophilic clusters in the polar regions of the cells indicating an increased activity of ACCase. Our results suggest that YqhY controls the activity of ACCase and that this control results in inhibition of ACCase activity. Hyperactivity of the enzyme complex in the absence of YqhY does then provoke mutations that cause reduced ACCase activity

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 Km 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

Selection-Driven Accumulation of Suppressor Mutants in <i>Bacillus subtilis</i>: The Apparent High Mutation Frequency of the Cryptic <i>gudB</i> Gene and the Rapid Clonal Expansion of <i>gudB⁺</i> Suppressors Are Due to Growth under Selection

<div><p>Soil bacteria like <i>Bacillus subtilis</i> can cope with many growth conditions by adjusting gene expression and metabolic pathways. Alternatively, bacteria can spontaneously accumulate beneficial mutations or shape their genomes in response to stress. Recently, it has been observed that a <i>B. subtilis</i> mutant lacking the catabolically active glutamate dehydrogenase (GDH), RocG, mutates the cryptic <i>gudB^CR</i> gene at a high frequency. The suppressor mutants express the active GDH GudB, which can fully replace the function of RocG. Interestingly, the cryptic <i>gudB^CR</i> allele is stably inherited as long as the bacteria synthesize the functional GDH RocG. Competition experiments revealed that the presence of the cryptic <i>gudB^CR</i> allele provides the bacteria with a selective growth advantage when glutamate is scarce. Moreover, the lack of exogenous glutamate is the driving force for the selection of mutants that have inactivated the active <i>gudB</i> gene. In contrast, two functional GDHs are beneficial for the cells when glutamate was available. Thus, the amount of GDH activity strongly affects fitness of the bacteria depending on the availability of exogenous glutamate. At a first glance the high mutation frequency of the cryptic <i>gudB^CR</i> allele might be attributed to stress-induced adaptive mutagenesis. However, other loci on the chromosome that could be potentially mutated during growth under the selective pressure that is exerted on a GDH-deficient mutant remained unaffected. Moreover, we show that a GDH-proficient <i>B. subtilis</i> strain has a strong selective growth advantage in a glutamate-dependent manner. Thus, the emergence and rapid clonal expansion of the active <i>gudB</i> allele can be in fact explained by spontaneous mutation and growth under selection without an increase of the mutation rate. Moreover, this study shows that the selective pressure that is exerted on a maladapted bacterium strongly affects the apparent mutation frequency of mutational hot spots.</p></div