RESUMEN
Biofilms can be viewed as tissue-like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut-shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA-binding mode suggests that RemA organizes its target DNA into nucleosome-like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic-stress adjustment.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Lepidópteros , Animales , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Factores de Transcripción/metabolismoRESUMEN
Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrhoeas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt-stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of l-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress.
Asunto(s)
Clostridioides difficile , Salinidad , Adaptación Fisiológica , Betaína/metabolismo , Prolina/metabolismoRESUMEN
The Gram-positive bacterium Bacillus subtilis is frequently exposed to hyperosmotic conditions. In addition to the induction of genes involved in the accumulation of compatible solutes, high salinity exerts widespread effects on B. subtilis physiology, including changes in cell wall metabolism, induction of an iron limitation response, reduced motility and suppression of sporulation. We performed a combined whole-transcriptome and proteome analysis of B. subtilis 168 cells continuously cultivated at low or high (1.2 M NaCl) salinity. Our study revealed significant changes in the expression of more than one-fourth of the protein-coding genes and of numerous non-coding RNAs. New aspects in understanding the impact of high salinity on B. subtilis include a sustained low-level induction of the SigB-dependent general stress response and strong repression of biofilm formation under high-salinity conditions. The accumulation of compatible solutes such as glycine betaine aids the cells to cope with water stress by maintaining physiologically adequate levels of turgor and also affects multiple cellular processes through interactions with cellular components. Therefore, we additionally analysed the global effects of glycine betaine on the transcriptome and proteome of B. subtilis and revealed that it influences gene expression not only under high-salinity, but also under standard growth conditions.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Betaína/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Proteoma , Salinidad , Cloruro de Sodio/farmacologíaRESUMEN
The ABC-transporters OpuB and OpuC from Bacillus subtilis function as osmoprotectant import systems. Their structural genes have most likely evolved through a duplication event but the two transporters are remarkably different in their substrate profile. OpuB possesses narrow substrate specificity, while OpuC is promiscuous. We assessed the functionality of hybrids between these two ABC-transporters by reciprocally exchanging the coding regions for the OpuBC and OpuCC substrate-binding proteins between the corresponding opuB and opuC operons. Substantiating the critical role of the binding protein in setting the substrate specificity of ABC transporters, OpuB::OpuCC turned into a promiscuous system, while OpuC::OpuBC now exhibited narrow substrate specificity. Both hybrid transporters possessed a high affinity for their substrates but the transport capacity of the OpuB::OpuCC system was moderate due to the synthesis of only low amounts of the xenogenetic OpuCC protein. Suppressor mutations causing single amino acid substitutions in the GbsR repressor controlling the choline to glycine betaine biosynthesis pathway greatly improved OpuB::OpuCC-mediated compatible solute import through transcriptional up-regulation of the hybrid opuB::opuCC operon. Collectively, we demonstrate for the first time that one can synthetically switch the substrate specificity of a given ABC transporter by combining its core components with a xenogenetic ligand-binding protein.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Betaína/metabolismo , Colina/metabolismo , Operón , Osmorregulación , Especificidad por Sustrato , Transcripción Genética , Regulación hacia ArribaRESUMEN
Arsenic, a highly cytotoxic and cancerogenic metalloid, is brought into the biosphere through geochemical sources and anthropogenic activities. A global biogeochemical arsenic biotransformation cycle exists in which inorganic arsenic species are transformed into organoarsenicals, which are subsequently mineralized again into inorganic arsenic compounds. Microorganisms contribute to this biotransformation process greatly and one of the organoarsenicals synthesized and degraded in this cycle is arsenobetaine. Its nitrogen-containing homologue glycine betaine is probably the most frequently used compatible solute on Earth. Arsenobetaine is found in marine and terrestrial habitats and even in deep-sea hydrothermal vent ecosystems. Despite its ubiquitous occurrence, the biological function of arsenobetaine has not been comprehensively addressed. Using Bacillus subtilis as a well-understood platform for the study of microbial osmostress adjustment systems, we ascribe here to arsenobetaine both a protective function against high osmolarity and a cytoprotective role against extremes in low and high growth temperatures. We define a biosynthetic route for arsenobetaine from the precursor arsenocholine that relies on enzymes and genetic regulatory circuits for glycine betaine formation from choline, identify the uptake systems for arsenobetaine and arsenocholine, and describe crystal structures of ligand-binding proteins from the OpuA and OpuB ABC transporters complexed with either arsenobetaine or arsenocholine.
Asunto(s)
Arsenicales/metabolismo , Bacillus subtilis/metabolismo , Presión Osmótica , Temperatura , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico , Biotransformación , Colina/metabolismo , Citoprotección , Concentración OsmolarRESUMEN
Ectoine and 5-hydroxyectoine are widely synthesized microbial osmostress protectants. They are also versatile nutrients but their catabolism and the genetic regulation of the corresponding genes are incompletely understood. Using the marine bacterium Ruegeria pomeroyi DSS-3, we investigated the utilization of ectoines and propose a seven steps comprising catabolic route that entails an initial conversion of 5-hydroxyectoine to ectoine, the opening of the ectoine ring, and the subsequent degradation of this intermediate to l-aspartate. The catabolic genes are co-transcribed with three genes encoding a 5-hydroxyectoine/ectoine-specific TRAP transporter. A chromosomal deletion of this entire gene cluster abolishes the utilization of ectoines as carbon and nitrogen sources. The presence of ectoines in the growth medium triggers enhanced expression of the importer and catabolic operon, a process dependent on a substrate-inducible promoter that precedes this gene cluster. EnuR, a member of the MocR/GabR-type transcriptional regulators, controls the activity of this promoter and functions as a repressor. EnuR contains a covalently bound pyridoxal-5'-phosphate, and we suggest that this co-factor is critical for the substrate-mediated induction of the 5-hydroxyectoine/ectoine import and catabolic genes. Bioinformatics showed that ectoine consumers are restricted to the Proteobacteria and that EnuR is likely a central regulator for most ectoine/5-hydroxyectoine catabolic genes.
Asunto(s)
Aminoácidos Diaminos/metabolismo , Proteínas Bacterianas/metabolismo , Rhodobacteraceae/metabolismo , Factores de Transcripción/metabolismo , Ácido Aspártico/metabolismo , Proteínas Bacterianas/genética , Carbono/metabolismo , Medios de Cultivo , Proteínas de Transporte de Membrana/metabolismo , Familia de Multigenes , Nitrógeno/metabolismo , Factores de Transcripción/genéticaRESUMEN
The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Prolina/biosíntesis , Pirrolina Carboxilato Reductasas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , Glutamato-5-Semialdehído Deshidrogenasa/genética , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Mutación Puntual/genética , Regiones Promotoras Genéticas/genética , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
Ectoine and hydroxyectoine are effective microbial osmostress protectants, but can also serve as versatile nutrients for bacteria. We have studied the genetic regulation of ectoine and hydroxyectoine import and catabolism in the marine Roseobacter species Ruegeria pomeroyi and identified three transcriptional regulators involved in these processes: the GabR/MocR-type repressor EnuR, the feast and famine-type regulator AsnC and the two-component system NtrYX. The corresponding genes are widely associated with ectoine and hydroxyectoine uptake and catabolic gene clusters (enuR, asnC), and with microorganisms predicted to consume ectoines (ntrYX). EnuR contains a covalently bound pyridoxal-5'-phosphate as a co-factor and the chemistry underlying the functioning of MocR/GabR-type regulators typically requires a system-specific low molecular mass effector molecule. Through ligand binding studies with purified EnuR, we identified N-(alpha)-L-acetyl-2,4-diaminobutyric acid and L-2,4-diaminobutyric acid as inducers for EnuR that are generated through ectoine catabolism. AsnC/Lrp-type proteins can wrap DNA into nucleosome-like structures, and we found that the asnC gene was essential for use of ectoines as nutrients. Furthermore, we discovered through transposon mutagenesis that the NtrYX two-component system is required for their catabolism. Database searches suggest that our findings have important ramifications for an understanding of the molecular biology of most microbial consumers of ectoines.
Asunto(s)
Aminoácidos Diaminos/metabolismo , Elementos Reguladores de la Transcripción/genética , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Transactivadores/genética , Aminobutiratos/química , Proteínas Bacterianas/metabolismo , Transporte Biológico/genética , Señales (Psicología) , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/genética , Familia de MultigenesRESUMEN
The development of a semi-permeable cytoplasmic membrane was a key event in the evolution of microbial proto-cells. As a result, changes in the external osmolarity will inevitably trigger water fluxes along the osmotic gradient. The ensuing osmotic stress has consequences for the magnitude of turgor and will negatively impact cell growth and integrity. No microorganism can actively pump water across the cytoplasmic membrane; hence, microorganisms have to actively adjust the osmotic potential of their cytoplasm to scale and direct water fluxes in order to prevent dehydration or rupture. They will accumulate ions and physiologically compliant organic osmolytes, the compatible solutes, when they face hyperosmotic conditions to retain cell water, and they rapidly expel these compounds through the transient opening of mechanosensitive channels to curb water efflux when exposed to hypo-osmotic circumstances. Here, we provide an overview on the salient features of the osmostress response systems of the ubiquitously distributed bacterium Bacillus subtilis with a special emphasis on the transport systems and channels mediating regulation of cellular hydration and turgor under fluctuating osmotic conditions. The uptake of osmostress protectants via the Opu family of transporters, systems of central importance for the management of osmotic stress by B. subtilis, will be particularly highlighted.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Presión Osmótica , Bacillus subtilis/citología , Transporte Biológico , Homeostasis , HumanosRESUMEN
Dimethylsulfoniopropionate (DMSP) is an abundant osmolyte and anti-stress compound produced primarily in marine ecosystems. After its release into the environment, microorganisms can exploit DMSP as a source of sulfur and carbon, or accumulate it as an osmoprotectant. However, import systems for this ecophysiologically important compatible solute, and its stress-protective properties for microorganisms that do not produce it are insufficiently understood. Here we address these questions using a well-characterized set of Bacillus subtilis mutants to chemically profile the influence of DMSP import on stress resistance, the osmostress-adaptive proline pool and on osmotically controlled gene expression. We included in this study the naturally occurring selenium analogue of DMSP, dimethylseleniopropionate (DMSeP), as well as a set of synthetic DMSP derivatives. We found that DMSP is not a nutrient for B. subtilis, but it serves as an excellent stress protectant against challenges conferred by sustained high salinity or lasting extremes in both low and high growth temperatures. DMSeP and synthetic DMSP derivatives retain part of these stress protective attributes, but DMSP is clearly the more effective stress protectant. We identified the promiscuous and widely distributed ABC transporter OpuC as a high-affinity uptake system not only for DMSP, but also for its natural and synthetic derivatives.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Presión Osmótica/fisiología , Estrés Fisiológico/fisiología , Compuestos de Sulfonio/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Calor , Prolina/metabolismo , Salinidad , Azufre/metabolismoRESUMEN
The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ(1)-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na(+)-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means.
Asunto(s)
Aminoácidos/metabolismo , Bacillus subtilis/metabolismo , Biotransformación , Presión Osmótica , Bacillus subtilis/crecimiento & desarrollo , Viabilidad Microbiana/efectos de los fármacosRESUMEN
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 γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant 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.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas de Transporte de Membrana/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Bacillus subtilis/metabolismo , Transporte Biológico Activo , Genoma Bacteriano , Cinética , Proteínas de Transporte de Membrana/genética , Mutación , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The gamma-glutamyl-phosphate reductase (ProA) interlinks both the anabolic and osmostress adaptive proline biosynthetic routes of Bacillus subtilis. Because no paralogous protein to ProA exists in this microorganism, proA mutants should exhibit a tight proline auxotrophic growth phenotype. Contrary to expectations, proA mutants formed microcolonies on agar plates lacking proline and faster growing Pro(+) suppressor mutants arose. These mutants carried alterations in the rocR-rocDEF region encoding enzymes of the arginine degradation pathway and its transcriptional activator RocR. They were of two types: (i) mutants carrying single amino acid substitutions in RocR resulting in partial inducer-independent variants and (ii) mutants carrying single base-pair changes in the vicinity of the SigL/Sig-54-dependent -12/-24 class rocDEF promoter that activate a cryptic SigA-type promoter. Consequently, enhanced rocDEF transcription should lead to increased cellular amounts of the RocD ornithine aminotransferase, an enzyme that synthesizes the same reaction product as ProA, gamma-glutamic-semialdehyde/delta-1-pyrroline-5-carboxylate. This compound can be enzymatically converted into proline. The Pro(+) suppressors also exhibited a new regulatory pattern by allowing enhanced rocDEF transcription in response to proline availability when ammonium is present. Our work provides an example how flexibly bacteria can genetically develop routes to bypass constraints imposed on their biosynthetic networks and evolve new regulatory mechanisms.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mutación , Prolina/biosíntesis , Transactivadores/genética , Sustitución de Aminoácidos , Arginina/biosíntesis , Arginina/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Prolina/metabolismo , Regiones Promotoras Genéticas/genética , Pirroles , Alineación de Secuencia , Transactivadores/metabolismoRESUMEN
The Gram-positive bacterium Bacillus subtilis encounters nutrient limitations and osmotic stress in its natural soil ecosystem. To ensure survival and sustain growth, highly integrated adaptive responses are required. Here, we investigated the system-wide response of B. subtilis to different, simultaneously imposed stresses. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses of the transcriptome, proteome, metabolome and fluxome. Surprisingly, the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. Specifically, increased production of several enzymes of central carbon metabolism compensates for their reduced activity in the presence of high salt. A major response of the cell during osmotic stress is the production of the compatible solute proline. This is achieved through the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways.
Asunto(s)
Bacillus subtilis/metabolismo , Tolerancia a la Sal , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Betaína/metabolismo , Metabolismo de los Hidratos de Carbono , Análisis por Conglomerados , Metabolismo Energético , Regulación Bacteriana de la Expresión Génica , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas , Presión Osmótica , Proteoma/genética , Proteoma/metabolismo , TranscriptomaRESUMEN
L-Proline is a widely used compatible solute and is employed by Bacillus subtilis, through both synthesis and uptake, as an osmostress protectant. Here, we assessed the stress-protective potential of the plant-derived L-proline derivatives N-methyl-L-proline, L-proline betaine (stachydrine), trans-4-L-hydroxproline and trans-4-hydroxy-L-proline betaine (betonicine) for cells challenged by high salinity or extremes in growth temperature. l-Proline betaine and betonicine conferred salt stress protection, but trans-4-L-hydroxyproline and N-methyl-L-proline was unable to do so. Except for L-proline, none of these compounds served as a nutrient for B. subtilis. L-Proline betaine was a considerably better osmostress protectant than betonicine, and its import strongly reduced the l-proline pool produced by B. subtilis under osmotic stress conditions, whereas a supply of betonicine affected the L-proline pool only modestly. Both compounds downregulated the transcription of the osmotically inducible opuA operon, albeit to different extents. Mutant studies revealed that L-proline betaine was taken up via the ATP-binding cassette transporters OpuA and OpuC, and the betaine-choline-carnitine-transporter-type carrier OpuD; betonicine was imported only through OpuA and OpuC. L-Proline betaine and betonicine also served as temperature stress protectants. A striking difference between these chemically closely related compounds was observed: L-proline betaine was an excellent cold stress protectant, but did not provide heat stress protection, whereas the reverse was true for betonicine. Both compounds were primarily imported in temperature-challenged cells via the high-capacity OpuA transporter. We developed an in silico model for the OpuAC-betonicine complex based on the crystal structure of the OpuAC solute receptor complexed with L-proline betaine.
Asunto(s)
Bacillus subtilis/fisiología , Calor , Presión Osmótica , Fitoquímicos/metabolismo , Plantas/química , Prolina/análogos & derivados , Estrés Fisiológico , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/efectos de la radiación , Prolina/metabolismoRESUMEN
Glycine betaine is a potent osmotic and thermal stress protectant of many microorganisms. Its synthesis from glycine results in the formation of the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG), and these compounds are also produced when it is catabolized. Bacillus subtilis does not produce sarcosine or DMG, and it cannot metabolize these compounds. Here we have studied the potential of sarcosine and DMG to protect B. subtilis against osmotic, heat, and cold stress. Sarcosine, a compatible solute that possesses considerable protein-stabilizing properties, did not serve as a stress protectant of B. subtilis. DMG, on the other hand, proved to be only moderately effective as an osmotic stress protectant, but it exhibited good heat stress-relieving and excellent cold stress-relieving properties. DMG is imported into B. subtilis cells primarily under osmotic and temperature stress conditions via OpuA, a member of the ABC family of transporters. Ligand-binding studies with the extracellular solute receptor (OpuAC) of the OpuA system showed that OpuAC possesses a moderate affinity for DMG, with a Kd value of approximate 172 µM; its Kd for glycine betaine is about 26 µM. Docking studies using the crystal structures of the OpuAC protein with the sulfur analog of DMG, dimethylsulfonioacetate, as a template suggest a model of how the DMG molecule can be stably accommodated within the aromatic cage of the OpuAC ligand-binding pocket. Collectively, our data show that the ability to acquire DMG from exogenous sources under stressful environmental conditions helps the B. subtilis cell to cope with growth-restricting osmotic and temperature challenges.
Asunto(s)
Bacillus subtilis/fisiología , Sarcosina/análogos & derivados , Cloruro de Sodio/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Betaína/metabolismo , Presión Osmótica , Sarcosina/metabolismo , Estrés Fisiológico , TemperaturaRESUMEN
In bacteria, the availability of environmental inorganic phosphate is typically sensed by the conserved PhoR-PhoB two-component signal transduction pathway, which uses the flux through the PstSCAB phosphate transporter as a readout of the extracellular phosphate level to control phosphate-responsive genes. While the sensing of environmental phosphate is well-investigated, the regulatory effects of cytoplasmic phosphate are unclear. Here, we disentangle the physiological and transcriptional responses of Caulobacter crescentus to changes in the environmental and cytoplasmic phosphate levels by uncoupling phosphate uptake from the activity of the PstSCAB system, using an additional, heterologously produced phosphate transporter. This approach reveals a two-pronged response of C. crescentus to phosphate limitation, in which PhoR-PhoB signaling mostly facilitates the utilization of alternative phosphate sources, whereas the cytoplasmic phosphate level controls the morphological and physiological adaptation of cells to growth under global phosphate limitation. These findings open the door to a comprehensive understanding of phosphate signaling in bacteria.
Asunto(s)
Proteínas Bacterianas , Caulobacter crescentus , Citoplasma , Regulación Bacteriana de la Expresión Génica , Fosfatos , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/crecimiento & desarrollo , Fosfatos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Citoplasma/metabolismo , Transducción de Señal , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Transporte de Fosfato/genéticaRESUMEN
Glycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the buildup of large pools, whose size was sensitively determined by the degree of the osmotic stress imposed. The amassing of glycine betaine caused repression of the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was upregulated in response to both sudden and sustained increases in the external osmolarity. Nonionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. opuA expression was downregulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless of whether this osmoprotectant was imported or was newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was upregulated in a B. subtilis mutant that was unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Betaína/farmacología , Regulación Bacteriana de la Expresión Génica/fisiología , Prolina/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Bacillus subtilis/clasificación , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/genética , Betaína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Concentración Osmolar , Presión Osmótica , Plásmidos , Prolina/metabolismo , Cloruro de SodioRESUMEN
Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute l-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (Dpp, Opp, App, and DtpT) operating in B. subtilis and relies on their hydrolysis to liberate proline. The effectiveness with which proline-containing peptides confer osmoprotection varies considerably, and this can be correlated with the amount of the liberated and subsequently accumulated free proline by the osmotically stressed cell. Through gene disruption experiments, growth studies, and the quantification of the intracellular proline pool, we have identified the PapA (YqhT) and PapB (YkvY) peptidases as responsible for the hydrolysis of various types of Xaa-Pro dipeptides and Xaa-Pro-Xaa tripeptides. The PapA and PapB peptidases possess overlapping substrate specificities. In contrast, osmoprotection by peptides of various lengths and compositions with a proline residue positioned at their N terminus was not affected by defects in the PapA and PapB peptidases. Taken together, our data provide new insight into the physiology of the osmotic stress response of B. subtilis. They illustrate the flexibility of this ubiquitously distributed microorganism to effectively exploit environmental resources in its acclimatization to sustained high-osmolarity surroundings through the accumulation of compatible solutes.
Asunto(s)
Bacillus subtilis/fisiología , Presión Osmótica , Péptidos/metabolismo , Prolina/metabolismo , Estrés Fisiológico , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Técnicas de Inactivación de Genes , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Transporte de Proteínas , ProteolisisRESUMEN
OBJECTIVE: This scoping review aims to identify and map the empirical literature on the implementation strategies and outcomes of school-based programs for adolescent suicide prevention (SBASP). INTRODUCTION: School-based programs are preferred interventions for preventing suicide in adolescents, and their effectiveness has been well-systematized in several reviews. Implementation research is a growing field for prevention programs, making it possible to understand the nature of success or failure outcomes and maximize intervention benefits. However, there is a knowledge gap in the implementation research applied to adolescent suicide prevention in the educational context. We conduct a scoping review to provide the first overview of the scope of implementation research applied to adolescent suicide prevention programs in the school setting to know what implementation strategies and outcomes are reported by these programs and how they are evaluated. METHODS: The proposed scoping review will be conducted following six stages, including the definition of objectives. Studies must be empirical and address implementation strategies or implementation outcomes of school-based programs for adolescent suicide prevention. Studies that focused exclusively on clinical efficacy or effectiveness evaluation will be excluded. A preliminary search of PubMed was conducted to refine the initial search strings, followed by a final search of several other electronic databases. Finally, a gray literature search will identify unpublished literature and reduce location bias. There will be no limits to a specific date. Two independent reviewers will screen, select, and extract the retrieved records. The results will be presented using tabular forms and a narrative summary with attention to the review objectives and research questions and their implications for research and practice of school-based programs for adolescent suicide prevention.