RESUMEN
Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii, light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptor-independent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle.IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur.
Asunto(s)
Acinetobacter baumannii/fisiología , Acinetobacter baumannii/efectos de la radiación , Luz , Redes y Vías Metabólicas/efectos de la radiación , Antioxidantes/metabolismo , Metabolismo de los Lípidos/efectos de la radiación , Fenilacetatos/metabolismo , Tensoactivos/metabolismo , Trehalosa/biosíntesis , Sistemas de Secreción Tipo VI/efectos de la radiaciónRESUMEN
Thermosensors detect temperature changes and trigger cellular responses crucial for survival at different temperatures. The thermosensor DesK is a transmembrane (TM) histidine kinase which detects a decrease in temperature through its TM segments (TMS). Here, we address a key issue: how a physical stimulus such as temperature can be converted into a cellular response. We show that the thickness of Bacillus lipid membranes varies with temperature and that such variations can be detected by DesK with great precision. On the basis of genetic studies and measurements of in vitro activity of a DesK construct with a single TMS (minimal sensor DesK [MS-DesK]), reconstituted in liposomes, we propose an interplay mechanism directed by a conserved dyad, phenylalanine 8-lysine 10. This dyad is critical to anchor the only transmembrane segment of the MS-DesK construct to the extracellular water-lipid interphase and is required for the transmembrane segment of MS-DesK to function as a caliper for precise measurement of membrane thickness. The data suggest that positively charged lysine 10, which is located in the hydrophobic core of the membrane but is close to the water-lipid interface, pulls the transmembrane region toward the water phase to localize its charge at the interface. Nevertheless, the hydrophobic residue phenylalanine 8, located at the N-terminal extreme of the TMS, has a strong tendency to remain in the lipid phase, impairing access of lysine 10 to the water phase. The outcome of this interplay is a fine-tuned sensitivity to membrane thickness that elicits conformational changes that favor different signaling states of the protein. IMPORTANCE: The ability to sense and respond to extracellular signals is essential for cell survival. One example is the cellular response to temperature variation. How do cells "sense" temperature changes? It has been proposed that the bacterial thermosensor DesK acts as a molecular caliper measuring membrane thickness variations that would occur as a consequence of temperature changes and activates a pathway to restore membrane fluidity at low temperature. Here, we demonstrated that membrane thickness variations do occur at physiological temperatures by directly measuring Bacillus lipid membrane thickness. We also dissected the N-terminal sensing motif of MS-DesK at the molecular-biophysical level and found that the dyad phenylalanine-lysine at the water-lipid phase is critical for achievement of a fine-tuned sensitivity to temperature.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Membrana Celular/enzimología , Proteínas Quinasas/metabolismo , Secuencias de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Membrana Celular/química , Membrana Celular/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/genética , TemperaturaRESUMEN
BACKGROUND: The DesK-DesR two-component system regulates the order of membrane lipids in the bacterium Bacillus subtilis by controlling the expression of the des gene coding for the delta 5-acyl-lipid desaturase. To activate des transcription, the membrane-bound histidine kinase DesK phosphorylates the response regulator DesR. This covalent modification of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator. However, the mechanism of regulation of DesR activity by phosphorylation and oligomerization is not well understood. METHODS: We employed deletion analysis and reporter fusions to study the role of the N-terminal domain on DesR activity. In addition, electromobility shift assays were used to analyze the binding capacity of the transcription factor to deletion mutants of the des promoter. RESULTS: We show that DesR lacking the N-terminal domain is still able to bind to the des promoter. We also demonstrate that if the RA site is moved closer to the -35 region of Pdes, the adjacent site RB is dispensable for activation. GENERAL SIGNIFICANCE: Our results indicate that the unphosphorylated regulatory domain of DesR obstructs the access of the recognition helix of DesR to its DNA target. In addition, we present evidence showing that RB is physiologically relevant to control the activation of the des gene when the levels of DesR-P reach a critical threshold.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Ácido Graso Desaturasas/metabolismo , Fluidez de la Membrana , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión/genética , delta-5 Desaturasa de Ácido Graso , Ensayo de Cambio de Movilidad Electroforética , Ácido Graso Desaturasas/química , Ácido Graso Desaturasas/genética , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Secuencias Reguladoras de Ácidos Nucleicos/genética , Activación TranscripcionalRESUMEN
To address the mechanism of thermosensing and its implications for molecular engineering, we previously deconstructed the functional components of the bacterial thermosensor DesK, a histidine kinase with a five-span transmembrane domain that detects temperature changes. The system was first simplified by building a sensor that consists of a single chimerical transmembrane segment that retained full sensing capacity. Genetic and biophysical analysis of this minimal sensor enabled the identification of three modular components named determinants of thermodetection (DOTs). Here we combine and tune the DOTs to determine their contribution to activity. A transmembrane zipper represents the master DOT that drives a reversible and activating dimerization through the formation of hydrogen bonds. Our findings provide the mechanism and insights to construct a synthetic transmembrane helix based on a poly-valine scaffold that harbors the DOTs and regulates the activity. The construct constitutes a modular switch that may be exploited in biotechnology and genetic circuitry.
Asunto(s)
Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Sensación Térmica/fisiología , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Membrana Celular/metabolismo , Enlace de Hidrógeno , Proteínas de la Membrana/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , TemperaturaRESUMEN
Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. We are investigating how Bacillus subtilis cells sense and transduce low-temperature signals to adjust its gene expression. One important step has been accomplished in the dissection of a novel pathway for the adjustment of unsaturated fatty acid synthesis in B.subtilis, termed the Des pathway. It responds to a decrease in growth temperature by enhancing the expression of the des gene, coding for an acyl-lipid desaturase. The Des pathway is uniquely and stringently regulated by a tw-component system composed of a membrane-associated kinase, DesK, and a soluble transcriptional activator, DesR. The temperature sensing ability of the DesK protein is regulated by the extent of disorder within the membrane lipid bilayer. In this work, we present the mechanism by which the sensor protein DesK controls the signal decay of its cognate partner, DesR, and how this response regulator activates transcription of its target promoter. The results of these analysis will be presented and discussed in the context of transcriptional regulation of membrane fluidity homeostasis.
Asunto(s)
Bacillus subtilis/fisiología , Frío , Fluidez de la Membrana/fisiología , Transducción de Señal/fisiologíaRESUMEN
The Des pathway of Bacillus subtilis regulates the expression of the acyl-lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids from saturated phospholipid precursors. Activation of this pathway takes place when cells are shifted to low growth temperature or when they are grown in minimal media in the absence of isoleucine supplies. The master switch for the Des pathway is a two-component regulatory system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene. We propose that both, a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism, involving the ability of DesK to sense a decrease in membrane fluidity.
Asunto(s)
Bacillus subtilis/metabolismo , Ácidos Grasos/metabolismo , delta-5 Desaturasa de Ácido Graso , Ácido Graso Desaturasas/metabolismo , Lípidos , Modelos Biológicos , Temperatura , Transcripción GenéticaRESUMEN
A laboratory exercise was designed to illustrate how physical stimuli such as temperature and light are sensed and processed by bacteria to elaborate adaptive responses. In particular, we use the well-characterized Des pathway of Bacillus subtilis to show that temperature modulates gene expression, resulting ultimately in modification of the levels of unsaturated fatty acids required to maintain proper membrane fluidity at different temperatures. In addition, we adapt recent findings concerning the modulation by light of traits related to virulence such as motility and biofilm formation in the chemotropic bacterium Acinetobacter baumannii. Beyond the theoretical background that this activity provides regarding sensing of environmental stimuli, the experimental setup includes approaches derived from classic genetics, microbiology, and biochemistry. The incorporation of these kind of teaching and training activities in middle-advanced Microbiology or Bacterial Genetics courses promotes acquisition of general and specific techniques and improves student's comprehension of scientific literature and research.
Asunto(s)
Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Genética Microbiana/métodos , Enseñanza/métodos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/fisiología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiología , Bacterias/genética , Bacterias/crecimiento & desarrollo , Bacteriología/educación , Biopelículas/efectos de la radiación , Genética Microbiana/educación , Humanos , Luz , Reproducibilidad de los Resultados , Investigación/educación , TemperaturaRESUMEN
Bacillus subtilis responds to a sudden decrease in temperature by transiently inducing the expression of the des gene encoding for a lipid desaturase, Δ5-Des, which introduces a double bond into the acyl chain of preexisting membrane phospholipids. This Δ5-Des-mediated membrane remodeling is controlled by the cold-sensor DesK. After cooling, DesK activates the response regulator DesR, which induces transcription of des. We show that inhibition of fatty acid synthesis by the addition of cerulenin, a potent and specific inhibitor of the type II fatty acid synthase, results in increased levels of short-chain fatty acids (FA) in membrane phospholipids that lead to inhibition of the transmembrane-input thermal control of DesK. Furthermore, reduction of phospholipid synthesis by conditional inactivation of the PlsC acyltransferase causes significantly elevated incorporation of long-chain FA and constitutive upregulation of the des gene. Thus, we provide in vivo evidence that the thickness of the hydrophobic core of the lipid bilayer serves as one of the stimulus sensed by the membrane spanning region of DesK.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Cerulenina/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Proteínas de la Membrana/metabolismo , Bacillus subtilis/efectos de la radiación , Membrana Celular/metabolismo , Frío , Ácido Graso Desaturasas/metabolismo , Transducción de SeñalRESUMEN
Membrane proteins are abundant in nature and play a key role in many essential life processes. They typically span the membrane with one or more hydrophobic segments. Temporal changes in properties of such transmembrane (TM) segments often are a prerequisite for functional activity of membrane proteins. However, very little is known about the molecular nature of this important step in signaling. In a recent published work, we report the finding that both the sensing and transmission of DesK, a bacterial cold sensor, which has five TM segments, can be captured into a chimerical single membrane-spanning minimal sensor. Thus, the DesK system allows minimization of a complex phenomenon to a perfect functional system. This "minimalist" approach helped to uncover the modus operandis of a receptor for environmental cold, but also explores the use of a novel approach to study how the TM domains of a sensor protein transmit signals across membranes.
RESUMEN
The influence of the lipid environment on the function of membrane proteins is increasingly recognized as crucial. Nevertheless, the molecular mechanisms underlying protein-lipid interactions remain obscure. Membrane lipid composition has a regulatory effect on membrane protein activity, and for a number of membrane proteins a clear correlation was found between protein activity and properties of the membrane bilayer such as fluidity. Membrane thickness is an important property of a lipid bilayer. It is expected that hydrophobic thickness match the hydrophobic thickness of transmembrane segments of integral membrane proteins. Any mismatch between the hydrophobic thicknesses of the lipid bilayer and the protein would lead to some modification in either the structure of the protein or the structure of the bilayer, or both. Consequent rearrangements may result in changes in protein activity. Here we review the behavior of several transmembrane proteins whose activity is altered by hydrophobic core thickness.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Membrana Dobles de Lípidos/metabolismoRESUMEN
Thermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in many physiological roles, including membrane remodeling, chemotaxis, touch, and pain [1-3], but, the mechanism by which their transmembrane (TM) domains transmit temperature signals is largely unknown. The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below approximately 30°C [1, 4] providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. Here we show that the multimembrane-spanning domain from DesK can be simplified into a chimerical single-membrane-spanning minimal sensor (MS) that fully retains, in vivo and in vitro, the sensing properties of the parental system. The MS N terminus contains three hydrophilic amino acids near the lipid-water interface creating an instability hot spot. Mutational analysis of this boundary-sensitive beacon revealed that membrane thickness controls the signaling state of the sensor by dictating the hydration level of the metastable hydrophilic spot. Guided by these results we biochemically demonstrated that the MS signal transmission activity is sensitive to bilayer thickness. Membrane thickness could be a general cue for sensing temperature in many organisms.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/fisiología , Frío , Proteínas de la Membrana/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Membrana Celular , Histidina Quinasa , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Proteínas Quinasas/química , Proteínas Quinasas/fisiologíaRESUMEN
The Des pathway of Bacillus subtilis regulates the synthesis of the cold-shock induced membrane-bound enzyme Delta5-fatty acid desaturase (Delta5-Des). A central component of the Des pathway is the response regulator, DesR, which is activated by a membrane-associated kinase, DesK, in response to a decrease in membrane lipid fluidity. Despite genetic and biochemical studies, specific details of the interaction between DesR and the DNA remain unknown. In this study we show that only the phosphorylated form of protein DesR is able to bind to a regulatory region immediately upstream of the promoter of the Delta5-Des gene (Pdes). Phosphorylation of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P DNA binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator. Subsequently, this phosphorylation signal propagation leads to the activation of the des gene through recruitment of RNA polymerase to Pdes. This is the first dissected example of a transcription factor functioning as a phosphorylation-activated switch for a cold-shock gene, allowing the cell to optimize the fluidity of membrane phospholipids.
Asunto(s)
Bacillus subtilis/enzimología , Ácido Graso Desaturasas/metabolismo , Fluidez de la Membrana/fisiología , Bacillus subtilis/genética , Secuencia de Bases , Membrana Celular/enzimología , Huella de ADN , ADN Bacteriano/metabolismo , delta-5 Desaturasa de Ácido Graso , Dimerización , Ácido Graso Desaturasas/química , Ácido Graso Desaturasas/genética , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas/fisiología , Activación Transcripcional/fisiologíaRESUMEN
The Des pathway of Bacillus subtilis regulates the expression of the acyl-lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids (UFAs) from saturated phospholipid precursors. Previously, we showed that the master switch for the Des pathway is a two-component regulatory system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene. Activation of this pathway takes place when cells are shifted to low growth temperature. Here, we report on the mechanism by which isoleucine regulates the Des pathway. We found that exogenous isoleucine sources, as well as its alpha-keto acid derivative, which is a branched-chain fatty acid precursor, negatively regulate the expression of the des gene at 37 degrees C. The DesK-DesR two-component system mediates this response, as both partners are required to sense and transduce the isoleucine signal at 37 degrees C. Fatty acid profiles strongly indicate that isoleucine affects the signalling state of the DesK sensor protein by dramatically increasing the incorporation of the lower-melting-point anteiso-branched-chain fatty acids into membrane phospholipids. We propose that both a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism. Thus, the Des pathway would provide a novel mechanism to optimize membrane lipid fluidity at a constant temperature.