Larisa E. Cybulski

Molecular basis of thermosensing: a two‐component signal transduction thermometer in Bacillus subtilis

Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock‐induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two‐component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Δ5‐lipid desaturase from Bacillus subtilis. We found that DesR binds to a DNA sequence extending from position −28 to −77 relative to the start site of the temperature‐regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Δ5‐desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK–DesR two‐component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.

Control of membrane lipid fluidity by molecular thermosensors.

Bacteria can encounter a wide range of environments and must adapt to new conditions in order to survive. As the selective barrier between living cells and their environment, the plasma membrane plays a key role in cell viability. The barrier function of the cytoplasmic membrane is known to depend

Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis acyl‐lipid desaturase

Summary 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 α‐keto acid derivative, which is a branched‐chain fatty acid precursor, negatively regulate the expression of the des gene at 37°C. The DesK–DesR two‐component system mediates this response, as both partners are required to sense and transduce the isoleucine signal at 37°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.

Membrane Thickness Cue for Cold Sensing in a Bacterium

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.

A lipid-mediated conformational switch modulates the thermosensing activity of DesK

Significance Environmental temperature variations affect most biological processes and reactions, and cells must adapt accordingly. One such example is the fine-tuned regulation of membrane fluidity and its impact on a large array of cell physiological processes. How do cells “sense” membrane fluidity? Here, we discovered that the bacterial histidine-kinase, DesK, transmits temperature information through lipid-mediated conformational changes of a region of the protein that links its membrane sensor domain with its catalytic domain and activates the expression of a desaturase, which allows membrane fluidity to be recovered at this lower temperature. Because many thermosensors and other types of sensors contain similar linker domains, the mechanism we describe here could prove a general theme. The thermosensor DesK is a multipass transmembrane histidine-kinase that allows the bacterium Bacillus subtilis to adjust the levels of unsaturated fatty acids required to optimize membrane lipid fluidity. The cytoplasmic catalytic domain of DesK behaves like a kinase at low temperature and like a phosphatase at high temperature. Temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane (TM) segment. These residues are buried in the lipid phase at low temperature and partially “buoy” to the aqueous phase at higher temperature with the thinning of the membrane, promoting the required conformational change. Nevertheless, the core question remains poorly understood: How is the information sensed by the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain to control DesK activity? Here, we identify a “linker region” (KSRKERERLEEK) that connects the TM sensor domain with the cytoplasmic catalytic domain involved in signal transmission. The linker adopts two conformational states in response to temperature-dependent membrane thickness changes: (i) random coiled and bound to the phospholipid head groups at the water-membrane interface, promoting the phosphatase state or (ii) unbound and forming a continuous helix spanning a region from the membrane to the cytoplasm, promoting the kinase state. Our results uphold the view that the linker is endowed with a helix/random coil conformational duality that enables it to behave like a transmission switch, with helix disruption decreasing the kinase/phosphatase activity ratio, as required to modulate the DesK output response.

Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. IMPORTANCE The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways. The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness.

Significance The ability to sense and respond to environmental signals is essential for cell survival. Unraveling the molecular mechanisms underlying signaling processes remains a challenge, however. Here we present a model for the mode of action of a bacterial thermosensor. The physical stimulus for activation appears to be a temperature-induced increase in membrane thickness, to which the sensor responds by elongation of its transmembrane helix. This leads to exposure of three serine residues on one side of the helix, inducing reorientation of adjacent helices to allow the formation of a serine zipper, which then acts as trigger for kinase activation. The reversible formation of a serine zipper represents a novel mechanism by which membrane-embedded sensors may detect and transmit signals. DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called “minimal sensor DesK” (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein–lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.

Cerulenin inhibits unsaturated fatty acids synthesis in Bacillus subtilis by modifying the input signal of DesK thermosensor

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.

Regulation of fatty acid desaturation in Bacillus subtilis

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.

Oligomerization of Bacillus subtilis DesR is required for fine tuning regulation of membrane fluidity.

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.