Interhelical H-Bonds Modulate the Activity of a Polytopic Transmembrane Kinase.

DesK is a Histidine Kinase that allows to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify.

A Transmembrane Histidine Kinase Functions as a pH Sensor.

The two-component system DesK-DesR regulates the synthesis of unsaturated fatty acids in the soil bacteria . This system is activated at low temperature and maintains membrane lipid fluidity upon temperature variations. Here, we found that DesK-the transmembrane histidine kinase-also responds to pH and studied the mechanism of pH sensing.

Driving the catalytic activity of a transmembrane thermosensor kinase.

DesK is a Bacillus thermosensor kinase that is inactive at high temperatures but turns activated when the temperature drops below 25 °C. Surprisingly, the catalytic domain (DesKC) lacking the transmembrane region is more active at higher temperature, showing an inverted regulation regarding DesK. How does the transmembrane region control the catalytic domain, repressing activity at high temperatures, but allowing activation at lower temperatures? By designing a set of temperature minimized sensors that share the same catalytic cytoplasmic domain but differ in number and position of hydrogen-bond (H-bond) forming residues along the transmembrane helix, we are able to tune, invert or disconnect activity from the input signal.

Reverse Engineering of a Thermosensing Regulator Switch.

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).

Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii.

Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen , 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.

The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper.

Unlabelled: 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.

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

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.

Using a microbial physiologic and genetic approach to investigate how bacteria sense physical stimuli.

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.

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.

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

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.

Bilayer hydrophobic thickness and integral membrane protein function.

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.

Playing with transmembrane signals.

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.

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.

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.

Molecular mechanisms of low temperature sensing bacteria.

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.

Bacillus subtilis DesR functions as a phosphorylation-activated switch to control membrane lipid fluidity.

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.

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.

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

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.