Bacteria can encounter a wide range of environments and must adapt to new conditions in order to survive. As the se- lective 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 critically on the physical state of lipid bilayers (17), making it susceptible to changes in environmental temperature. In fact, it has been established that normal cell function requires mem- brane lipid bilayers that are largely fluid; indeed, the bilayers of most organisms are entirely or mostly fluid at physiological temperatures. However, at lower temperatures, membrane lipid bilayers undergo a reversible change of state from a fluid (disordered) to a nonfluid (ordered) array of the fatty acyl chains (21, 56). The temperature at the midpoint of this tran- sition is called the transition temperature, and the change of state accompanying an increase in temperature is called the lipid phase transition, the gel-liquid crystalline transition, or most properly, the order-disorder transition. The transition temperature is a function of the membrane lipid composition and, in organisms deficient in cholesterol, mainly depends on the fatty acid composition of the membrane lipids (21, 56). The (overly simplified) rule of thumb is that phospholipids that contain unsaturated fatty acids (UFAs) have much lower tran- sition temperatures than those lipids made of saturated fatty acids (SFAs). The effect is due to different packing of the two types of phospholipid acyl chains. SFA acyl chains can pack tightly, but the steric hindrance imparted by the rigid kink of the cis double bond results in much poorer chain packing of UFAs, even below the phase transition temperature (16, 17). From these considerations, it seems clear that bacteria and most (if not all) poikilothermic organisms must regulate their phase transition in response to temperature. Without regula- tion, an organism shifted from a high to a low temperature would have membrane lipids with suboptimal fluidity, result- ing in subnormal membrane function. The mechanism of reg- ulation in all of the cases examined seems to occur via the incorporation of proportionally more UFAs (or fatty acids of analogous properties) as the temperature decreases. This reg- ulatory mechanism system, called thermal control of fatty acid synthesis, seems to be a universally conserved adaptation re- sponse allowing cells to maintain the appropriate fluidity of membrane lipids regardless of the ambient temperature. This means that cells must process temperature signals to adjust enzyme activities or to activate unique genes necessary to adapt the membranes to the new temperature. The question arises, how do cells sense a change in temperature and adjust the fluidity of the membrane lipid bilayer accordingly? Here, we discuss the basic features of thermal regulation of membrane lipid fluidity in Escherichia coli and Bacillus subtilis, in which the proposed mechanisms are firmly based on both genetic and biochemical evidence. Although the physiological consequences of this regulation are the same in both organ- isms, the mechanisms involved are entirely different.

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

The Bacillus subtilis DesK/DesR two-component system regulates the expression of the des gene coding for the Δ5 acyl lipid desaturase. It is believed that a decrease in membrane lipid fluidity activates the DesK/DesR signal transduction cascade, which results in synthesis of the Δ5 acyl lipid desaturase and desaturation of membrane phospholipids. These newly synthesized unsaturated fatty acids then act as negative signals of des transcription, thus generating a regulatory metabolic loop that optimizes membrane fluidity. We previously suggested that DesK is a bifunctional enzyme with both kinase and phosphatase activities that could assume different signaling states in response to changes in the fluidity of membrane lipids. However, no direct experimental evidence supported this proposed model. In this study, we show that the C-terminal fragment of the DesK protein (DesKC) indeed acts as an autokinase. Addition of the response regulator DesR to phosphorylated DesKC resulted in rapid transfer of the phosphoryl group to DesR. Further, phosphorylated DesR can be dephosphorylated in the presence of DesKC, thus demonstrating that the sensor kinase has the ability to covalently modify DesR through both kinase and phosphatase activities. We also present evidence that DesKC might be locked in a kinase-dominant state in vivo and that its activities are not affected either in vivo or in vitro by unsaturated fatty acids. These findings provide the first direct evidence that the transmembrane segments of DesK are essential to sense changes in membrane fluidity and for regulating the ratio of kinase to phosphatase activities of the cytoplasmic C-terminal domain.