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A key event in the evolution of primordial cells on earth was the development of the cytoplasmic membrane since it created a protected reaction chamber for the performance of life’s vital attributes. However, the invention of the semi-permeable cytoplasmic membrane also created a severe problem for the microbial cells because this protected environment with its high concentrations of nucleic acids, proteins, organic metabolites and inorganic ions possesses a very substantial osmotic potential.
This, in turn, creates an osmotic driving force that triggers water influx into the cell and thereby generates an intracellular hydrostatic pressure, the turgor, whose magnitude can greatly exceed that of the pressure present in a car tire. Turgor is considered essential for cell expansion and viability, but its magnitude is affected by fluctuations in the osmotic conditions of the varied habitats of microorganism. Low osmotic conditions trigger water influx and high osmotic conditions trigger water efflux from the microbial cell. When microorganisms face low osmotic environments, they expel ions and organic compounds through the transient opening of mechanosensitive channels to prevent cell rupture. When they face high osmotic conditions, the microbial cell escapes cellular dehydration and collapse of turgor by amassing ions (e.g., potassium) and a selected class of organic osmolytes, the so-called compatible solutes. We are studying the genetic regulatory circuits that permit microbial cells to detect osmotic changes in their environment and study the cellular and biochemical response systems that allow microbial cells to cope with fluctuating osmolarities. - In the long run, efforts to create synthetic microbial cells must incorporate into their molecular design devices to detect and alleviating osmotic stress. Without such systems, synthetic cells cannot survive and strive!
Microbial cells possess a considerable osmotic potential caused by the high concentrations of nucleic acids, proteins, organic metabolites and inorganic ions in the cytoplasm. This, in turn, creates an osmotic driving force that triggers water influx into the cell and thereby generates an intracellular hydrostatic pressure, the turgor, whose magnitude can greatly exceed that of a car tire. Turgor is considered essential for cell expansion and viability, but its magnitude is affected by fluctuations in the osmotic conditions of the varied habitats of microorganism. Microorganisms never developed the ability to actively pump water in or out of the cell to compensate for water fluxes across their cytoplasmic membrane that are elicited by variations in the external osmolarity. Instead, microorganisms learned in the course of evolution to determine the extent of cellular hydration and magnitude of turgor indirectly by dynamically modulating the osmotic potential of their cytoplasm in a direct response to cues emanating from osmotic changes in the environment. Microorganisms are threatened by cell rupture as a consequence of an undue rise in turgor triggered by water influx under hypoosmotic conditions. They are susceptible to dehydration of the cytoplasm and a collapse of turgor that follows water efflux in response to increases in the external osmolarity. Hence, an effective water management is a cornerstone for all free-living microbial cells to cope with fluctuations in the external osmotic conditions. - In the long run, efforts to create synthetic microbial cells must incorporate into their molecular design devices to detect osmotic changes in the environment and cellular response systems alleviating osmotic stress. Without such systems, synthetic cells cannot survive and thrive!
Our research effort within SYNMIKRO focuses on hyperosmotic growth conditions and aim to explore systems for the synthesis and uptake of compatible solutes to synthetically engineer an enhanced osmo-stress resistance of microbial cells. We use the Gram-positive soil bacterium Bacillus subtilis as our primary model system for these studies. To escape cellular dehydration and collapse of turgor under sustained high osmolarity conditions, B. subtilis and taxonomically closely species, amass a selected class of organic osmolytes, the so-called compatible solutes, to counteract the efflux of water and offset the collapse of turgor. These compounds have been selected in the course of evolution as highly effective cytoprotectants and chemical chaperones that provide not only stress resistance to high osmolarity challenges but also provide stress resistance to extremes in growth temperature (both low and high). Important compatible solutes for Bacilli are the amino acid L-proline, the trimethylammonium compound glycine betaine and the tetrahydropyrimidine ectoine and its derivative hydroxyectoine. These compounds can either be synthesized in response to osmotic stress or can be taken up from the environment through osmotically controlled high-affinity uptake systems. We assemble genetic building blocks (“bio-bricks”) for the synthesis of L-proline and ectoine/hydroxyectoine from various microorganisms and synthetic gene constructs to streamline and maximize the high-level cellular production or import of these compatible solutes. By synthetically promoting increased accumulation of compatible solutes we aim at enhancing resistance of microbial cells against severe osmotic stress, and high- and low temperature challenges.