Cyclic di‐AMP targets the cystathionine beta‐synthase domain of the osmolyte transporter OpuC

Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c‐di‐AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c‐di‐AMP content and that c‐di‐AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c‐di‐AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.     

Osmotic relations of the drought-tolerant shrub Artemisia tridentata in response to water stress

Turgor maintenance, solute content and recovery from water stress were examined in the drought-tolerant shrub Artemisia tridentata. Predawn water potentials of shrubs receiving supplemental water remained above ?2 MPa throughout summer, while predawn water potentials of untreated shrubs decreased to ?5 MPa. Osmotic potentials decreased in conjunction with water potentials maintaining turgor pressures above 0 MPa. The decreases in osmotic potentials were not the result of osmotic adjustment (i.e. solute accumulation). Leaf solute contents decreased during drought, but leaf water volumes decreased more than 75% from spring to summer, thereby passively concentrating solutes within the leaves. The maintenance of positive turgor pressures despite decreases in leaf water volumes is consistent with other studies of species with elastic cell walls. Inorganic ion, organic acid, and carbohydrate contents of leaves declined during drought. The only solutes accumulating in leaves of A. tridentata with water stress were proline and a cyclitol, both considered compatible solutes. Total and osmotic potentials recovered rapidly following rewatering of shrubs; solute contents did not change except for a decrease in proline. Maintaining turgor through the passive concentration of solutes may be advantageous compared to synthesis of new solutes for osmotic adjustment in arid environments.     

中度嗜盐菌相容性溶质机制的研究进展

生活在高盐环境中的中度嗜盐菌不仅能抗衡外界的高渗透压胁迫,而且还能迅速适应短时间内的渗透冲击。为适应该环境,中度嗜盐菌依赖于一种被称为相容性溶质的物质,以执行渗透保护功能。这类物质属于极性的、易溶的和低分子量的有机化合物,其中包括糖类、氨基酸类、甜菜碱类和四氢嘧啶类等。中度嗜盐菌主要采用相容性溶质机制来适应盐环境。在此,就中度嗜盐菌的盐适应机理、相容性溶质的种类和特点,以及其作用的分子机制进行了阐述和讨论。     

Stress responses ofBacillus subtilis to high osmolarity environments: Uptake and synthesis of osmoprotectants

A decrease in the water content of the soil imposes a considerable stress on the voil-living bacteriumBacillus subtilis: water exits from the cells, resulting in decreased turgor and cessation of growth. Under these adverse circumstances,B. subtilis actively modulates the osmolarity of its cytoplasm to maintain turgor within acceptable boundaries. A rapid uptake of potassium ions via turgor-responsive transport systems is the primary stress response to a sudden increase in the external osmolarity. This is followed by the massive accumulation of the so-called compatible solutes, i.e., organic osmolytes that are highly congruous with cellular functions and hence can be accumulated by bacterial cells up to molar concentrations. Initially, the compatible solute proline is accumulated viade novo synthesis, butB. subtilis can also acquire proline from the environment by an osmoregulated transport system, OpuE. The preferred compatible solute ofB. subtilis is the potent osmoprotectant glycine betaine. This trimethylammonium compound can be taken up by the cell through three high-affinity transport systems: the multicomponent ABC transporters OpuA and OpuC, and the single-component transporter OpuD. The OpuC systems also mediates the accumulation of a variety of naturally occurring betaines, each of which can confer a considerable degree of osmotic tolerance. In addition to the uptake of glycine betaine from the environment,B. subtilis can also synthesize this osmoprotectant but it requires exogenously provided choline as its precursor. Two evolutionarily closely related ABC transport systems, OpuB and OpuC, mediate the uptake of choline which is then converted by the GbsA and GbsB enzymes in a two-step oxidation process into glycine betaine. Our data show that the intracellular accumulation of osmoprotectants is of central importance for the cellular defence ofB. subtilis against high osmolarity stress.     

Enteric bacteria and osmotic stress: intracellular potassium glutamate as a secondary signal of osmotic stress?

Enteric bacteria have evolved an impressive array of mechanisms that allow the cell to grow at widely different external osmotic pressures. These serve two linked functions; firstly, they allow the cell to maintain a relatively constant turgor pressure which is essential for cell growth; and secondly they permit changes in cytoplasmic composition such that the accumulation of intracellular osmolytes required to restore turgor pressure does not impair enzyme function. The primary event in turgor regulation is the controlled accumulation of potassium and its counterion glutamate. At high external osmolarities the cytoplasmic levels of potassium glutamate can impair enzyme function. Rapid growth is therefore dependent upon secondary responses, principally the accumulation of compatible solutes, betaine (N-trimethylglycine), proline and trehalose. The accumulation of these solutes is achieved by the controlled activity of transport systems and enzymes in response to changes in external osmotic pressure. It has been proposed that the accumulation of potassium glutamate during turgor regulation acts as a signal for the activation of these systems [1,2]. This brief review will examine the evidence that control over the balance of cytoplasmic osmolytes is achieved by sensing of the intracellular potassium (and glutamate) concentration.     

Enteric bacteria and osmotic stress: Intracellular potassium glutamate as a secondary signal of osmotic stress?

Abstract Enteric bacteria have evolved an impressive array of mechanisms that allow the cell to grow at widely different external osmotic pressures. These serve two linked functions; firstly, they allow the cell to maintain in relatively constant turgor pressure which is essential for cell growth; and secondly they permit changes in cytoplasmic composition such that the accumulation of intracellular osmolytes required to restore turgor pressure does not impair enzyme function. The primary event in turgor regulation is the controlled accumulation of potassium and its counterion glutamate. At high external osmolarities the cytoplasmic levels of potassium glutamate can impair enzyme function. Rapid growth is therefore dependent upon secondary responses, principally the accumulation of compatible solutes, betaine ( N -trimethylglycine), proline and trehalose. The accumulation of these solutes is achieved by the controlled activity of transport systems and enzymes in response to changes in external osmotic pressure. It has been proposed that the accumulation of potassium glutamate during turgor regulation acts as a signal for the activation of these systems [1,2]. This brief review will examine the evidence that control over the balance of cytoplasmic osmolytes is achieved by sensing of the intracellular potassium (and glutamate) concentration.     

The choline dehydrogenase BetA of Acinetobacter baumannii: a flavoprotein responsible for osmotic stress protection

Acinetobacter baumannii is outstanding for its ability to cope with low water activities which significantly contributes to its persistence in hospital environments. The vast majority of bacteria are able to prevent loss of cellular water by amassing osmoactive compatible solutes or their precursors into the cytoplasm. One such precursor of an osmoprotectant is choline that is taken up from the environment and oxidized to the compatible solute glycine betaine. Here, we report the identification of the osmotic stress operon betIBA in A. baumannii. This operon encodes the choline oxidation pathway important for the production of the solute glycine betaine. The salt-sensitive phenotype of a betA deletion strain could not be rescued by addition of choline, which is consistent with the role of BetA in choline oxidation. We found that BetA is a choline dehydrogenase but also mediates in vitro the oxidation of glycine betaine aldehyde to glycine betaine. BetA was found to be associated with the membrane and to contain a flavin, indicative for BetA donating electrons into the respiratory chain. The choline dehydrogenase activity was not salt dependent but was stimulated by the compatible solute glutamate.     

Adaptation of Escherichia coli to high osmolarity environments: Osmoregulation of the high-affinity glycine betaine transport system ProU

Abstract: A sudden increase in the osmolarity of the environment is highly detrimental to the growth and survival of Fscherichia coli and Salmonella typhimurium since it triggers a rapid efflux of water from the cell, resulting in a decreased turgor. Changes in the external osmolarity must therefore be sensed by the microorganisms and this information must be converted into an adaptation process that aims at the restoration of turgor. The physiological reaction of the cell to the changing environmental condition is a highly coordinated process. Loss of turgor triggers a rapid influx of K⁺ ions into the cell via specific transporters and the concomitant synthesis of counterions, such as glutamate. The increased intracellular concentration of K⁺-glutamate allows the adaptation of the cell to environments of moderately high osmolarities. At high osmolarity, K⁺-glutamate is insufficient to ensure cell growth, and the bacteria therefore replace the accumulated K⁺ ions with compounds that are less d eleterious for the cell's physiology. These compatible solutes include polyoles such as trehalose, amino acids such as proline, and methyl-amines such as glycine betaine. One of the most important compatible solutes for bacteria is glycine betaine. This potent osmoprotectant is widespread in nature, and its intracellular accumulation is achieved through uptake from the environment or synthesis from its precursor choline. In this overview, we discuss the properties of the high-affinity glycine betaine transport system ProU and the osmotic regulation of its structural genes.     

Solute compatibility with enzyme function and structure: rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates.

The major nitrogenous osmolytes present in the cells of marine invertebrates, notably the free amino acids glycine, alanine and proline, and trimethylamine oxide and betaine, are highly compatible with proper enzyme function and structure. These nitrogenous osmolytes display either non-perturbing or, in some cases, favorable effects on enzyme-substrate and enzyme-cofactor complex formation, catalytic velocity and protein structural stability. In contrast, inorganic salts (KCl and NaCl) and certain of the free amino acids which play only a minor osmotic role, e.g., arginine and lysine, have strongly perturbing effects on one or more of these enzymic parameters. The compatible nitrogenous solutes therefore are suitable for use at high (several tenths molar) concentrations and at widely varying concentrations in osmo-conforming species. Certain nitrogenous solutes, especially trimethylamine oxide, betaine and glutamate, offset some of the perturbing effects of inorganic ions on enzyme function. The selective accumulation of osmolytes thus involves not only the concentration of non-perturbing solutes, but also a balanced accumulation of solutes with opposing effects on enzymes. The selection of end-products of anaerobic metabolism also appears to be based, in part, on considerations of solute compatibility with enzyme function. Octopine is a non-perturbing solute, whereas arginine, which is condensed with pyruvate to form octopine, is very strongly perturbing. Succinate has marked stabilizing effects on protein structure. We conclude that the composition of the intracellular fluids of marine invertebrates reflects selection for osmolytes and end-products whose net effects create a cellular microenvironment which is conducive to optimal enzyme function and structure. The accumulation of compatible solutes may preclude the necessity for widespread changes in protein structure in adapting to concentrated or highly variable osmotic environments.     

Ecological significance of compatible solute accumulation by micro-organisms: from single cells to global climate

The osmoadaptation of most micro-organisms involves the accumulation of K(+) ions and one or more of a restricted range of low molecular mass organic solutes, collectively termed 'compatible solutes'. These solutes are accumulated to high intracellular concentrations, in order to balance the osmotic pressure of the growth medium and maintain cell turgor pressure, which provides the driving force for cell extension growth. In this review, I discuss the alternative roles which compatible solutes may also play as intracellular reserves of carbon, energy and nitrogen, and as more general stress metabolites involved in protection of cells against other environmental stresses including heat, desiccation and freezing. Thus, the evolutionary selection for the accumulation of a specific compatible solute may not depend solely upon its function during osmoadaptation, but also upon the secondary benefits its accumulation provides, such as increased tolerance of other environmental stresses prevalent in the organism's niche or even anti-herbivory or dispersal functions in the case of dimethylsulfoniopropionate (DMSP). In the second part of the review, I discuss the ecological consequences of the release of compatible solutes to the environment, where they can provide sources of compatible solutes, carbon, nitrogen and energy for other members of the micro-flora. Finally, at the global scale the metabolism of specific compatible solutes (betaines and DMSP) in brackish water, marine and hypersaline environments may influence global climate, due to the production of the trace gases, methane and dimethylsulfide (DMS) and in the case of DMS, also couple the marine and terrestrial sulfur cycles.