Impact of transport processes in the osmotic response of Corynebacterium glutamicum

Osmoregulation, the adaptation of cells to changes in the external osmolarity, is an important aspect of the bacterial stress response, in particular for a soil bacterium like Corynebacterium glutamicum. Consequently, this organism is equipped with several redundant systems for coping with both hyper- and hypoosmotic stress. For the adaptation to hypoosmotic stress C. glutamicum possesses at least three different mechanosensitive (MS) channels. To overcome hyperosmotic stress C. glutamicum accumulates so-called compatible solutes either by means of biosynthesis or by uptake. Uptake of compatible solutes is in general preferred to de novo synthesis because of lower energy costs. Noticeable, only secondary transporters belonging to the MHS (ProP) or the BCCT-family (BetP, EctP and LcoP) are involved in the uptake of proline, betaine and ectoine. In contrast to Escherichia coli or Bacillus subtilis no ABC-transporters were found catalyzing uptake of compatible solutes. BetP was one of the first examples of the growing group of osmosensory proteins to be analyzed in detail. This transporter is characterized, besides the catalytic activity of betaine uptake, by the ability to sense osmotic changes (osmosensing) and to respond to the extent of osmotic stress by adaptation of transport activity (osmoregulation). BetP detects hyperosmotic stress via an increase in the internal K(+) concentration following a hyperosmotic shift, and thus acts as a chemosensor.     

Chill activation of compatible solute transporters in Corynebacterium glutamicum at the level of transport activity

The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K(+) and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10 degrees C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K(+) in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K(+) concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K(+), was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K(+)-mediated process.     

Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation

As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake. Beside the instant activation of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation. In order to adapt the pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial osmotic stress is compensated. Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in Corynebacterium glutamicum, in this adaptation process. For this purpose, betP was expressed in cells (C. glutamicum and Escherichia coli), which lack all known uptake systems for compatible solutes. Betaine uptake mediated by BetP as well as by a truncated form of BetP, which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake, unidirectional efflux and net uptake. We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase. The observed decrease in net uptake was thus mainly due to a decrease of Vmax of BetP and not a consequence of the presence of separate efflux system(s). These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also different from previously described adaptation mechanisms in other organisms. Cytoplasmic K+, which was shown to be responsible for activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process. Our results thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.     

BetP of Corynebacterium glutamicum, a transporter with three different functions: betaine transport, osmosensing, and osmoregulation

In order to circumvent deleterious effects of hypo- and hyperosmotic conditions in its environment, Corynebacterium glutamicum has developed a number of mechanisms to counteract osmotic stress. The first response to an osmotic upshift is the activation of uptake mechanisms for the compatible solutes betaine, proline, or ectoine, namely BetP, EctP, ProP, LcoP and PutP. BetP, the most important uptake system responds to osmotic stress by regulation at the level of both protein activity and gene expression. BetP was shown to harbor three different properties, i.e. catalytic activity (betaine transport), sensing of appropriate stimuli (osmosensing) and signal transduction to the catalytic part of the carrier protein which adapts its activity to the extent of osmotic stress (osmoregulation). BetP is comprised of 12 transmembrane segments and carries N- and C-terminal domains, which are involved in osmosensing and/or osmoregulation. Recent results on molecular properties of these domains indicate the significance of particular amino acids within the terminal 25 amino acids of the C-terminal domain of BetP for the process of osmosensing and osmoregulation.     

Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (K_m, 48 μM) to ectoine (K_m, 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (K_m, 63 μM) to betaine (K_m, 333 μM) and proline (K_m, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.     

Structure and function of the betaine uptake system BetP of Corynebacterium glutamicum: strategies to sense osmotic and chill stress

The soil bacterium Corynebacterium glutamicum has to cope with frequent fluctuations of the external osmolarity and temperature. The consequences of hyperosmotic and chill stress seem to differ, either causing dehydration of the cytoplasm or leading to impairment of cellular functions due to low temperature. Nevertheless, a particular type of regulatory response, namely the accumulation of so-called compatible solutes, is induced under both conditions. Compatible solutes are known to stabilize the native conformation of enzymes, which may be affected by osmotic and chill stress. BetP is a high-affinity uptake carrier for the compatible solute glycine betaine in C. glutamicum. BetP includes, besides its catalytic function, the ability to sense hyperosmotic conditions and chill stress. As a consequence, the carrier is activated in dependence of the extent of these types of stress. The signal input related to these changes of the environmental conditions is based on at least two different mechanisms. In case of hyperosmotic stress, BetP responds to the internal potassium concentration as a measure for hypertonicity, whereas chill stress is detected by an independent signal, most probably changes of the physical state of the membrane.     

Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation

As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake. Beside the instant activation of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation. In order to adapt the pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial osmotic stress is compensated. Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in Corynebacterium glutamicum, in this adaptation process. For this purpose, betP was expressed in cells (C. glutamicum and Escherichia coli), which lack all known uptake systems for compatible solutes. Betaine uptake mediated by BetP as well as by a truncated form of BetP, which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake, unidirectional efflux and net uptake. We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase. The observed decrease in net uptake was thus mainly due to a decrease of V_max of BetP and not a consequence of the presence of separate efflux system(s). These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also different from previously described adaptation mechanisms in other organisms. Cytoplasmic K⁺, which was shown to be responsible for activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process. Our results thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.     

Growth,cell volume,and fine structure of Porphyra umbilicalis in relation to osmotic tolerance

Porphyra umbilicalis, a marine red alga occurring in the intertidal zone of the cold North Sea, tolerates a wide range of osmotic conditions from 0.2 x to 6 x artificial seawater medium ASP₁₂. In cells osmotically adapted for two weeks, photosynthesis and respiration are progressively inhibited in media more concentrated than 2 x. In both hypo- and hyperosmotic stress ranges, the most striking fine structural change is the development of vacuoles. In comparison to 1 x medium, where vacuoles are virtually lacking, the vacuolar part of the protoplasm increases 6-fold in 0.2 x and 10-fold in 3.5 x medium, respectively. However, at extreme hyperosmotic stress (6 x medium) the vacuolar part is extremely small. The largest cell volumes are found in 0.2 x and 3.5 x media, the smallest one in 6 x medium. In the osmotically regulated range (0.2–3.5 x medium), the regulated parameter is the volume of the protoplasm without the vacuolar system. It is suggested that at hyperosmotic stress the vacuoles may serve as osmotically active compartment, probably by accumulation of inorganic ions. The intracellular content of Floridean starch granules decreases with increasing osmotic pressure, possibly indicating the significance of soluble organic constituents as osmotically active solutes.Member of the Arbeitsgemeinschaft für Elektronenmikroskople un der Ticrärztlichen Hochschule Hannover     

Impact of osmotic stress on volume regulation,cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B

The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.     

Osmolality, temperature, and membrane lipid composition modulate the activity of betaine transporter BetP in Corynebacterium glutamicum

The gram-positive soil bacterium Corynebacterium glutamicum, a major amino acid-producing microorganism in biotechnology, is equipped with several osmoregulated uptake systems for compatible solutes, which is relevant for the physiological response to osmotic stress. The most significant carrier, BetP, is instantly activated in response to an increasing cytoplasmic K(+) concentration. Importantly, it is also activated by chill stress independent of osmotic stress. We show that the activation of BetP by both osmotic stress and chill stress is altered in C. glutamicum cells grown at and adapted to low temperatures. BetP from cold-adapted cells is less sensitive to osmotic stress. In order to become susceptible for chill activation, cold-adapted cells in addition needed a certain amount of osmotic stimulation, indicating that there is cross talk of these two types of stimuli at the level of BetP activity. We further correlated the change in BetP regulation properties in cells grown at different temperatures to changes in the lipid composition of the plasma membrane. For this purpose, the glycerophospholipidome of C. glutamicum grown at different temperatures was analyzed by mass spectrometry using quantitative multiple precursor ion scanning. The molecular composition of glycerophospholipids was strongly affected by the growth temperature. The modulating influence of membrane lipid composition on BetP function was further corroborated by studying the influence of artificial modulation of membrane dynamics by local anesthetics and the lack of a possible influence of internally accumulated betaine on BetP activity.     

Glycine betaine transport in Lactococcus lactis is osmotically regulated at the level of expression and translocation activity

Microorganisms react upon hyperosmotic stress by accumulating compatible solutes. Here we report that Lactococcus lactis uses a transport system for glycine betaine that, contrary to earlier observations (D. Molenaar et al., J. Bacteriol. 175:5438-5444, 1993), is osmotically regulated at the levels of both expression and transport activity.     

外源精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下生长和发酵特性的影响

【目的】探究外源添加不同氨基酸和相容性溶质对谷氨酸棒杆菌(Corynebacterium glutamicum)在高糖胁迫环境下生长的影响及可能的作用机理。【方法】通过在培养基中外源添加各种氨基酸和相容性溶质,研究其对谷氨酸棒杆菌在高葡萄糖和高蔗糖胁迫下生长的影响,并分析添加精氨酸对高葡萄糖胁迫下菌株糖转运和代谢途径中关键酶转录水平的影响,以及对菌株发酵产氨基酸的影响。进一步探究了碱性氨基酸在其它棒状杆菌属中抵御高葡萄糖胁迫的潜在作用。【结果】在高葡萄糖胁迫条件下,外源添加赖氨酸、精氨酸和组氨酸后谷氨酸棒杆菌的生物量分别提高54.7%、50.0%和37.6%;而在高蔗糖胁迫条件下,添加脯氨酸和四氢嘧啶后菌株生物量增加20%以上。进一步研究表明,在高葡萄糖胁迫下,外源添加精氨酸后谷氨酸棒杆菌的葡萄糖利用速率提高约2.5倍,谷氨酸的发酵产量也增加了127.5%。此外,碱性氨基酸对其它4种棒状杆菌也具有一定的渗透保护效应。【结论】精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下具有良好的渗透保护作用,可能归因于其能促进葡萄糖的转运和代谢能力,同时发现碱性氨基酸的渗透保护效应对棒状杆菌属具有一定的普遍性。     

Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine

In microorganisms, members of the binding-protein-dependent ATP-binding cassette transporter superfamily constitute an important class of transport systems. Some of them are involved in osmoprotection under hyperosmotic stress by facilitating the uptake of “compatible solutes”. Currently, the molecular mechanisms used by these transport systems to recognize compatible solutes are limited to transporters specific for glycine betaine and proline betaine. Therefore, this study reports a detailed analysis of the molecular principles governing substrate recognition in the Ehu system from Sinorhizobium meliloti, which is responsible for the uptake of the compatible solutes ectoine and hydroxyectoine. To contribute to a broader understanding of the molecular interactions underlying substrate specificity, our study focused on the substrate-binding protein EhuB because this protein binds the ligand selectively, delivers it to the translocation machinery in the membrane and is thought to be responsible for substrate specificity. The crystal structures of EhuB, in complex with ectoine and hydroxyectoine, were determined at a resolution of 1.9 Å and 2.3 Å, respectively, and allowed us to assign the structural principles of substrate recognition and binding. Based on these results, site-directed mutagenesis of amino acids involved in ligand binding was employed to address their individual contribution to complex stability. A comparison with the crystal structures of other binding proteins specific for compatible solutes revealed common principles of substrate recognition, but also important differences that might be an adaptation to the nature of the ligand and to the demands on protein affinity imposed by the environment.     

Protection of Bacillus subtilis against cold stress via compatible-solute acquisition

Accumulation of compatible solutes is a strategy widely employed by bacteria to achieve cellular protection against high osmolarity. These compounds are also used in some microorganisms as thermostress protectants. We found that Bacillus subtilis uses the compatible solute glycine betaine as an effective cold stress protectant. Glycine betaine strongly stimulated growth at 15°C and permitted cell proliferation at the growth-inhibiting temperature of 13°C. Initial uptake of glycine betaine at 15°C was low but led eventually to the buildup of an intracellular pool whose size was double that found in cells grown at 35°C. Each of the three glycine betaine transporters (OpuA, OpuC, and OpuD) contributed to glycine betaine accumulation in the cold. Protection against cold stress was also accomplished when glycine betaine was synthesized from its precursor choline. Growth of a mutant defective in the osmoadaptive biosynthesis for the compatible solute proline was not impaired at low temperature (15°C). In addition to glycine betaine, the compatible solutes and osmoprotectants l-carnitine, crotonobetaine, butyrobetaine, homobetaine, dimethylsulfonioactetate, and proline betaine all served as cold stress protectants as well and were accumulated via known Opu transport systems. In contrast, the compatible solutes and osmoprotectants choline-O-sulfate, ectoine, proline, and glutamate were not cold protective. Our data highlight an underappreciated facet of the acclimatization of B. subtilis to cold environments and allow a comparison of the characteristics of compatible solutes with respect to their osmotic, heat, and cold stress-protective properties for B. subtilis cells.     

The BCCT family of carriers: from physiology to crystal structure

Increases in the environmental osmolarity are key determinants for the growth of microorganisms. To ensure a physiologically acceptable level of cellular hydration and turgor at high osmolarity, many bacteria accumulate compatible solutes. Osmotically controlled uptake systems allow the scavenging of these compounds from scarce environmental sources as effective osmoprotectants. A number of these systems belong to the BCCT family (betaine-choline-carnitine-transporter), sodium- or proton-coupled transporters (e.g. BetP and BetT respectively) that are ubiquitous in microorganisms. The BCCT family also contains CaiT, an L-carnitine/γ-butyrobetaine antiporter that is not involved in osmotic stress responses. The glycine betaine transporter BetP from Corynebacterium glutamicum is a representative for osmoregulated symporters of the BCCT family and functions both as an osmosensor and osmoregulator. The crystal structure of BetP in an occluded conformation in complex with its substrate glycine betaine and two crystal structures of CaiT in an inward-facing open conformation in complex with L-carnitine and γ-butyrobetaine were reported recently. These structures and the wealth of biochemical data on the activity control of BetP in response to osmotic stress enable a correlation between the sensing of osmotic stress by a transporter protein with the ensuing regulation of transport activity. Molecular determinants governing the high-affinity binding of the compatible solutes by BetP and CaiT, the coupling in symporters and antiporters, and the osmoregulatory properties are discussed in detail for BetP and various BCCT carriers.     

Adaptation of the filamentous fungus Ashbya gossypii to hyperosmotic stress: different osmoresponse to NaCl and mannitol stress

Osmoadaption mechanisms of the biotechnologically important hemiascomycete Ashbya gossypii were investigated, thereby distinguishing between halo- and osmotolerance by exposure to NaCl and mannitol stress. We studied the growth and ultrastructure of differently treated cells and quantified the intracellular contents of compatible solutes and inorganic ions. Mannitol affected growth of A. gossypii at concentrations above 0.8 M, whereas NaCl inhibited growth at 0.2 M. NaCl-treated cells differed from control cells in having smaller vacuoles, which occupied a smaller part of the cell volume. Glycerol was found to be the predominant compatible solute in A. gossypii; accumulation of inorganic ions could not be detected. Measurement of glycerol uptake under isosmotic conditions as well as upon hyperosmotic stress revealed the existence of a highly active glycerol-uptake system, which, however, was down-regulated under hyperosmotic stress. Investigation of glycerol biosynthesis by measuring glycerol-3-phosphate dehydrogenase activity under hyperosmotic conditions indicated that accumulation of glycerol in A. gossypii is almost solely due to biosynthesis. Received: 13 January 1998 / Received revision: 7 April 1998 / Accepted: 13 April 1998     

Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.     

Use of exogenous glycine betaine and its precursor choline as osmoprotectants in Antarctic sea‐ice diatoms1

Wide salinity ranges experienced during the seasonal freeze and melt of sea ice likely constrain many biological processes. Microorganisms generally protect against fluctuating salinities through the uptake, production, and release of compatible solutes. Little is known, however, about the use or fate of glycine betaine (GBT hereafter), one of the most common compatible solutes, in sea‐ice diatoms confronted with shifts in salinity. We quantified intracellular concentrations and used [¹⁴C]‐labeled compounds to track the uptake and fate of the nitrogen‐containing osmolyte GBT and its precursor choline in three Antarctic sea‐ice diatoms Nitzschia lecointei, Navicula cf. perminuta, and Fragilariopsis cylindrus at ?1°C. Experiments show that these diatoms have effective transporters for GBT, but take up lesser amounts of choline. Neither compound was respired. Uptake of GBT protected cells against hyperosmotic shock and corresponded with reduced production of extracellular polysaccharides in N. lecointei cells, which released 85% of the retained GBT following hypoosmotic shock. The ability of sea‐ice diatoms to rapidly scavenge and release compatible solutes is likely an important strategy for survival during steep fluctuations in salinity. The release and recycling of compatible solutes may play an important role in algal–bacterial interactions and nitrogen cycling within the semi‐enclosed brines of sea ice.