The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus

The glycine betaine carrier BetP of Corynebacterium glutamicum was recently shown to function both as an osmosensor and as an osmoregulator in proteoliposomes by sensing changes in the internal K(+) concentration as a measure of hyperosmotic stress. In vivo analysis of mutants carrying deletions at the C-terminal extension of BetP indicated that this domain participates in osmostress-dependent activity regulation. To address the question, whether a putative K(+) sensor is located within the C-terminal domain, several mutants with truncations in this domain were purified and reconstituted in proteoliposomes of Escherichia coli phospholipids, since this in vitro system allowed variation of the K(+) concentration at the lumenal side. Truncation of 12 amino acids led to a partly deregulated BetP in terms of osmoregulation; however, K(+) sensitivity was not impaired in this mutant. The deletion of 25 amino acid residues at the C-terminal end of BetP led to both deregulation of the carrier activity, i.e., high activity independent of external osmolality, and loss of K(+)-dependent transport stimulation, indicating that this region of the C-terminal domain is necessary for K(+) sensing and/or K(+)-dependent carrier activation. Immunological and proteolysis analyses showed that BetP and its recombinant forms were reconstituted in a right-side-out orientation, i.e., the C-terminal domain faces the lumen of the proteoliposomes and is thus able to detect the K(+) signal at the inside. This is the first experimental demonstration of a direct connection between an osmotic stimulus, i.e., the change in internal K(+), and a putative sensor domain.     

Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum

The glycine betaine carrier BetP from Corynebacterium glutamicum was recently shown to function as both an osmosensor and osmoregulator in proteoliposomes made from Escherichia coli phospholipids by sensing changes in the internal K+ concentration as a measure of hyperosmotic stress (Rübenhagen, R., Morbach, S., and Kr?mer, R. (2001) EMBO J. 20, 5412-5420). Furthermore, evidence was provided that a stretch of 25 amino acids of the C-terminal domain of BetP is critically involved in K+ sensing. This K+-sensitive region has been further characterized. Glu572 turned out to be important for osmosensing in E. coli cells and in proteoliposomes made from E. coli phospholipids. BetP mutants E572K, E572P, and E572A/H573A/R574A were unable to detect an increase in the internal K+ concentration in this membrane environment. However, these BetP variants regained their ability to detect osmotic stress in membranes with increased phosphatidylglycerol content, i.e. in intact C. glutamicum cells or in proteoliposomes mimicking the composition of the C. glutamicum membrane. Mutants E572P and Y550P were still insensitive to osmotic stress also in this membrane background. These results led to the following conclusions. (i) The K+ sensor in mutants E572Q, E572D, and E572K is only partially impaired. (ii) Restoration of activity regulation is not possible if the correct conformation or orientation of the C-terminal domain is compromised by a proline residue at position 572 or 550. (iii) Phosphatidylglycerol in the membrane of C. glutamicum seems to stabilize the inactive conformation of BetP C252T and other mutants.     

Cation specificity of osmosensing by the betaine carrier BetP of Corynebacterium glutamicum

The Na(+)/betaine carrier BetP from Corynebacterium glutamicum was purified and reconstituted in Escherichia coli phospholipid liposomes and its osmosensory properties were studied with respect to the cation specificity of osmotic activation. To dissect the influence of the co-substrate Na(+) on the energetics of uptake from its possible role as a putative trigger of osmolality-dependent BetP activation, the internal Na(+) concentration was varied without changing DeltapNa(+). Studying betaine uptake at increasing luminal Na(+) or K(+) revealed that BetP activity was triggered by Na(+) only to a negligible extent compared to activation by K(+). We conclude that activation of BetP in proteoliposomes depends solely on K(+), both in mechanistic and in physiological terms.     

Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes

The secondary glycine betaine uptake system BetP of Corynebacterium glutamicum was purified from Escherichia coli membranes in strep-tagged form after heterologous expression of the betP gene and was reconstituted in E. coli lipids. BetP retained its kinetic properties (V(max) and K(m) for betaine and Na(+)) as compared with intact cells. The influence of driving forces (Na(+) gradient and/or electrical potential) on betaine uptake was quantified in proteoliposomes. BetP was effectively regulated by the external osmolality and was stimulated by the local anesthetic tetracaine. A shift of the optimum of osmotic stimulation to higher osmolalities was linearly correlated with an increasing share of phosphatidyl glycerol, the major lipid of the C. glutamicum plasma membrane in the E. coli lipid proteoliposomes. This finding correlates with results demonstrating an identical shift when betP was expressed in E. coli instead of C. glutamicum. These data indicate that (i) BetP comprises all elements of osmosensing and osmoregulatory mechanisms of betaine uptake, (ii) osmoregulation of BetP is directly related to protein/membrane interactions, (iii) the turgor pressure presumably plays no major role in osmoregulation of BetP, and (iv) the regulatory properties of BetP may be related to the physical state of the surrounding membrane.     

Structural asymmetry in a trimeric Na+/betaine symporter, BetP, from Corynebacterium glutamicum

The Na⁺-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.     

Conformational changes of the betaine transporter BetP from Corynebacterium glutamicum studied by pulse EPR spectroscopy

The betaine transporter BetP from Corynebacterium glutamicum is activated by hyperosmotic stress critically depending on the presence and integrity of its sensory C-terminal domain. The conformational properties of the trimeric BetP reconstituted in liposomes in the inactive state and during osmotic activation were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Comparison of intra- and intermolecular inter spin distance distributions obtained by double electron-electron resonance (DEER) EPR with the crystal structure of BetP by means of a rotamer library analysis suggest a rotation of BetP protomers within the trimer by about 15° as compared to the X-ray structure. Furthermore, we observed conformational changes upon activation of BetP, which are reflected in changes of the distances between positions 545 and 589 of different protomers in the trimer. Introduction of proline at positions 550 and 572, both leading to BetP variants with a permanent (low level) transport activity, caused changes of the DEER data similar to those observed for the activated and inactivated state, respectively. This indicates that not only displacements of the C-terminal domain in general but also concomitant interactions of its primary structure with surrounding protein domains and/or lipids are crucial for the activity regulation of BetP.     

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.     

Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum

The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum, was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in beta-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E.coli polar lipids produced 2D crystals with unit cell parameters of 182A x 154A, gamma=90 degrees exhibiting p22(1)2(1) symmetry. Electron cryo-microscopy yielded a projection map at 7.5A. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane alpha-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.     

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.     

Regulatory properties and interaction of the C- and N-terminal domains of BetP, an osmoregulated betaine transporter from Corynebacterium glutamicum

The glycine betaine carrier BetP from Corynebacterium glutamicum responds to changes in external osmolality by regulation of its transport activity, and the C-terminal domain was previously identified to be involved in this process. Here we investigate the structural requirements of the C-terminal domain for osmoregulation as well as interacting domains that are relevant for intramolecular signal transduction in response to osmotic stress. For this purpose, we applied a proline scanning approach and amino acid replacements other than proline in selected positions. To analyze the impact of the surrounding membrane, BetP mutants were studied in both C. glutamicum and Escherichia coli, which strongly differ in their phospholipid composition. A region of approximately 25 amino acid residues within the C-terminal domain with a high propensity for alpha-helical structure was found to be essential in terms of its conformational properties for osmodependent regulation. The size of this region was larger in E. coli membranes than in the highly negatively charged C. glutamicum membranes. As a novel aspect of BetP regulation, interaction of the C-terminal domain with one of the cytoplasmic loops as well as with the N-terminal domain was shown to be involved in osmosensing and/or osmoregulation. These results support a functional model of BetP activation that involves the C-terminal domain shifting from interaction with the membrane to interaction with intramolecular domains in response to osmotic stress.     

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.     

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.     

The role of lipids and salts in two-dimensional crystallization of the glycine-betaine transporter BetP from Corynebacterium glutamicum

The osmoregulated and chill-sensitive glycine–betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for 2D crystallization were compared. Only the use of lipids extracted from C. glutamicum cells led to the formation of large, well-ordered crystalline areas. To understand the lipid-derived influence on crystallinity, lipid extracts from different stages of the crystallization process were analyzed by quantitative multiple-precursor ion scanning mass spectroscopy (MS). Results show that BetP has a preference for fatty acid moieties 16:0–18:1, and that a phosphatidyl glycerol (PG) 16:0–18:1 rich preparation prevents formation of pseudo crystals.     

Isoleucine excretion in Corynebacterium glutamicum: evidence for a specific efflux carrier system

Summary Corynebacterium glutamicum effectively secretes isoleucine when the precursor 2-ketobutyrate is added to the medium. Isoleucine secretion was studied under different conditions with respect to various parameters, i.e. rate of isoleucine excretion and uptake, concentration gradients of isoleucine, other amino acids and ions, and membrane potential. By comparing these parameters in the presence and absence of the amino acid precursor it has been shown that the efflux of isoleucine in C. glutamicum can neither be explained by a passive diffusion mechanism nor by a process involving functional inversion of the isoleucine uptake carrier. Based on our results concerning the distribution of metabolites and the kinetics of excretion we conclude that isoleucine is excreted in C. glutamicum by a separate, presumably active efflux carrier system.     

Structure of a GTP-dependent bacterial PEP-carboxykinase from Corynebacterium glutamicum

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2₁ with four molecules per asymmetric unit. The 2.3 Å resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.     

The role of Ca2+ ions in a phage infection of Corynebacterium glutamicum

It was shown that neither uncouplers of oxidative phosphorylation, nor lack of Ca2+ ions affected the normal MC-2 phage absorption on Corynebacterium glutamicum cells, while the phage development was repressed under these conditions. Simultaneous measurement of Ca2+, K+ and H+ ion flows, as well as measurement of membrane potential showed that the addition of the phage into the experimental medium led to significant depolarization of the membrane from -160 mV to -100 mV due to the penetration of Ca2+ ions into the cells followed by K+ and H+ efflux. The (Ca2+) to (K+ + H+) ratio was shown to be 1 : 1. Phage DNA is supposed to be injected into the host cells as a positively charged (Ca2+-DNA) complex.     

Corynebacterium glutamicum superoxide dismutase is a manganese-strict non-cambialistic enzyme in vitro

Superoxide dismutase (SOD) of Corynebacterium glutamicum was purified and characterized. The enzyme had a native molecular weight of about 80kDa, whereas a monomer with molecular weight of 24kDa was found on SDS-PAGE suggesting it to be homotetramer. The native SOD activity stained gel revealed a unique cytosolic enzyme. Supplementing growth media with manganese increased the specific activity significantly, while adding iron did not result in significant difference. No growth perturbation was observed with the supplemented media. In vitro metal removal and replacement studies revealed conservation of about 85% of the specific activity by substitution with manganese, while substitution with copper, iron, nickel or zinc did not restore any significant specific activity. Manganese was identified by atomic absorption spectrometer, while no signals corresponding to fixing other metallic elements were detected. Thus, C. glutamicum SOD could be considered a strict (non-cambialistic) manganese superoxide dismutase (MnSOD).     

Lysine excretion by Corynebacterium glutamicum. 1. Identification of a specific secretion carrier system

Corynebacterium glutamicum effectively excretes lysine when the internal lysine concentration is elevated. Lysine efflux was investigated using selected mutants which are not able to regulate lysine biosynthesis by feedback inhibition. Secretion of lysine is not the consequence of unspecific permeability of the plasma membrane but is mediated by a secretion carrier which is specific for lysine. Lysine export is characterized by high activation energy and follows Michaelis-Menten type kinetics with an internal Km of 20 mM and a Vmax of 12 nmol.min-1.mg dry cells-1. Excretion can proceed against a preexisting chemical gradient and against the electrical potential, which rules out a previously suggested pore model. Lysine excretion can also be observed in the wild-type strain especially under conditions of peptide uptake. Its possible physiological function may be related to regulation of internal amino acid concentrations under special growth conditions.     

Glutamate is excreted across the cytoplasmic membrane through the NCgl1221 channel of Corynebacterium glutamicum by passive diffusion

The NCgl1221 gene, which encodes a mechanosensitive channel, has been reported to be critically involved in glutamate (Glu) overproduction by Corynebacterium glutamicum, but direct evidence of Glu excretion through this channel has not yet been provided. In this study, by electrophysiological methods, we found direct evidence of Glu excretion through this channel by passive diffusion. We found that the introduction into Phe-producing Escherichia coli of mutant NCgl1221 genes that induce Glu overproduction by C. glutamicum improved productivity. This suggests a low-substrate preference of this channel, indicates its potential as a versatile exporter, and more broadly, indicates the potential of exporter engineering.