The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation.

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichiacoli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress.

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.

Osmosensing properties of the histidine protein kinase MtrB from Corynebacterium glutamicum.

The MtrB-MtrA two component system of Corynebacterium glutamicum was recently shown to be in involved in the osmostress response as well as cell wall metabolism. To address the question of whether the histidine protein kinase MtrB is an osmosensor, the kinase was purified and reconstituted into liposomes in a functionally active form. The activity regulation was investigated by varying systematically physicochemical parameters, which are putative stimuli that could be used by the bacterial cell to detect osmotic conditions. Membrane shrinkage was ruled out as a stimulus for activation of MtrB. Instead, MtrB was shown to be activated upon the addition of various chemical compounds, like sugars, amino acids, and polyethylene glycols. Because of the different chemical nature of the solutes, it seems unlikely that they bind to a specific binding site. Instead, they are proposed to act via a change of the hydration state of the protein shifting MtrB into the active state. For MtrB activation it was essential that these solutes were added at the same side as the cytoplasmic domains of the kinase were located, indicating that hypertonicity is sensed by MtrB via cytoplasmatically located protein domains. This was confirmed by the analysis of two MtrB mutants in which either the large periplasmic loop or the HAMP domain was deleted. These mutants were regulated similar to wild type MtrB. Thus, we postulate that MtrB belongs to a class of histidine protein kinases that sense environmental changes at cytoplasmatic protein domains independently of the periplasmic loop and the cytoplasmic HAMP domain.

The key role of the mycolic acid content in the functionality of the cell wall permeability barrier in Corynebacterineae.

Recently, it has been shown that trehalose and mycolic acids are essential for the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium smegmatis, and important but not indispensable to the survival of Corynebacterium glutamicum. Therefore, to investigate the function of mycolic acids in both the permeability of the cell wall to small nutrients and antibiotics, and the excretion of amino acids by C. glutamicum, a trehalose-deficient mutant of the L-lysine producer ATCC 21527, designated LP Delta treS Delta otsA Delta treY, was constructed. By using different carbon sources in either the presence or the absence of external trehalose, a set of endogenously trehalose-free LP Delta treS Delta otsA Delta treY cells that exhibited various mycolate contents was generated. The results showed that the structure of the arabinogalactan of these different cell types of LP Delta treS Delta otsA Delta treY was not affected when the mycolic acid layer was either missing or impaired. Nevertheless, cells were more susceptible to antibiotics, and the permeability of their cell walls to glycerol was increased. Interestingly, a concomitant increase in the excretion of both L-lysine and L-glutamate was also observed, indicating that the mycolic acid content of the permeability barrier (and not only the peptidoglycan and/or the arabinogalactan) is implicated in the glutamate excretion process.

Characterization of compatible solute transporter multiplicity in Corynebacterium glutamicum.

The soil bacterium Corynebacterium glutamicum is efficiently protected against hyperosmotic stress by a high redundancy of uptake systems and biosynthesis pathways for compatible solutes. We have previously identified and analyzed four osmoregulated uptake systems for betaine, ectoine, and proline. Because of overlapping substrate specificities, it is not possible to quantify their individual contribution to the stress response in wild-type cells. Using a set of strains in which only one uptake system for compatible solutes is present, we investigated the expression regulation at their transcriptional and translational level. The carrier ectP was found to be regulated at the level of transcription, but the already high maximal uptake capacity of approx. 30 nmol/(min mg cell dry mass, cdm) was not further elevated if the medium osmolality was severely increased, indicating that the amount of EctP is not changed. Thus, EctP may represent the rescue system for C. glutamicum. The betP, lcoP, and proP genes were induced upon hyperosmotic conditions, resulting in a 3-10-fold increase of their transport activity. These systems are thus used to fine-tune the uptake capacity for compatible solutes to the actual demands of the cell. ProP represents the most strongly regulated compatible solute uptake system in C. glutamicum.

In vitro analysis of the two-component system MtrB-MtrA from Corynebacterium glutamicum.

The two-component system MtrBA is involved in the osmostress response of Corynebacterium glutamicum. MtrB was reconstituted in a functionally active form in liposomes and showed autophosphorylation and phosphatase activity. In proteoliposomes, MtrB activity was stimulated by monovalent cations used by many osmosensors for the detection of hypertonicity. Although MtrB was activated by monovalent cations, they lead in vitro to a general stabilization of histidine kinases and do not represent the stimulus for MtrB to sense hyperosmotic stress.

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 Krämer, 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.

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.

The crucial role of trehalose and structurally related oligosaccharides in the biosynthesis and transfer of mycolic acids in Corynebacterineae.

Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.

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 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.

Deletion of the genes encoding the MtrA-MtrB two-component system of Corynebacterium glutamicum has a strong influence on cell morphology, antibiotics susceptibility and expression of genes involved in osmoprotection.

The MtrAB two-component signal transduction system is highly conserved in sequence and genomic organization in Mycobacterium and Corynebacterium species, but its function is completely unknown. Here, the role of MtrAB was studied with C. glutamicum as model organism. In contrast to M. tuberculosis, it was possible to delete the mtrAB genes in C. glutamicum. The mutant cells showed a radically different cell morphology and were more sensitive to penicillin, vancomycin and lysozyme but more resistant to ethambutol. In order to identify the molecular basis for this pleiotropic phenotype, the mRNA profiles of mutant and wild type were compared with DNA microarrays. Three genes showed a more than threefold increased RNA level in the mutant, i.e. mepA (NCgl2411) encoding a putative secreted metalloprotease, ppmA (NCgl2737 ) encoding a putative membrane-bound protease modulator, and lpqB encoding a putative lipoprotein of unknown function. Expression of plasmid-encoded mepA in Escherichia coli led to elongated cells that were hypersensitive to an osmotic downshift, supporting the idea that peptidoglycan is the target of MepA. The mRNA level of two genes was more than fivefold decreased in the mutant, i.e. betP and proP which encode transporters for the uptake of betaine and proline respectively. The microarray results were confirmed by primer extension and RNA dot blot experiments. In the latter, the transcript level of genes involved in osmoprotection was tested before and after an osmotic upshift. The mRNA level of betP, proP and lcoP was strongly reduced or undetectable in the mutant, whereas that of mscL (mechanosensitive channel) was increased. The changes in cell morphology, antibiotics susceptibility and the mRNA levels of betP, proP, lcoP, mscL and mepA could be reversed by expression of plasmid-encoded copies of mtrAB in the DeltamtrAB mutant, confirming that these changes occurred as a consequence of the mtrAB deletion.

LcoP, an osmoregulated betaine/ectoine uptake system from Corynebacterium glutamicum.

In Corynebacterium glutamicum, four uptake systems for compatible solutes have been characterized so far. DHPE (DeltabetPDeltaputPDeltaproPDeltaectP), a derivative of the C. glutamicum type strain ATCC 13032 carrying deletions in the corresponding genes, still showed a low betaine uptake rate of 1.4 nmol/(min mg cdm). Genome analyses revealed the presence of a putative carrier, named low capacity osmoregulated permease (LcoP), which shows similarities to compatible solute transporters of the betaine/carnitine/choline transporter (BCCT)-family. Deletion of lcoP in DHPE resulted in betaine and ectoine uptake deficiency. LcoP, a betaine and ectoine permease is regulated at the expression and the activity level by the external osmolality. Addition of local anesthetics modulated the activity of BCCT-family members BetP, EctP, and LcoP in a different manner, indicating a different type of lipid-protein interaction.

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.

Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

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.

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.

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.

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.