Susanne Morbach

Three pathways for trehalose metabolism in Corynebacterium glutamicum ATCC13032 and their significance in response to osmotic stress

Genome scanning of Corynebacterium glutamicum ATCC13032 revealed the presence of five different genes encoding enzymes belonging to three putative trehalose biosynthesis pathways (OtsAB, TreYZ, TreS). The function of the different pathways and of trehalose as an osmoprotectant was studied by characterizing several strains defective for individual trehalose biosynthetic routes. Trehalose synthesis was shown to increase upon hyperosmotic conditions. Cytoplasmic trehalose levels varied considerably depending on kind and accessibility of carbon and nitrogen sources. In contrast to other organisms, osmoregulated trehalose synthesis in C. glutamicum is mediated by the TreYZ and not by the OtsAB pathway. Irrespective of their significance for the osmotic response, otsA and treS were upregulated at the transcriptional level after hyperosmotic shock. In vivo, TreS‐mediated trehalose synthesis only occurred if maltose was used as the carbon source. In vitro, TreS catalysed the conversion of maltose into trehalose and, conversely, trehalose into maltose. As the reaction seems to be near equilibrium, TreS appears to be important for trehalose degradation rather than synthesis because a 1000‐fold excess of trehalose to maltose was detected in the cytoplasm. Also, evidence is given that both the OtsAB and the TreYZ pathways are involved, but not essential, in supplying trehalose for mycolic acid biosynthesis.

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 ΔmtrAB mutant, confirming that these changes occurred as a consequence of the mtrAB deletion.

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 fromEscherichia 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 theC. glutamicum plasma membrane in the E. colilipid proteoliposomes. This finding correlates with results demonstrating an identical shift when betP was expressed inE. 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.

The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K

The isolated glycine betaine uptake carrier BetP from Corynebacterium glutamicum was reconstituted in Escherichia coli phospholipid liposomes and its response to osmotic stress studied. The transport activity of BetP, which was previously shown to comprise both osmosensory and osmoregulatory functions, was used to identify the nature of the physicochemical stimulus related to hyperosmotic stress. Putative factors modulating transport activity in response to osmotic stress were dissected. These include type, osmolality and concentration of solutes in the internal and/or external compartment (cationic, anionic, zwitterionic, neutral), as well as membrane strain as a response to increased osmolality. Osmoresponsive activation of BetP was independent of any external factor and of physical alterations of the membrane, but was triggered by a change in the internal K+ concentration. Activation did not depend on the type of anion present and was K+ (or Cs+ and Rb+) specific, as choline and NH4+ did not trigger BetP activity. The half‐maximal activation of BetP in E.coli phospholipid liposomes was correlated to an internal concentration of 221 ± 23 mM K+.

Body Shaping under Water Stress: Osmosensing and Osmoregulation of Solute Transport in Bacteria

Fluctuation of external osmolarity is one of the most common types of environmental stress factors for all kind of cells, both of prokaryotic and of eukaryotic origin. Cells try to keep their volume and/or turgor pressure constant; consequently, both a decrease (hypoosmotic stress) and an increase (hyperosmotic stress) of the solute concentration (correctly: increase or decrease in water activity) in the surrounding area, respectively, are challenges for cellular metabolism and survival. A common example from the prokaryotic world is the fate of a soil bacterium that, after a sunny day has dried out the soil (hyperosmotic stress), is suddenly exposed to a drop of distilled water from a rain cloud (hypoosmotic stress). The immediate and inevitable passive response to the sudden osmotic shift in the surroundings is fast water efflux out of the cell in the former situation and water influx in the latter. In the worst case, these responses may lead to either loss of cell turgor and plasmolysis or to cell burst. In order to overcome such drastic consequences cells have developed effective mechanisms, namely osmoadaptation, to cope with the two different types of osmotic stress. For a graded reaction to osmotic shifts, cells must be able (1) to sense stimuli related to osmotic stress, (2) to transduce corresponding signals to those systems that properly respond (3) by activating transport or enzymatic functions or (4) by changing gene expression profiles. In this review, membrane proteins involved in the cells active response to osmotic stress are described. Molecular details of structure, function, and regulation of mechanosensitive efflux channels from various organisms, as well as of osmoregulated uptake systems are discussed.

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.

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.

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.

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.

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.