UniversityFacultyInstitute
Prof. Reinhard Krämer

Prof. Reinhard Krämer

tel.: 0221 / 470 64 64

fax.: 0221 / 470 50 91

email.: r.kraemer"at"uni-koeln.de

Stress Response and Signal Transduction in Corynebacterium glutamicum

Prof. Dr. Reinhard Krämer, Dr. S. Nicklisch, K. Börngen, S. Maksimov, E. Glees, U. Meyer

The pictures on this page are taken from recent presentations or lectures. Please feel free to contact us for further information.

Scientific Background

Soil bacteria, such as Corynebacterium glutamicum, possess efficient mechanisms to cope with many different types of external stress factors, e.g. osmotic stress, heat stress, chill stress, and others. A major external stress in the natural habitat, i.e. the soil, is osmotic stress due to high external osmolality causing dehydration (hyperosmotic stress) or low external solute concentration causing massive water influx (hypoosmotic stress). To cope with hyperosmotic stress, C. glutamicum accumulates so-called compatible solutes by uptake or by synthesis thereby increasing the intracellular osmolality. The response to osmotic stress can be divided into several aspects.

  1. Physical stimuli related to osmotic stress have to be somehow sensed by the bacterial cell (stimulus perception, osmosensing).
  2. Stimuli related to osmotic stress have to be transformed into intracellular signals (signal tansduction).
  3. Signal transduction leads within very short periods of time (ms to s) to cellular responses at the level of protein activity (osmoregulation).
  4. In parallel to these events at the the level of protein activity, signals related to osmotic stress cause alterations of transcription and translation (osmoregulation) of specific genes, as well as modulation of RNA and/or protein stability.
    For a fine-tuned physiological response, signal transduction and integration is necessary for regulation at all different levels of protein activity, gene expression and posttranslational modification.

    AIMS

    • Molecular understanding of osmosensing and activity regulation of osmoregulated secondary carriers for compatible solutes.
    • Molecular dissection of osmosensing by two component sensory systems.
    • Osmostress dependent regulation of transport and biosynthesis processes at different levels (protein activity, transcription, translation, posttranslational modification).
    • Physiological understanding of osmoregulation and cold shock regulation of a soil bacterium.
    • Structure-function relation of transport systems for compatible solutes during sensing and regulation events.
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Selected Projects

Analysis of osmosensing and osmoregulation by recombinant betaine carrier (BetP) proteins.

The physiological response of C. glutamicum to hyperosmotic stress is investigated by using a combination of biochemical, molecular and biotechnological techniques. We have characterized five uptake systems (BetP, EctP, ProP, LcoP, PutP) and biosynthetic pathways (proline, trehalose) for compatible solutes, which are involved in the response to hyperosmotic stress. Within this ensemble of carriers, in particular the most active and highly regulated betaine transporter BetP is studied in depth. The characterization of wild type C. glutamicum and deletion mutants revealed that the uptake of betaine, ectoine and proline is preferred over the synthesis of trehalose or proline. The various systems are redundant, since the loss of either all uptake systems or of the biosynthetic pathway for trehalose or proline has no significant influence on the viability of the cells upon osmotic stress. Expression profiling with respect to osmodependent genes (RNA hybridization, Northern hybridization, RT-PCR, RACE-PCR) and of the global response to osmotic stress was used to obtain a better understanding of the signal transduction cascade related to osmotic challenge. Two corynebacterial carriers, BetP and EctP, are mainly responsible for the uptake of betaine after a hyperosmotic shock. Both are regulated at the level of activity and in each case the hydrophilic N- and C-terminal domains of these transporters are involved in activity regulation. The activity of BetP was shown to be modulated by both the concentration of cytoplasmic potassium as well as by the nature of the surrounding phospholipid membrane (see also projects 3 and 4). Beside its catalytic (transport) activity BetP is shown to harbor also osmosensing and osmoregulatory functions. The functional properties of the purified carriers (e.g. BetP) as well as recombinant transporter forms are characterized by using reconstitution in proteoliposomes, transport measurements and spectroscopic methods like CD, SPR (surface plasmon resonance), and EPR (electron paramagnetic resonance). The combination of activity data (sensing properties) and spectroscopy (structural data) is used to obtain more information on osmosensing and osmoregulation.

Project 1_1 Project 1_2

Fig. 1: Membrane topology of BetP and factors affecting BetP activity: (1) two, N- and C-terminal (regulatory) domains, (2) concentration of internal (cytoplasmic) K+, (3) membrane surface charge.

Fig. 2: Activity regulation of wildtype BetP and recombinant carrier proteins in dependence of the external osmolality. The C-terminal truncation mutant has lost its regulatory dependence on osmotic stress.

BetP comprises both catalytic and regulatory domains. The state of activity of BetP is influenced by (1) the presence and integrity of the regulatory terminal domains, (2) the concentration of cytoplasmic K+, which represents the physiological trigger related to hyperosmotic stress, and (3) the surface charge of the surrounding membrane (Fig. 1). Truncations indicate regulatory functions of the terminal domains, leading to modulation (N-terminal truncation) as well as loss (C-terminal domain) of the regulatory response to hyperosmotic stress (Fig. 2).


Analysis of osmosensing and signal transduction by the two component system MtrBA from C. glutamicum.

C. glutamicum harbors 13 different two component systems and one of them (MtrBA) is directly related to transcription regulation of target genes involved in the response to hyperosmotic stress. Two component systems are in general comprised of a membrane bound sensor histidine kinase and a soluble response regulator protein located in the cytoplasm. A physical stimulus, related to a particular external stress factor, is percieved by the sensor kinase and transduced to an intracellular signal transduction cascade via (auto)phosphorylation of the sensor kinase, (trans)phosphorylation of the response regulator, and regulatory interaction with specific binding sites near target genes at the DNA.
We are interested in the kind of physical stimulus/stimuli related to external (osmotic) stress which is perceived by the sensor kinase MtrB as well as on the intramolecular signal transduction to the response regulator MtrA. For this purpose, MtrB and MtrA are isolated, purified and MtrB is reconstituted into proteoliposomes. Sensing and signal transduction is then analyzed both in vitro (proteoliposomes), and in vivo (intact cells) by using wild type and recombinant proteins. We are interested in the molecular understanding of stimulus perception and signal transduction of the MtrBA system, which is also compared to mechanism of other two component systems not involved in osmoregulation (e.g. DcuRS of E. coli), and other osmoresponsive systems (e.g. BetP of C. glutamicum).

Project 2_1 Project 2_2

Fig. 3: Structural model and intramolecular signal transduction by the two component system MtrBA from C. glutamicum.

Fig. 4: Signal transduction capacity of wild type and recombinant forms of MtrBA lacking either the transmembrane part (A) or the external loop (B).


Analysis of chill sensing and regulation by the betaine transporter BetP from C. glutamicum.

Besides osmotic stress, we are also interested in the response of C. glutamicum to chill stress both in terms of mechanism of signal transduction and of physiological significance of the stress response. C. glutamicum was found to respond to chill stress at several levels of regulation, namely (1) protein activity (instant response), (2) transcription regulation of target genes, and (3) complete rearrangement of plasme membrane composition.
The response of C. glutamicum at all three levels of regulation is currently investigated.

  1. We found that osmoresponsive carrier proteins react to exposure to low temperatures in a specific way, leading to significant activation of the betaine carrier BetP around 10 – 15°C, whereas other carrier proteins are not or not significantly activated.
  2. Transcription of genes related to the response to chill stress in C. glutamicum is regulated via the two-component system MtrBA.
  3. When grown at different temperatures, C. glutamicum adapts the lipid composition of its plasma membrane to the altered conditions. Both the distribution of fatty acids and of phospholipid head groups varies significantly in response to varying growth temperatures, which in turn strongly influences the activity regulation of the betaine carrier BetP.

    These investigations further indicate a strong dependence of the functional properties of BetP on the physical state of the surrounding phospholipid membrane (see also projects 1 and 4, description of cell wall composition of C. glutamicum).

    Project 3_1 Project 3_2

    Fig. 5: Response of the three carriers for compatible solutes BetP, EctP and LcoP to chill stress in C. glutamicum. The betaine carrier BetP is activated at low temperature.

    Fig. 6: Variation of lipid composition (fatty acids (18:1 and 16:0) and phospholipid headgroups (PG, PI and CL)) of the C. glutamicum plasma membrane (see figure in project 4) in response to temperature variation during cell growth.


    Significance of cell wall composition for physiological and biotechnological aspects of C. glutamicum.

    The cell wall of the grampositive bacterium C. glutamicum is a complicated structure comprising layers of peptidoglycan and arabinogalactan as well as a mycolic acid double layer representing an additional permeability barrier besides the plasma membrane, which resembles the situation in gramnegative bacteria. For this reason, C. glutamicum (and related organisms) harbor porin proteins, similar as observed in gramnegative bacteria. Mycolates, the presence of which in the cell wall is characteristic for bacteria from the genus Corynebacteriaceae, e.g. Corynebacteria or Mycobacteria, are synthesized from mycolic acids and trehalose, consequently, trehalose is an important compound of the cell wall in these organisms. Besides this function, trehalose may in addition be used in C. glutamicum as compatible solute in response to hyperosmotic stress under particular metabolic conditions. We are interested in several aspects of cell wall function and biosynthesis in C. glutamicum.

    1. Enzymes and pathways involved in the biosynthesis of mycolates from trehalose and mycolic acids.
    2. The dual role of trehalose for both cell wall biosynthesis and osmoprotection.
    3. Significance of cell wall intactness for transmembrane solute transport, both with respect to basic (cell wall structure) and applied aspects (amino acid production).
    4. The particular connection between interference with cell wall structure and glutamate excretion.
    5. The basic significance of the physical state of the plasma membrane, possibly related to the structure of the cell wall, for the functional activity of membrane embedded transport proteins (see also projects 1 and 3).

      Project 4_1 Project 4_2

      Fig. 7: Structure of cell envelope (cell wall and plasma membrane) of C. glutamicum containing phospholipids (violet color) and mycolates (orange color) as well as transport proteins in the plasma membrane, e.g. BetP.

      Fig. 8: Three different possible pathways for the biosynthesis, resp. metabolism of trehalose in C. glutamicum (TreYZ, OtsAB and TreS).

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Funding

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Cooperation

  • Prof. E. Agosin, Universidad de Santiago, Chile
  • Prof. K. Altendorf, Universität Osnabrück
  • Prof. E. Bremer, Universität Marburg
  • Prof. R. Benz, Universität Würzburg
  • Prof. M. Bott, Forschungszentrum Jülich
  • Prof. M. Daffe, Institut de Pharmacologie et de Biologie Structurale, France
  • Prof. Dr. S. Hoening, Universität zu Köln
  • Prof. Dr. K. Jung, Ludwig-Maximilian-Universität München
  • Prof. W. Mäntele, Universität Frankfurt
  • Prof. B. Martinac, University of Queensland, Brisbane (Australien)
  • Dr. A. Shevchenko, C. Ejsing, MPI f. Molekulare Zellbiologie, Dresden
  • Prof. Dr. L. Schmitt, Heinrich-Heine-Universität Düsseldorf
  • Prof. Dr. H.-J. Steinhoff, Dr. I. Borovykh, Universität Osnabrück
  • Prof. Dr. R. Tampé, Dr. J. Koch Universität Frankfurt
  • Prof. J. Wood, University of Guelph (Kanada)
  • Dr. C. Ziegler, Prof. W. Kühlbrandt, MPI f. Biophysik, Frankfurt
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Selected Papers

  • Möker, N., Kramer, J., Unden, G., Krämer, R. und Morbach, S. (2007) In vitro analysis of the two-component system MtrB-MtrA from Corynebacterium glutamicum. J. Bacteriol., [Epub ahead of print] [Abstract]

  • Schiller, D., Ott, V., Krämer R. und Morbach, S. (2006) Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum. J. Biol. Chem. 281, 7737-7746 [Abstract]

  • Tropis, M., Meniche, X., Wolf, A., Gebhardt, H., Strelkov, S., Chami, M., Schomburg, D., Krämer, R., Morbach, S. und Daffe, M. (2005) The crucial role of trehalose and structurally related oligosaccharides in the biosynthesis and transfer of mycolic acids in Corynebacterineae. J. Biol. Chem. 280, 26573-26585 [Abstract]

  • Morbach, S. und Krämer, R. (2005) Structure and function of the betaine uptake system BetP of Corynebacterium glutamicum: strategies to sense osmotic and chill stress. J. Mol. Microbiol. Biotechnol. 10, 143-153 [Abstract]

  • Özcan N., Krämer, R. und Morbach, S. (2005) Chill activation of compatible solute transporters in Corynebacterium glutamicum at the level of transport activity. J. Bacteriol., 187, 4752-4759 [Abstract]

  • Morbach S. und Krämer, R. (2005) Osmoregulation. In: Handbook of Corynebacterium glutamicum. Eggeling, L. und Bott, M. (Ed.), CRC-Press

  • Möker, N., Brocker, M., Schaffer, S., Krämer, R., Morbach, S. und Bott, M. (2004) 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. Mol. Microbiol. 54, 420-438 [Abstract]

  • Krämer, R. und Morbach, S. (2004) BetP of Corynebacterium glutamicum, a transporter with three different functions: betaine transport, osmosensing, and osmoregulation. Biochim. Biophys. Acta 1658, 31-36 [Abstract]

  • Schiller, D., Krämer R. und Morbach, S. (2004) The C-terminal domain of the betaine carrier BetP of Corynebacterium glutamicum is directly involved in sensing K+ as an osmotic stimulus. Biochemistry 43, 5583-5591 [Abstract]

  • Wolf, A., Krämer, R. and S. Morbach (2003) Three different pathways for trehalose metabolism in Corynebacterium glutamicum and their significance in response to osmotic stress. Mol. Microbiol. 49, 1119-1134 [Abstract]

  • Morbach S. and R. Krämer (2002) Body shaping under water stress: Osmosensing and osmoregulation of solute transport in bacteria. Chembiochem 3(5): 384-397 [Abstract]

  • Rübenhagen R, Morbach S, Krämer R (2001). The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K+. EMBO J. 20, 5412-5420. [Abstract]

  • Rübenhagen R, Rönsch H, Jung H, Krämer R, Morbach S, (2000). Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes. J. Biol. Chem. 275, 735-741. [Abstract]

  • complete list

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