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.

[1]  R. Krämer,et al.  Identification of Mechanosensitive Ion Channels in the Cytoplasmic Membrane of Corynebacterium glutamicum , 1999, Journal of bacteriology.

[2]  Sergei Sukharev,et al.  Straightening and sequential buckling of the pore-lining helices define the gating cycle of MscS , 2007, Nature Structural &Molecular Biology.

[3]  Wendy Bartlett,et al.  A role for mechanosensitive channels in survival of stationary phase: Regulation of channel expression by RpoS , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Hanahan,et al.  Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Pavel Strop,et al.  Crystal Structure of Escherichia coli MscS, a Voltage-Modulated and Mechanosensitive Channel , 2002, Science.

[6]  I. Booth,et al.  Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity , 1999, The EMBO journal.

[7]  Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins. , 1999, FEMS microbiology letters.

[8]  Klaus Schulten,et al.  Ion conduction through MscS as determined by electrophysiology and simulation. , 2007, Biophysical journal.

[9]  H. Rottenberg,et al.  Non-equilibrium thermodynamics of energy conversion in bioenergetics. , 1979, Biochimica et biophysica acta.

[10]  Boris Martinac,et al.  A large-conductance mechanosensitive channel in E. coli encoded by mscL alone , 1994, Nature.

[11]  L. Barton,et al.  Protoporphyrinogen oxidation coupled to nitrite reduction with membranes from Desulfovibrio gigas , 1989 .

[12]  B. Poolman,et al.  Glycine Betaine Fluxes in Lactobacillus plantarum during Osmostasis and Hyper- and Hypo-osmotic Shock (*) , 1996, The Journal of Biological Chemistry.

[13]  S. Morbach,et al.  Osmosensor and Osmoregulator Properties of the Betaine Carrier BetP from Corynebacterium glutamicum in Proteoliposomes* , 2000, The Journal of Biological Chemistry.

[14]  R. Krämer,et al.  Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum. , 2003, FEMS microbiology letters.

[15]  R. Krämer,et al.  Structure and Function of the Betaine Uptake System BetP of Corynebacterium glutamicum: Strategies to Sense Osmotic and Chill Stress , 2006, Journal of Molecular Microbiology and Biotechnology.

[16]  E. Perozo Gating prokaryotic mechanosensitive channels , 2006, Nature Reviews Molecular Cell Biology.

[17]  C Kung,et al.  Pressure-sensitive ion channel in Escherichia coli. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Bowie,et al.  Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel , 2005, Nature Structural &Molecular Biology.

[19]  C. Lambert,et al.  Uptake of glutamate in Corynebacterium glutamicum. 2. Evidence for a primary active transport system. , 1990, European journal of biochemistry.

[20]  J. Naismith,et al.  The Structure of an Open Form of an E. coli Mechanosensitive Channel at 3.45 Å Resolution , 2008, Science.

[21]  A. Coulombe,et al.  Multiple Mechanosensitive Ion Channels from Escherichia coli, Activated at Different Thresholds of Applied Pressure , 1996, The Journal of Membrane Biology.

[22]  S. Morbach,et al.  The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K+ , 2001, The EMBO journal.

[23]  B. Riemann,et al.  Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy , 1994 .

[24]  Hisao Ito,et al.  Mutations of the Corynebacterium glutamicum NCgl1221 Gene, Encoding a Mechanosensitive Channel Homolog, Induce l-Glutamic Acid Production , 2007, Applied and Environmental Microbiology.

[25]  B. Martinac 3.5 Billion Years of Mechanosensory Transduction: Structure and Function of Mechanosensitive Channels in Prokaryotes , 2007 .

[26]  Sergei Sukharev,et al.  Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes. , 2002, Biophysical journal.

[27]  Structural plasticity in MS channels , 2005, Nature Structural &Molecular Biology.

[28]  S. Morbach,et al.  Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation. , 2004, Biochimica et biophysica acta.

[29]  C Kung,et al.  Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. , 1993, Biophysical journal.

[30]  A. Strøm,et al.  Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli , 1986, Journal of bacteriology.

[31]  D C Rees,et al.  Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. , 1998, Science.

[32]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[33]  H. Kunte,et al.  New Type of Osmoregulated Solute Transporter Identified in Halophilic Members of the Bacteria Domain: TRAP Transporter TeaABC Mediates Uptake of Ectoine and Hydroxyectoine in Halomonas elongata DSM 2581T , 2002, Journal of bacteriology.

[34]  P. Blount,et al.  Correlating a Protein Structure with Function of a Bacterial Mechanosensitive Channel* 210 , 2000, The Journal of Biological Chemistry.

[35]  Sergei Sukharev,et al.  The “Dashpot” Mechanism of Stretch-dependent Gating in MscS , 2005, The Journal of general physiology.

[36]  B. Martinac Mechanosensitive Channels in Prokaryotes , 2001, Cellular Physiology and Biochemistry.

[37]  O. Hamill,et al.  Molecular basis of mechanotransduction in living cells. , 2001, Physiological reviews.