The MscCG protein from the Grampositive Corynebacterium glutamicum ATCC 13032 is a member of the MscS-type of mechanosensitive efflux channels, relevant for the response to hypoosmotic challenge. The major difference of C. glutamicum MscCG from other MscS-type proteins, e.g. MscS from Escherichia coli, is the presence of an additional, 247 amino acid long C-terminal domain, which we showed to carry a transmembrane segment. Evidence was provided that the NCgl1221 gene product from C. glutamicum ATCC 13869, an orthologue of MscCG, is responsible for glutamate efflux under particular physiological conditions. C. glutamicum is used in microbial biotechnology for the production of amino acids, in particular glutamate. The mechanism of glutamate excretion, however, is not yet fully clear.
We have generated selected C-terminal point mutations, sequence modifications, and truncations of MscCG in C. glutamicum, gain-of-function and loss-of-function constructs of both E. coli MscS and C. glutamicum MscCG, as well as fusion constructs of these two proteins. We have investigated the properties of these constructs with respect to mechanosensitive efflux, electrical conductance, gating properties, impact on cell growth and on the membrane potential of the cell, as well as glutamate excretion. The electrophysiological properties of MscCG turned out to be comparable to those of MscS from E. coli, showing some differences in gating behaviour. Various recombinant forms of MscCG were shown to be closely similar with respect to gating and conductivity, but were found significantly different concerning glutamate excretion. Most interestingly, the characteristic additional C-terminal domain of MscCG was shown to exert a strong and specific influence on the functional properties of MscCG with respect to both mechanosensation and, in particular, to glutamate excretion. Consequently, the results obtained are relevant for both fundamental functional properties of the MscS-type channel MscCG as well as for biotechnological application.