Abstract
MscCG, a mechanosensitive channel of Corynebacterium glutamicum provides a major export mechanism for glutamate in this Gram-positive bacterium, which has for many years been used for industrial production of glutamate and other amino acids. The functional characterization of MscCG is therefore, of great significance to understand its conductive properties for different amino acids. Here we report the first successful giant spheroplast preparation of C. glutamicum amenable to the patch clamp technique, which enabled us to investigate mechanosensitive channel activities of MscCG in the native membrane of this bacterium. Single channel recordings from these spheroplasts revealed the presence of three types of mechanosensitive channels, MscCG, MscCG2, and CgMscL, which differ largely from each other in their conductance and mechanosensitivity. MscCG has a relatively small conductance of ~340 pS followed by an intermediate MscCG2 conductance of ~1.0 nS and comparably very large conductance of 3.7 nS exhibited by CgMscL. By applying Laplace’s law, we determined that very moderate membrane tension of ~5.5 mN/m was required for half activation of MscCG compared to ~12 mN/m required for half activation of both MscCG2 and CgMscL. Furthermore, by combining the micropipette aspiration technique with molecular dynamics simulations we measured mechanical properties of the C. glutamicum membrane, whose area elasticity module of KA ≈ 15 mN/m is characteristic of a very soft membrane compared to the three times larger area expansion modulus of KA ≈ 44 mN/m of the more elastic E. coli membrane. Moreover, we demonstrate that the “soft” properties of the C. glutamicum membrane have a significant impact on the MscCG gating characterized by a strong voltage-dependent hysteresis in the membrane of C. glutamicum compared to a complete absence of the hysteresis in the E. coli cell membrane. We thus propose that MscCG has evolved and adapted as an MscS-like channel to the mechanical properties of the C. glutamicum membrane enabling the channel to specialize in transport of amino acids such as glutamate, which are major osmolytes helping the bacterial cells survive extreme osmotic stress.
Highlights
Bacterial cells possess two types of mechanosensitive channels, the MscS-like and the MscL-like channels[1]
Given that most of the MscS-like channels have complex structure by having more than three transmembrane helices characteristic of the canonical MscS of E. coli and were shown to be involved in more specialized processes such as calcium signaling in fission yeast cellular signaling (Msy[1] and Msy2)[9] and apoptosis signaling in plants (MSL10)[42], MscCG channels have four transmembrane helices and seem to have acquired more complex structure and gating characteristics for their more specialized function in C. glutamicum
MscCG exhibited a pronounced gating hysteresis in the C. glutamicum membrane characterized by a significant delay in the channel closing upon release of negative pressure applied to a spheroplast patch at low membrane potentials around −100 mV
Summary
Bacterial cells possess two types of mechanosensitive channels, the MscS-like and the MscL-like channels[1]. When E. coli cells are exposed to hypoosmotic shock, MscS is activated first at the moderate membrane tension, and MscL opens close to the membrane lytic tension to prevent the cells from bursting[3] This osmoregulation system is evolutionarily highly adapted for bacterial survival upon the osmotic. E. coli possesses one MscL channel and six MscS-like channel proteins: MscS, MscK, YbdG, YnaI, YbiO, and YjeP4 These mechanosensitive channels have different activation thresholds and channel conductance allowing the bacterial cells to adapt to a wide range of changes in their osmotic environment. The characteristic structural feature of MscCG is a large carboxy-terminal extended domain, which is absent in the canonical E. coli MscS24 Another MscS-like channel, MscCG2, has been reported as a minor accessory glutamate exporter in a C. glutamicum strain[25]. We propose an explanation why only MscCG, and not MscCG2 and CgMscL, acts as a major glutamate exporter in C. glutamicum cells
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