P148: Electrophysiological analysis of the protein functions as a mechanosensitive channel which encoded by NCgl1221 in Corynebacterium glutamicum

 Corynebacterium glutamicum is used worldwide for the industrial fermentative production of L-glutamate. However, the mechanism by which this bacterium secretes glutamate remains unclear. It was recently reported that a mutation in NCgl1221 results in spontaneous glutamate secretion, and disruption of this gene essentially inhibits glutamate secretion. NCgl1221 encodes a homolog of the mechanosensitive channel of small conductance (MscS).

 In this study, we used an electrophysical technique to analyze the functions of this protein in Bacillus subtilis cell expressing the MscS-encoding gene of C. glutamicum. We selected B. subtilis because giant protoplasts required for electrophysiological studies have not yet been derived from C. glutamicum. B. subtilis possesses 4 genes encoding mechanosensitive channel homologs. Disruption of mscL and ykuT is reported to reduce cell survival during osmotic downshock. C. glutamicum possesses 2 genes (mscL and mscS) that encode mechanosensitive channel homologs. Therefore, a double-disruptant strain of B. subtilis with mscL and ykuT disrupted, was constructed; further, a C. glutamicum gene (mscS) was expressed in this strain, under the control of the xylose promoter. The double-disruptant B. subtilis strain expressing the mscS genge of C. glutamicum showed increased cell survival rate during osmotic downshock as compared to the double-disruptant strain not expressing this gene.

 We tried to analyze the mechanosensitive channel activity of MscS by using an electrophysiological technique; the patch-clamp method. Our findings showed that the giant provacuole prepared from the mscL ykuT double-disruptant strain of B. subtilis, expressing Ncgl1221, showed significantly higher pressure-dependent conductance than the control vacuole.