Improved electrotransformation frequencies of Corynebacterium glutamicum using cell-surface mutants

The electrotransformation efficiency for homologously- and heterologously-d erived plasmid DNA was determined for two families of Corynebacterium gluta micum strains derived from ATCC13059 (AS019 and auxotrophic, cell surface m utants MLB133 and MLB194) and ATCC13032 (parent strain and restriction-minu s mutants RM3 and RM4), following their growth in LBG supplemented with gly cine plus isonicotinic acid hydrazide (INH). Electrotransformation efficien cies of MLB133 were up to 100-fold higher than for strain ASO19 and, when u sing heterologously-derived plasmid DNA, MLB133 showed efficiencies compara ble to or better than strains RM3 and RM4, demonstrating the relative impor tance of cell surface structures in impeding DNA uptake in C. glutamicum. T ransmission electron microscopy analysis of cell surface structures showed that strain MLB133 has a thin cell wall relative to AS019 and growth in eit her glycine or INH further diminished its thickness. Both RM3 and RM4 were more sensitive to INH than ATCC13032 and growth in glycine plus INH further improved transformation efficiency. The mycolic acid composition of these strains is described and the impact of glycine and INH on this is reported.