Optimizing M9 minimal medium for chitin-induced natural transformation. V. cholerae A1552 was induced for natural competence by growth on chitin flakes. Panel A: Transformation frequencies (y-axis) using the expedite transformation protocol and 2 ug of gDNA of strain A1552-LacZ-Kan as transforming material. The medium used was DASW (lane 1), standard M9 medium (lane 2), and MgSO4 /CaCl2 enriched M9 medium (lane 3; see text for details), respectively. Average of at least three independent experiments. Panel B: Commercially available M9 medium was used as base and alternated with respect to NaCl, HEPES, MgSO4 and CaCl2. White columns represent average transformation frequencies of all low concentration samples (lane 1: 9 mM NaCl; lane 3: 0 mM HEPES; lane 5: 2 mM MgSO4; lane 7: 0.1 mM CaCl2) comparable to standard ingredients of M9 minimal medium. Black columns represent average transformation frequencies of high concentration samples mimicking DASW concentrations (lane 2: 259 mM NaCl; lane 4: 50 mM HEPES; lane 6: 32 mM MgSO4; lane 8: 5.1 mM CaCl2). Statistically significant differences are indicated by asterisks (*p < 0.05; **p < 0.01). Panel C: Magnitude of main effects and interactions of factors influencing natural transformation. Half-normal plot of the absolute estimated values (Y-axis) versus their positive normal score (X-axis) are shown as white circles. Black circles indicate statistically significant effects due to addition of MgSO4, CaCl2 as well as both together (MgSO4 × CaCl2).