The ctpE gene was knocked out by introducing a premature stop codon at Gln16 (CAG to TAG) using the CTBEevolve plasmid. The strains were grown in Middlebrook 7H9 broth to the mid-log phase. The cells were 1:100 diluted and cultured in Middlebrook 7H9 broth containing 1.0 mM EGTA or 0 mM EGTA. The cells were grown at 37°C for 48 h with shaking at 200 rpm and then the cells were left undisturbed for 1.0 h at room temperature for cell aggregation.