Insertion Mutagenesis of wca Reduces Acid and Heat Tolerance of Enterohemorrhagic Escherichia coli O157:H7

Strains of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 produce under stress copious amounts of exopolysaccharide (EPS) composed of colanic acid (CA). Studies were performed to evaluate the association of production of CA with survival of EHEC under adverse environmental conditions. A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain, E. coli O157:H7 W6-13, by inserting a kanamycin resistance gene cassette (kan) into wcaD and wcaE, 2 of the 21 genes required for CA biosynthesis. M4020 was defective in CA production as determined from the ratio of uronic acid to protein (UA/P) of cells grown from 1 to 4 days at 25°C on minimal glucose agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony morphology on MGA. The results of stress treatment revealed that M4020 was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60°C) in comparison to W6-13, indicating that CA confers on E. coli O157:H7 a protective effect from the environmental stresses of acid and heat.