In Vitro Evolution of an Archetypal Enteropathogenic Escherichia coli Strain

Abstract

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal r ) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nal r strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str r ) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK . Another, in gyrA , is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD , the lysogenization regulator, to purB . The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal r clone has a lower growth rate than the Str r isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB . Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal r clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro .