title: Rapid in vivo evolution of a beta-lactamase using phagemids.
abstract: RNA viruses are capable of undergoing extremely rapid evolution due to their high rates of reproduction, small genome size, and a high frequency of spontaneous mutagenesis. Here we demonstrate that a virus-like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coli in the presence of a helper phage. This enables one to subject individual phagemid-encoded genes to rapid in vivo evolution. We applied this approach to TEM-1 beta-lactamase which confers resistance to 0.05 mg/L of the antibiotic cefotaxime. After 3 weeks of in vivo evolution we were able to isolate a double mutant, E104K/G238S, of the enzyme which confers a 500-fold increased level of resistance to cefotaxime compared to the starting enzyme. In two independent experiments we obtained a triple mutant, E104K/G238S/T263M, which confers a 1000-fold increase in resistance compared to the wild type enzyme. The same three mutations have been previously observed in TEM-4 beta-lactamase which was discovered in a highly cefotaxime-resistant clinical isolate. The probability of randomly obtaining a beta-lactamase carrying three identical point mutations is less than 10(-10). This indicates that phagemid evolution can rapidly reproduce evolution occurring in nature.
authors.affiliation: Genencor International, Inc., 925 Page Mill Rd., Palo Alto, California 94304, USA.
authors.forename: J
authors.initials: J
authors.lastname: Long-McGie
authors.forename: A D
authors.initials: AD
authors.lastname: Liu
authors.forename: V
authors.initials: V
authors.lastname: Schellenberger
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D008967:Molecular Biology / Q000379:methods*; D017354:Point Mutation; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001618:beta-Lactamases