Rapid in vivo evolution of a beta-lactamase using phagemids

RNA viruses are capable of undergoing extremely rapid evolution due to thei r high rates of reproduction, small genome size, and a high frequency of sp ontaneous mutagenesis. Here we demonstrate that a virus-like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coil in the presence of a helper phage. This enables one to subject individual phagemid-encoded genes to rapid in vivo evolutio n. We applied this approach to TEM-1 p-lactamase which confers resistance t o 0.05 mg/L of the antibiotic cefotaxime. After 3 weeks of in vivo evolutio n 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 m utant, E104K/G238S/T263M, which confers a 1000-fold increase in resistance compared to the wild type enzyme. The same three mutations have been previo usly observed in TEM-4 beta-lactamase which was discovered in a highly cefo ta-lactamase carrying three identical point mutations isxime-resistant clin ical isolate. The probability of randomly obtaining a p-lactamase carrying three identical point mutations is less than 10(-10). This indicates that p hagemid evolution can rapidly reproduce evolution occurring in nature. (C) 2000 John Wiley & Sons, Inc.