Mechanisms of Adaption: Genotype to Molecular Phenotype to Growth Rate

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DESCRIPTION (provided by applicant): For 40 years evolutionary genetics has been purely statistical but recently manipulative methods (e.g. reverse genetics) have been joined with biochemistry and structural biology to dissect the mechanistic basis of adaptive changes. The broad, long-term objective of my research is to take advantages of these manipulative changes and take a mechanistic approach to study adaptation. For my postdoctoral research, I am developing a model system suited to my long term research program. The model system is a single strain of E.coli and 16 alleles of beta-lactamase that confer resistance to ampicillin. Specific Aim 1. Develop and test a novel model describing how whole-organism growth rate varies across temperature and antibiotic concentration. Characterize intra and inter allelic variation in growth rate across temperatures and antibiotic concentrations. For each allele, measure growth rate across a wide range of temperatures and antibiotic concentrations. Assuming growth rate depends on temperature according to the Arrhenius equation, combine the growth rate data with the model of bacterial growth rate to calculate each alleles activation energy. Specific Aim 2. At multiple temperatures between 15 and 60 degrees Celcious, measure the maximum reaction velocity (vmax) for each of the 16 beta-lactamase alleles. Measure the thermostability of these same alleles with circular dichroism. Use the kinetic data to calculate the activation energy for each allele. Test the hypothesis that the in vitro measures of activation energy for each allele agree with the estimates of activation energy made in Aim 1. Test the hypothesis that trade-offs commonly occur between enzyme activity and stability and that these trade-offs explain whole-organism growth rates. Relevance: The mode system for these experiments, the beta-lactmase enzyme, confers resistance to beta- lactam antibiotics. Beta-lactam antibiotics represent 65% of all antibiotics and account for 15 billion dollars a year in business. By measuring the growth rate of these beta-lactamase alleles at different temperatuers and antibiotic concentrations, I will be in a position to assess the persistence of these beta-lactamase alleles in nature.