Experimental evolution recapitulating adaptation after horizontal gene transfer

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Horizontal gene transfer (HGT) is a ubiquitous process that has shaped microbial genomes and ecology. Many bacteria become infectious through the transfer of pathogenicity islands or become drug resistant through transfer of resistance markers. However, we do not fully understand how genes acquired through HGT are integrated into the host's existing networks. Possessing all the genes necessary for a phenotype is not always sufficient to generate that phenotype. The new genes must function appropriately in their new environment, and the host must be able to tolerate any stress associated with the new genes and phenotype. Experimental evolution of a microbe after it acquires a metabolic pathway that is both physiologically challenging but also potentially beneficial would allow us to study this process under carefully controlled conditions. Members of the genus Methylobacterium are model organisms for studying the metabolism of single carbon compounds, including the environmental toxin dichloromethane (DCM). Closely related strains of M. extorquens vary in their ability to grow on DCM, and genome sequences suggest that the ability to grow on DCM has recently been acquired through HGT. However, cloning the relevant pathway into a strain that lacks the ability to grow on dichloromethane is not sufficient to permit growth. Therefore, I will combine rational engineering with experimental evolution to enable a strain of M. extorquens to grow on previously toxic DCM. Initial characterization of M. extorquens strains that can and cannot grow on DCM will suggest possible phenotypical determinants of DCM growth, such as tolerance to by-products including formaldehyde and hydrochloric acid. After the DCM catabolic pathway is transferred into a strain that is unable to use the pathway, the engineered strain will be evolved to select for mutants that take advantage of their newly acquired catabolic potential. Different selection strategies, such as pre-adaptation to a source of stress like pH or gradual weaning on the challenging substrate DCM, will be compared. The relative success of these different strategies will provide insight into the natural processes by which adaptation might occur following HGT. Finally, a careful characterization of the evolved strains will uncover the mechanisms of adaptation and investigate potential epistasis and trade-offs along the evolutionary trajectories.