Experimental evolution recapitulating adaptation after horizontal gene transfer

描述（由申请人提供）：水平基因转移（HGT）是一种普遍存在的过程，它塑造了微生物基因组和生态。许多细菌通过致病岛的转移而变得具有感染性，或者通过抗性标记的转移而变得具有耐药性。然而，我们并不完全了解通过HGT获得的基因如何整合到宿主现有的网络中。拥有一个表型所需的所有基因并不总是足以产生该表型。新的基因必须在新的环境中发挥适当的功能，宿主必须能够忍受与新基因和表型相关的任何压力。微生物在获得一种既具有生理挑战性又具有潜在益处的代谢途径后的实验进化将使我们能够在仔细控制的条件下研究这一过程。甲基杆菌属的成员是研究单碳化合物代谢的模式生物，包括环境毒素二氯甲烷（DCM）。与M.扭脱体在DCM上生长的能力各不相同，基因组序列表明，最近通过HGT获得了在DCM上生长的能力。然而，将相关途径克隆到缺乏在二氯甲烷上生长的能力的菌株中不足以允许将相关途径克隆到细胞中。 增长因此，我将联合收割机的合理工程与实验进化相结合，使M.在之前有毒的DCM上生长。对M.可以和不能在DCM上生长的扭脱体菌株将提示DCM生长的可能的表型决定因素，例如对包括甲醛和盐酸的副产物的耐受性。在DCM分解代谢途径被转移到不能使用该途径的菌株中后，工程菌株将被进化以选择利用其新获得的分解代谢潜力的突变体。不同的选择策略，如预适应 与应激源如pH或在挑战性底物DCM上逐渐断奶进行比较。这些不同的策略的相对成功将提供深入了解的自然过程，适应可能发生以下HGT。最后，对进化菌株的仔细表征将揭示适应机制，并调查潜在的上位性和沿着进化轨迹的权衡。

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.