Investigating the interchangeability of a central protein in Escherichia coli’s chemotaxis network

The evolutionary trajectory of complex, multi-component macromolecular machineries such as the chemotaxis network remains poorly understood, particularly with regard to maintaining the network's functionality. This thesis presents an experimental case study investigating the robustness of the most complex networks in proteobacteria to evolutionary changes. Despite extensive research on the chemotaxis network in Escherichia coli, the stepwise adaptive evolution of its components and their impact on network robustness has yet to be fully explored. To address these gaps, our multidisciplinary approach integrates systems biology, molecular biology, phylogenetics, genomics, and biophysics. This study primarily investigates the CheY protein, which is highly conserved across proteobacteria and plays a central role in the chemotaxis network. The effects of replacing CheY with both ancient and modern homologs were examined, and the resulting changes in motility behavior were assessed. This approach enabled the quantification of how protein functionality within the network degrades across evolutionary distances, thereby shedding light on the limits of protein interchangeability of a core protein in a biological network. This study has demonstrated that the chemotaxis network is robust to alterations in its central node. However, this robustness is not without limits. The factors that influence the limit of interchangeability are the evolutionary limits of interaction partners and protein dosage. In the latter part of the study, we conducted experimental evolution to evaluate the network’s adaptability to these changes. Our findings revealed that the limits of protein interchangeability could be expanded through adaptive evolution. Typically, the network adapts to the changes of its central protein, which illustrates the evolvability of biological networks. Notably, expression-related changes often occur outside the network to overcome the protein dosage limitation. Our work underscores the value of studying the evolution of complex networks by establishing a link between the evolutionary changes of a central protein in the network and the network functionality. This approach could not only elucidate past evolutionary events but could also provide a framework for designing more robust biological systems.