Specificity and evolution of bacterial two-component signal transduction systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Cells possess a remarkable capacity to sense and process a diverse range of signals. Duplication and divergence of a relatively small number of gene families has provided the raw material enabling cells to quickly increase their signaling capacity. After duplication, however, all pathway components are identical in sequence and function. To evolve a new role, the pathways must become insulated at the level of signal transduction. Two-component signal transduction systems, consisting of a sensor histidine kinase and a cognate response regulator, are the main means by which bacteria sense and respond to their environment. These systems have undergone extensive duplication and lateral gene transfer such that most species encode dozens to hundreds of these pathways, yet there is little evidence of cross-talk at the level of signal transduction. Previous work has shown that interaction specificity is dictated by molecular recognition and determined by a small set of specificity residues. I begin by studying the evolutionary trajectories of specificity residues in a duplicated two-component system that lead to insulation of pathways while at the same time maintaining interaction between cognate kinases and regulators. I then examine specificity residues in orthologs of a single two-component system and show that specificity residues are typically under purifying selection, but, as a result of additions to the two-component signaling network, can undergo bursts of diversification followed by extended stasis. By reversing these mutations I demonstrate that avoidance of cross-talk is a major selective pressure. Finally, I show that covalent attachment of the response regulator to a kinase represents an alternative mechanism for enforcing specificity. In these cases, no changes are needed to accommodate a duplication; the high effective concentration of the covalently attached response regulator prevents cross-talk with other two component proteins in the cell. This may allow hybrid kinases to be duplicated or transferred between genomes more easily. This work sheds light on the apparent ease with which two-component systems have expanded to become the dominant signaling system in bacterial genomes and, more generally, how a small number of gene families can be responsible for signal transduction in all organisms.by Emily Jordan Capra.Ph.D

Spatial tethering of kinases to their substrates relaxes evolutionary constraints on specificity

Signal transduction proteins are often multi-domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two-component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so-called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross-talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems.National Science Foundation (U.S.) (CAREER Award)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Adaptive Mutations that Prevent Crosstalk Enable the Expansion of Paralogous Signaling Protein Families

Orthologous proteins often harbor numerous substitutions, but whether these differences result from neutral or adaptive processes is usually unclear. To tackle this challenge, we examined the divergent evolution of a model bacterial signaling pathway comprising the kinase PhoR and its cognate substrate PhoB. We show that the specificity-determining residues of these proteins are typically under purifying selection but have, in α-proteobacteria, undergone a burst of diversification followed by extended stasis. By reversing mutations that accumulated in an α-proteobacterial PhoR, we demonstrate that these substitutions were adaptive, enabling PhoR to avoid crosstalk with a paralogous pathway that arose specifically in α-proteobacteria. Our findings demonstrate that duplication and the subsequent need to avoid crosstalk strongly influence signaling protein evolution. These results provide a concrete example of how system-wide insulation can be achieved postduplication through a surprisingly limited number of mutations. Our work may help explain the apparent ease with which paralogous protein families expanded in all organisms.National Science Foundation (U.S.) (NSF Graduate Research Fellowship)National Science Foundation (U.S.) (NSF CAREER Award