Cells have the remarkable ability to process information and make decisions, helping them adapt to stress, execute developmental programs, and stably progress through the cell cycle. Information processing and regulation at the cellular level are rarely achieved by a single factor but instead require the coordinated action of multiple genes and proteins. How are regulatory molecules orchestrated at a systems level? What are the design principles underlying regulatory circuits, and what are the constraints that shape them? How do regulatory systems evolve while continuing to fulfill their critical cellular roles?
To address these questions, my lab studies the signal transduction pathways and information-processing abilities of bacteria. We focus on two particular areas: (1) We study the regulatory circuits that control cell cycle progression in Caulobacter crescentus, particularly the role of two-component signal transduction systems, the dominant signaling modality in the bacterial kingdom. Because these signaling proteins are absent in humans, they are also considered possible new antibiotic targets. (2) We examine the mechanisms by which bacterial cells enforce the specificity of signaling pathways to prevent unwanted crosstalk. These studies include efforts in protein engineering, the design of synthetic signaling circuits, and investigations of protein evolution.
Mapping the Complex Regulatory Circuitry Governing Cell Cycle Progression in Caulobacter
The cell cycle of C. crescentus is an ideal model system for the study of regulatory circuits, as this organism is amenable to a full range of genetic, biochemical, cytological, and genomic investigations. Because its cells are easily synchronized and exhibit distinguishable G1, S, and G2 phases (Figure 1), Caulobacter is particularly well suited to studies of temporal dynamics. Caulobacteris also a powerful system for understanding the mechanisms by which cells generate and maintain asymmetry, a fundamental aspect of developmental biology.
Although some key cell cycle regulators in Caulobacter are already known, it remains a challenge to identify the complete set of regulators and to establish their connectivity, i.e., the circuit topology. To this end, we have systematically deleted each of the 106 two-component signaling genes in the C. crescentus genome and identified those that contribute to cell cycle regulation. We have also developed phosphotransfer profiling, a new systematic biochemical approach, for the rapid and accurate mapping of connectivity between histidine kinases and response regulators. We are using these systematic approaches, along with bioinformatics, microarrays, genetics, and biochemistry, to map the core genetic oscillator that drives the cell cycle.
Progression through the Caulobacter cell cycle requires periodic changes in activity of the master regulator CtrA, a response regulator of the two-component signaling family. The cyclical changes in CtrA activity are governed mainly by regulated proteolysis and phosphorylation. Our lab recently mapped the two-component signaling pathways that control the timing of both phosphorylation and proteolysis of CtrA. CckA, a master kinase, simultaneously drives the phosphorylation and proteolytic stabilization of CtrA. As CtrA accumulates during the cell cycle, it drives the synthesis of DivK, another key regulator protein, which then feeds back to inhibit CckA, leading to dephosphorylation and degradation of CtrA, thereby resetting the circuit. This is the key feedback loop driving cell cycle oscillations. There are, however, additional, or auxiliary, feedback loops that influence CtrA, and we have begun using a combination of genetics and time-lapse microscopy of single cells to probe the role played by each feedback loop.
We have also recently explored the mechanisms by which Caulobacter cells sense and respond to DNA damage. Our results have led to the identification of the first bona fide cell cycle checkpoint in Caulobacter. This surveillance system recognizes DNA damage and responds by inducing the expression of critical genes, most notably one that directly inhibits the cell division machinery, thereby delaying cytokinesis until DNA damage is cleared.
Elucidating the Molecular Basis of Specificity and Evolution of Bacterial Signaling Systems
Two-component signaling pathways typically include a sensor histidine kinase that phosphorylates a cognate response regulator. Most bacteria employ dozens, if not hundreds, of these signaling systems to coordinate a vast range of cellular processes. How does a single cell coordinate the activity of so many highly related molecules? How do cells maintain the specificity of signal transduction and prevent harmful crosstalk? My laboratory has recently shown that the substrate (response regulator) specificity of each histidine kinase is an intrinsic property and based on molecular recognition rather than cellular factors such as scaffolds. An understanding of how this recognition is encoded within histidine kinases has, however, been largely refractory to structural approaches, necessitating alternative approaches.
We postulated that the amino acids dictating specificity must coevolve in cognate kinase-substrate pairs; i.e., if a specificity residue in the kinase mutates, it must be accompanied by a compensatory mutation in the substrate, or vice versa. To test this idea, we analyzed patterns of amino acid coevolution in large multiple-sequence alignments of kinase-substrate pairs. The amino acids showing strongest coevolution in this analysis map to the molecular surfaces that mediate phosphotransfer from a histidine kinase to its substrate, a response regulator (Figure 2). Guided by these results, we successfully rewired two-component signaling pathways by mutating the specificity residues in one histidine kinase to match those found in another histidine kinase. We have recently extended these results to rationally reprogram response regulators to accept phosphate from alternative histidine kinases. This ability to rewire two-component pathways presents a new opportunity to engineer synthetic signaling pathways, both as a stringent test of how well we understand molecular recognition and as a novel means through which to probe biological processes.
Our ability to rewire two-component signaling proteins also offers an opportunity to explore the selective pressures that impact the evolution of signaling pathways. For example, our work has demonstrated that kinase-regulator pairs coevolve, but what drives this coevolution to occur? One possibility is random drift; i.e., a specificity residue in one molecule happens to mutate, necessitating a compensatory change in the cognate molecule, with the changes providing no selective advantage. Alternatively, a kinase-regulator pair may change its specificity residues to avoid detrimental crosstalk with other systems, perhaps following a gene duplication or horizontal gene transfer event. By reconstructing the phylogenetic history of specific histidine kinases, we are tracing changes in specificity residues and can infer their identities before and after events such as gene duplication. These ancestral states can be reconstructed in the laboratory and their specificities empirically tested. The results are beginning to shed new light on how specificity evolves at the molecular level, such as the number, types, and order of mutations required to insulate pathways from one another.
Grants from the National Institutes of Health and the National Science Foundation provided partial support for these projects.
As of May 30, 2012