Current projects in our lab fall into four general areas, summarized below. For more information, please see our publications here or on PubMed.
Two-component signal transduction pathways
All cells harbor a relatively small number of signaling protein families. Through gene duplication and divergence, organisms have dramatically expanded a limited set of signaling proteins, giving rise to large, paralogous protein families that endow cells with sophisticated information processing capabilities. residues in histidine kinases and response regulators.
(Left) Schematic of a two-component signaling pathway. (Right) Structure of a histidine kinase and its cognate response regulator highlighting the coevolving residues that dictate interaction specificity.
Most bacteria encode dozens to hundreds of two-component signaling systems, usually comprised of a sensor histidine kinase that can respond to an environmental change or stimulus by phosphorylating a cognate response regulator that then triggers changes in cellular physiology or gene expression. We have shown that histidine kinases harbor an intrinsic ability to distinguish their cognate substrate from all possible non-cognate partners rather than relying on auxiliary factors like scaffolds. By studying patterns of amino-acid coevolution in cognate kinase-substrate pairs, we have identified the key specificity-determining residues. This work guided the rational rewiring of two-component signaling pathways and forms the foundation for current studies in three areas. (1) Protein evolution. We are studying the mechanisms by which two-component pathways diverge after duplication to create novel signaling pathways that are insulated from existing pathways. We use a combination of genetics, biochemistry, and phylogenetic studies, including ancestral reconstructions, to gain insight into the mutational trajectories that give rise to new signaling proteins. (2) Molecular recognition. Because partner specificity relies on only a handful of residues in each protein, two-component signaling pathways are amenable to systematic investigations into molecular recognition. By building large, comprehensive mutant libraries and with deep-sequencing as a readout, we are generating maps of the sequence space that underlies the kinase-substrate interaction in two-component pathways. The results are providing insight into the relationship of protein structure, function, and evolution. (3) Synthetic biology. The ability to rationally reprogram kinase and substrate specificity in two-component pathways is enabling new efforts in the design of synthetic signaling circuits in vivo.
Schematic of a canonical toxin-antitoxin system.
Another large set of paralogous proteins in bacteria are toxin-antitoxin systems. Typically, a toxin and antitoxin are co-produced from the same operon, binding each other to form an inert complex. However, in stressful conditions, such as nutrient starvation, the toxins are liberated to suppress cell growth. The induction of toxins underlies the phenomenon of persistence, in which a sub-population of bacteria avoid being killed by antibiotics that target actively growing cells. We are studying toxin-antitoxin systems from a variety of angles including efforts to discover novel systems in bacterial genomes and to identify both their mechanisms of action and the pathways that trigger their induction. We have also studied the specificity and evolution of toxin-antitoxin systems. As with two-component signaling proteins, we used analyses of amino-acid coevolution in cognate toxin-antitoxin pairs to pinpoint the residues crucial to interaction specificity. We are using these studies to map the sequence space underlying toxin-antitoxin systems and to elucidate fundamental principles of how protein-protein interactions evolve.
Cell cycle regulation
We have a long-standing interest in the molecular mechanisms that drive cell cycle progression and the establishment of cellular asymmetry in Caulobacter crescentus. Every cell division for Caulobacter is asymmetric, producing two different daughter cells – a swarmer cell and a stalked cell – that differ morphologically and, importantly, with respect to replicative capacity. Whereas the stalked cell can immediately initiate a new round of DNA replication, the swarmer cell is delayed in a G1 state and must first differentiate into a stalked cell before initiating S phase. Caulobacter cells are easily synchronized, cell cycle progression can be tracked by monitoring a series of morphological transitions, and a complete suite of genetic tools is available.
(Top) Schematic of the Caulobacter cell cycle and the subcellular localization of regulatory proteins that govern cellular asymmetry. (Bottom) Summary of the regulatory pathways controlling CtrA activity and cellular asymmetry.
Our current work focuses on understanding the genetic circuits that control two key cell cycle regulators, the highly conserved replication initiator DnaA and an essential response regulator, CtrA, that silences replication. We are mapping the complex signaling pathways that control the activities of these two regulators. For CtrA, our focus has been on a suite of two-component signaling proteins that orchestrate when and where CtrA is phosphorylated. This work has included the identification of basic mechanisms by which protein kinases are localized and activated within bacterial cells. For DnaA, we have focused on understanding how nutrient status and proteotoxic stress impact the levels and activity of DnaA. More recently, we have begun to explore the mechanisms that connect DnaA to CtrA and thereby ensure the orderly progression of cell cycle events and production of asymmetric daughter cells. Finally, we have identified and examined two novel checkpoint systems that allow Caulobacter to halt cell cycle progression in response to DNA damage.
Chromosome structure and organization
We have recently expanded our work on Caulobacter to understanding the role of chromosome structure in DNA replication, transcription, and DNA repair. We recently implemented Hi-C, a method based on chromosome-conformation-capture technology, to generate the first high-resolution map of a bacterial chromosome in vivo. This work revealed that the Caulobacter chromosome is organized into a series of topological domains. The boundaries between domains are, in most cases, established by the action of highly expressed genes and current work aims to better define how this works. We are also using Hi-C, in combination with fluorescence microscopy, genetics, and biochemical methods, to elucidate the role of DNA-binding proteins such as SMC (structural maintenance of chromosomes protein) in compacting and organizing the genome. We have also examined chromosome organization and spatial dynamics following DNA damage, particularly double-strand breaks. Additional efforts seek to determine how chromosome organization impacts other DNA-based processes such as DNA replication, transcription, and recombination.
Hi-C profile for the Caulobacter crescentus chromosome (top) with domains revealed through directional preference analysis (middle). A model of the global chromosome conformation (bottom).