Research

Cellular Asymmetry and Cell Cycle Regulation in Bacteria

Caulobacter crescentus is a powerful model for understanding the bacterial cell cycle as well as the mechanisms cells use to establish and enforce cellular asymmetry. 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. Every cell division 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 sessile stalked cell can immediately initiate a new round of DNA replication, the swarmer cell is delayed in a G1 state and must differentiation into a stalked cell before initiating S phase.

At the heart of the circuitry controlling the asymmetric fates of swarmer and stalked cells is the master regulator CtrA. This essential transcription factor controls nearly 100 genes and also binds directly to the origin of replication to silence initiation. We have recently mapped, for the first time, an integrated regulatory circuit that can account for the cell cycle- and cell type-dependent changes in CtrA activity. Crucial to the operation of this circuit is the histidine kinase CckA which ultimately drives CtrA phosphorylation. How CckA is regulated has been a long-standing mystery, as has the observation that CckA is maximally active when localized to the swarmer pole of predivisional cells. Recently, we demonstrated that CckA activity requires the non-canonical kinase DivL, but that phosphorylated DivK can inhibit the DivL:CckA complex. Localization of CckA and DivL is important as it brings them in proximity to a DivK phosphatase. The cell pole is thus used to create a protected zone within the cell to activate CckA.

In addition to functioning as a kinase at one pole, CckA also functions as a phosphatase at the opposite pole. The opposing polar activities of CckA lead to a gradient of phosphorylated CtrA across the predivisional cell, despite a homogeneous distribution of CtrA-GFP.

These studies now indicate that cell fate asymmetry in Caulobacter is established before cell division, contrary to previous models. More generally, our findings suggest that the uniform distribution of GFP-tagged proteins may belie spatially heterogeneous patterns of activity.

We are also probing the feedback structure of the cell cycle regulatory network. Why is the circuit so complex? What is the role of specific feedback loops to the reliability or robustness of the system? Although the network can appear irreducibly complex, our recent work suggests that the network is comprised of simpler and genetically separable modules. Finally, we have begun to examine the evolution of the bacterial cell cycle, to try and understand how the Caulobacter regulatory circuit relates to that used in E. coli and other bacteria.

Cell Division Control

We are also interested in understanding how cells sense and respond to DNA damage. We have characterized the complete gene expression responses and are using new high-throughput techniques to systematically identify genes required for responding to DNA damage. Our results have led to the identification of a novel cell cycle checkpoint system in Caulobacter that directly targets the cell division machinery. We are characterizing the molecular basis of this surveillance system and the means by which it inhibits cell division following DNA damage.

We have also uncovered evidence of a novel pathway that regulates the activity of CtrA, the master cell cycle regulator, following damage. Current work combines genetic, biochemical, and cytological studies to investigate these novel molecular mechanisms.

Specificity and Evolution of Signaling Pathways

Most bacteria encode dozens, if not hundreds, of two-component signal transduction systems which coordinate a wide 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? And, importantly, how do new pathways evolve?

We have found that the substrate specificity of a histidine kinase is dictated by molecular recognition rather than cellular factors such as scaffolds. An understanding of how substrate recognition is encoded within histidine kinases has, however, been refractory to structural approaches, necessitating alternative approaches. We postulated that the amino acids dictating specificity must coevolve in cognate kinase-substrate pairs; that is, 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 mapped to the molecular surfaces that mediate kinase-substrate interaction. Guided by these results, we have successfully rewired two-component signaling pathways by mutating these specificity residues thereby redirecting information flow in a rational manner. We can also rationally reprogram substrates to be phosphorylated by different kinases.

The ability to rewire two-component pathways presents a new opportunity to engineer synthetic signaling pathways, both for creating novel circuits and for probing biological processes of interest.

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 or neutral drift. Alternatively, a kinase-substrate pair may change its specificity residues to avoid detrimental cross-talk with other systems, perhaps following gene duplication or horizontal gene transfer events. By reconstructing the phylogenetic history of specific histidine kinases, we are tracing changes in specificity residues and inferring their identities before and after events such as gene duplication. These ancestral states can be reconstructed in the laboratory and their specificities empirically tested, both in vitro and in vivo. The results are beginning to shed new light on how specificity evolves at the molecular level.