Understanding the control logic in the bacterium Caulobacter crescentus has progressed to the point where we now have an integrated systems view of the operation of its entire cell cycle functioning as a state machine. Oscillating levels of a small number of temporally controlled master regulators drive cell cycle progression. Cell cycle regulation is, to a striking degree, a whole cell phenomenon with transcriptional circuitry interwoven with the 3-dimensional deployment of signaling proteins and proteases. Remarkably, this circuitry is integrated with the replication of the single circular chromosome. We are exploring the dynamic 3D localization of chromosomal elements, the specific proteins involved in chromosome segregation, and the components of the cell division machinery. The overall ‘wiring diagram’ incorporates changes in DNA methylation state that enhance system robustness.
Our lab takes an interdisciplinary approach to understand the systems biology of a living cell. We use:
- state-of the-art imaging technologies, including super resolution tracking of single molecules in living cells to manipulate and observe dynamic localization of structural and signaling proteins involved in phospho-signaling cascades and regulated proteolysis.
- crystal structures of critical proteins, and their mutant variants, that mediate the dynamically localized phospho-signaling pathway that controls asymmetric cell division.
- extensive ‘omics’ data bases for all elements of the Caulobacter genome, including ribosome profiling, global 5’race to identify all transcription start sites, RNA-seq, LC-MS proteomics, and saturation transposon insertion analysis to identify essential elements of the Caulobacter genome, all at single base pair resolution.
- biocomputational modeling of the circuitry that uses 4 global transcriptional regulators and an essential DNA methyltransferase that together drive the cell cycle. Superimposed on this circuit, is the spatial and temporal control provided by a small number of essential phosphokinases and proteases. The proteins of the Caulobacter cell cycle control system are co-conserved across many α-proteobacteria species, but there are great differences in the regulatory apparatus’ functionality and peripheral connectivity to other cellular subsystems from species to species. This pattern is similar to that observed for the ‘kernels’ of the regulatory networks that regulate development of the metazoan body plans. These finding emphasize that understanding a bacterial species requires a cell wide investigation of the organization of the regulatory logic in time and space.
- molecular genetic dissection and biochemical analysis of the components of the chromosome replication and segregation machinery. Recent discoveries have revealed the intricate subcellular organization of the Caulobacter chromosome, the mechanism used for chromosome segregation, and the protein complexes that direct chromosome movement along the long axis of the cell.
- full genome sequencing at multiple times in the cell cycle to identify changes in the pattern of DNA methylation as a function of replication fork progression. Changes in methylation state contribute to the time of transcription of critical global factors that control the expression of hundreds of cell cycle regulated genes, integrating the process of chromosome replication with the transient appearance of cell cycle-regulated transcription factors.