In the not-too-distant past, bacteria were thought to be relatively simple organisms, constantly engaged in DNA replication and binary fission, with cytosolic components distributed at random throughout the cell. We now know that bacterial cells can have complex life cycles and highly organized interior architectures. Bacteria contain cytoskeletal elements that influence their shape and mediate cell division. They place large macromolecular assemblies, such as flagella, stalks, and pili, at specific sites on the cell surface. GFP fusion proteins have revealed that individual signal transduction proteins can have subcellular addresses, and they can move as a function of the cell cycle or developmental status of the bacterium. I want to understand how spatial cues, such as protein localization, and temporal events, such as protein activation or degradation, are controlled and combined to produce an orderly bacterial cell cycle.
The gram negative bacterium Caulobacter crescentus is an ideal organism in which to ask these questions, because its cell cycle is easily studied and every cell division is asymmetric, resulting in two progeny with different morphologies, protein complements, and replicative fates. The life cycle of Caulobacter begins with the motile swarmer cell (SW), which cannot replicate its chromosome or undergo cell division. The swarmer cell differentiates into a stalked cell (ST), shedding its flagellum and building a stalk at the same site. During this transition, the cell gains the ability to initiate DNA replication and enter the division cycle. The stalked cell grows into an asymmetric predivisional cell (PD), with a new flagellum at the pole opposite the stalk. Every cell division produces a swarmer cell and a stalked cell. The stalked cell can immediately begin a new round of chromosome replication and cell division, while the swarmer cell must first differentiate into a stalked cell.
We can isolate pure populations of swarmer cells and observe many parameters during their synchronous progress through the cell cycle, including fluorescent protein localization, DNA content, and global transcriptional patterns. The Caulobacter genome has been sequenced, which expedites all genetic manipulations and allows us to search comprehensively for genes that affect processes of interest. We also pursue in vitro studies to determine how the biochemical properties of individual regulatory proteins contribute to cell cycle progression and cellular asymmetry.
