Bacterial cell cycle progression and development rely on a backbone of transcription factors and phosphorelay systems to temporally coordinate gene expression with events in the cell. The phosphorelays have been relatively well characterized in the model organism Caulobacter crescentus, but identifying the key factors that drive transcription has proven a major challenge. The transcription factors CtrA and DnaA have been implicated in many crucial steps of the cell cycle, but the factors driving expression of most cell cycle regulated genes remain unknown. Likewise, no master regulators have been identified for other developmental steps such as the entry into stationary phase. My long-term goal is to identify these master regulators and dynamically characterize how they orchestrate cell activities in response to environmental cues. The work proposed here has two specific aims, the first aim is to find novel master regulators using a systematic reverse genetics approach. A knock-out library of all the transcription factors in C. crescentus is being constructed, and will be screened for cell cycle defects such as growth changes or aberrant chromosome numbers. Candidate genes will then be prioritized and further analyzed by microarray methods to determine how they affect known master regulators and other genes. The library will also be screened for defects in the stationary phase response by reduced expression of reporter genes.
The second aim i s to characterize a mutant, iscR, found in the pilot screen for Aim 1. This gene is a homolog to the E. coli repressor of Fe-S cluster biosynthesis. In C. crescentus, the iscR mutant exhibits a dramatic G1-arrest, suggesting that it plays a critical role in the G1-S transition. Microarrays and a suppressor screen will be used to determine the mechanism by which IscR responds to environmental cues such as Fe availability and influences the G1 to S transition. The results from these experiments will clarify how organisms navigate cell-cycle progression and development by integrating multiple levels of regulation.
Research proposed here will identify novel regulators of cell-cycle and stationary phase genes, potentially discovering new targets for antibiotics. In addition, these studies will shed light on the strategies used by all organisms to regulate cell cycle progression. The C. crescentus cell cycle shares many features with the eukaryotic cell cycle and even though the individual proteins are different, the designs of the genetic circuits are strikingly similar.
