| I set out to search for new components of the genetic regulatory network that governs cell cycle progression in Caulobacter.;In Chapter 2, I describe a knockout screen of cell cycle regulated genes whose functions are unknown. In agreement with genome-wide studies of essential genes in E. coli (Baba et al., 2006) and Bacillus subtilis (Kobayashi et al., 2003), I found that the majority of these genes are non-essential and could be deleted easily. Furthermore, most of the deletion strains do not show a phenotype. Nevertheless, some of the non-essential genes may perform essential functions but are synthetically lethal with other genes.;From my screen, I discovered five novel essential loci, namely CC0307, CC0782, CC0903, CC1701, and CC1914. Depletion of CC0307 caused the cells to grow rounder and eventually lyse and die. CC0307 may play a role in phosphotransfer, as I showed that mutations in certain histidine residues reduced the ability of the protein to complement a deletion of the original wildtype gene. I also provided evidence that the first genome annotation of CC0782 is likely to be incorrect. In the second genome annotation, the CC0782 gene does not exist and in its place are two distinct genes. I further showed that the smaller of the two genes is probably the one that is essential.;Chapter 3 is devoted to an in-depth characterization of one of the essential cell cycle-regulated genes, CC0903, which I have named bomZ (bypass of mip Z). I showed that BomZ is a small protein whose cellular levels are tightly cell cycle regulated. Overexpression of bomZ uncouples DNA replication and cell division and causes the cells to grow filamentous. The effect of BomZ overproduction on cell division is indirect. BomZ serves as a cell cycle regulator and represses the transcription of the ctrA master regulator. BomZ overproduction also affects the localization and proteolysis mechanisms that regulate CtrA activity. I close the chapter by presenting two models that update our understanding of the regulatory network governing cell cycle progression in Caulobacter. The models incorporate several new feedback loops that I discovered and show how BomZ is linked to other important cell cycle proteins.;In Chapter 4, I summarize my PhD work and highlight several new avenues of research. First, I have investigated genes that are cell cycle regulated at the transcriptional level. However, many genes are cell cycle regulated at the post-transcriptional level. Hence, an important future direction is to study genes whose protein level, localization pattern, interacting partners, or post-translational modifications change over the course of the cell cycle. Second, a systems-level understanding of the updated genetic regulatory network governing Caulobacter cell cycle progression is lacking. A combined computational and experimental approach would help to identify critical feedback loops and to understand the key design principles of the network.;In sum, my PhD work has led to a better understanding of the genes encoded in the Caulobacter genome. The set of deletion strains that I created would serve as a useful tool for future study of Caulobacter cell biology. I also identified a new cell cycle regulator that functions at the core of the genetic circuitry that drives cell cycle progression. (Abstract shortened by UMI.)... |
