Proper spatial and temporal regulation of the cell cycle is crucial to the survival and proliferation of all organisms. Complex regulatory circuits have evolved to ensure stable, orderly progression through the cell cycle. Surprisingly, the molecular mechanisms and design principles underlying these circuits remain incompletely understood, particularly in bacteria. The alpha-proteobacterium Caulobacter crescentus is an experimentally tractable system for elucidating the fundamental mechanisms underlying cell cycle regulation in bacteria, and for understanding regulation at the systems level. The Caulobacter cell cycle is driven largely by two essential regulators: CtrA and DnaA. CtrA is a response regulator and two-component signal transduction protein that directly regulates the expression of nearly 100 genes, many of which coordinate cell division. CtrA also directly binds to and silences the origin of replication in daughter swarmer cells, but is inactivated and degraded in daughter stalked cells. CtrA thus governs the asymmetric replicative fates of daughter cells, a hallmark of the Caulobacter cell cycle. However, CtrA does not significantly influence the fundamental periodicity of DNA replication. Instead, DnaA, a nearly universal bacterial protein that initiates DNA replication, governs the periodicity of replication. In rich media, DnaA protein levels are constant, indicating that it is regulated predominantly at the level of activity. Although substantial progress has been made in understanding the regulation of CtrA and, to a lesser extent, DnaA, major gaps remain. The goal of this project is to define the complete molecular circuitry controlling CtrA and DnaA in both nutrient replete conditions and following starvation.
We aim to identify new components of this circuitry, to elucidate their connections to DnaA and CtrA, and to understand their dynamics using a combination of genetic, biochemical, bioinformatic, genomic, and cell biological approaches. Specifically, we aim to (i) determine how the activation of CtrA is coupled to DNA replication initiation, (ii) idenify and characterize factors that regulate DnaA activity during growth in rich media, and (iii) elucidate the molecular mechanisms governing CtrA and DnaA activity following nutrient starvation. These studies will unveil how bacterial cells tightly regulate DNA replication and modulate cell cycle progression in a range of growth conditions. A better understanding of how bacteria regulate their cell cycle will guide the development of new antibiotics that slow or halt the proliferation of pathogens. Finally, by comparing the regulatory strategies unveiled here to those employed in eukaryotes our work will help to reveal the design principles of regulatory circuits throughout biology.

Public Health Relevance

This project will aid efforts to develop new antibiotics by identifying and understanding essential components of the cell cycle machinery in bacteria. We have a specific focus on two-component signal transduction proteins, which have emerged as attractive drug targets because they are critical to the proliferation of bacteria but conspicuousl absent from metazoans. We are also focused on understanding DnaA, a nearly universal regulator of DNA replication in bacteria that is also, in principle, an ideal target for new antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM082899-07
Application #
8640191
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Hamlet, Michelle R
Project Start
2008-04-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
7
Fiscal Year
2014
Total Cost
$280,806
Indirect Cost
$85,187
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Le, Tung Bk; Laub, Michael T (2016) Transcription rate and transcript length drive formation of chromosomal interaction domain boundaries. EMBO J 35:1582-95
Lubin, Emma A; Henry, Jonathan T; Fiebig, Aretha et al. (2016) Identification of the PhoB Regulon and Role of PhoU in the Phosphate Starvation Response of Caulobacter crescentus. J Bacteriol 198:187-200
Wang, Xindan; Le, Tung B K; Lajoie, Bryan R et al. (2015) Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis. Genes Dev 29:1661-75
Badrinarayanan, Anjana; Le, Tung B K; Laub, Michael T (2015) Bacterial chromosome organization and segregation. Annu Rev Cell Dev Biol 31:171-99
Aakre, Christopher D; Herrou, Julien; Phung, Tuyen N et al. (2015) Evolving new protein-protein interaction specificity through promiscuous intermediates. Cell 163:594-606
Haakonsen, Diane L; Yuan, Andy H; Laub, Michael T (2015) The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters. Genes Dev 29:2272-86
Badrinarayanan, Anjana; Le, Tung B K; Laub, Michael T (2015) Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria. J Cell Biol 210:385-400
Salazar, Michael E; Laub, Michael T (2015) Temporal and evolutionary dynamics of two-component signaling pathways. Curr Opin Microbiol 24:7-14
Leslie, David J; Heinen, Christian; Schramm, Frederic D et al. (2015) Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA. PLoS Genet 11:e1005342
Le, Tung Bk; Laub, Michael T (2014) New approaches to understanding the spatial organization of bacterial genomes. Curr Opin Microbiol 22:15-21

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