Bacteria are infamous for their fast replication rates, which are in part possible because of robust cell cycle mechanisms that ensure that virtually every division produces progeny with a full complement of the genetic material. In this project, we seek to understand the mechanisms involved in DNA partitioning and cell cycle control with the long-term goal of generating new strategies to control bacterial growth as bacteria remain a serious threat to human health. Our bacterial model of choice is the genetically tractable bacterium Caulobacter crescentus, a well-established cell cycle model for which synchronized cell cycle populations are easily obtained and whose cell cycle is conveniently associated with morphological transitions. In C. crescentus, chromosome segregation is initiated by the motion of the parS DNA sequence near the origin of replication. This active motion is dependent on the broadly conserved proteins ParA and ParB.
Our first aim i s to build on our previous findings to dissect the mechanism underlying the ParABS-dependent segregation mechanism. For this, we will use a battery of in vitro biochemical assays to examine the dimerization and ATPase activity of wild-type and mutant ParA proteins in the presence of varying concentrations of DNA, ParB and nucleotides. In addition, we will use super-resolution microscopy techniques to image the process of DNA segregation inside cells. Since the cell polarization factors TipN and PopZ affect the ParABS system, our second aim is to elucidate the function of these two proteins. Our recent findings suggest that TipN affects the directionality and speed of DNA segregation by sequestering ParA monomers. We will test this model using different ParA mutants and a combination of established biochemical, cytological and genetic assays. PopZ is known to assemble into a high-order structure at the cell poles where it tethers the ParB/parS partition complex and recruits cell cycle signaling proteins. Using a mutagenesis approach, we will address how PopZ achieves these activities and whether one activity (e.g., assembly into a high-order structure) is required for another (e.g., polar localization and/or interaction with ParB/parS).
Our third aim i s to address the intrinsic cell cycle mechanisms that regulate cell size in C. crescentus. This directly relates to the first two aims as previous work from our lab shows that the segregation of the ParB/parS complex is regulated by cell size and not time. Because of the coupling between DNA segregation, growth and division, defects in TipN, PopZ or the ParABS system result in cell size aberrations. We propose to perform a comprehensive single-cell study of various strains with different cell size distributions using the powerful image analysis software MicrobeTracker that we developed. This study will examine the contribution of each cell cycle event in cell size compensation, and provide new insight into cell cycle control.

Public Health Relevance

Bacterial multiplication relies on robust cell cycle mechanisms that ensure that all progeny inherit a copy of the bacterial genome at each division. This project will provide new, quantitative insight into the poorly understood mechanisms by which bacteria can drive and regulate DNA segregation and cell cycle progression. Given the dramatic impact of bacteria on human health and activities, such a fundamental understanding of bacterial biology is critical as a basis for rational design of novel broad-spectrum antibacterial strategies

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065835-11
Application #
8705531
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Hamlet, Michelle R
Project Start
2003-05-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
11
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
New Haven
State
CT
Country
United States
Zip Code
06510
Campos, Manuel; Govers, Sander K; Irnov, Irnov et al. (2018) Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli. Mol Syst Biol 14:e7573
Surovtsev, Ivan V; Jacobs-Wagner, Christine (2018) Subcellular Organization: A Critical Feature of Bacterial Cell Replication. Cell 172:1271-1293
Irnov, Irnov; Wang, Zhe; Jannetty, Nicholas D et al. (2017) Crosstalk between the tricarboxylic acid cycle and peptidoglycan synthesis in Caulobacter crescentus through the homeostatic control of ?-ketoglutarate. PLoS Genet 13:e1006978
Arias-Cartin, Rodrigo; Dobihal, Genevieve S; Campos, Manuel et al. (2017) Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter. EMBO J 36:301-318
Surovtsev, Ivan V; Lim, Hoong Chuin; Jacobs-Wagner, Christine (2016) The Slow Mobility of the ParA Partitioning Protein Underlies Its Steady-State Patterning in Caulobacter. Biophys J 110:2790-9
Paintdakhi, Ahmad; Parry, Bradley; Campos, Manuel et al. (2016) Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis. Mol Microbiol 99:767-77
Huang, Fang; Sirinakis, George; Allgeyer, Edward S et al. (2016) Ultra-High Resolution 3D Imaging of Whole Cells. Cell 166:1028-1040
Surovtsev, Ivan V; Campos, Manuel; Jacobs-Wagner, Christine (2016) DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos. Proc Natl Acad Sci U S A 113:E7268-E7276
Campos, Manuel; Surovtsev, Ivan V; Kato, Setsu et al. (2014) A constant size extension drives bacterial cell size homeostasis. Cell 159:1433-46
Laloux, GĂ©raldine; Jacobs-Wagner, Christine (2014) How do bacteria localize proteins to the cell pole? J Cell Sci 127:11-9

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