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Bacterial Cellular Organization

Research Summary

Christine Jacobs-Wagner explores the internal organization of bacteria at every level, from the molecular mechanisms involved in its origin, maintenance, and replication in time and space to its function in cellular physiology and morphogenesis.

Life requires cellular organization in time and space, a principle that applies to all branches of the evolutionary tree. Even bacteria, once erroneously perceived as tiny bags of jumbled-up molecules, rely on temporal and spatial organization for processes as diverse as cell growth, development, pathogenesis, motility, cell cycle control, cell division, and chromosome segregation.

Bacterial cells are highly polarized, possess a cytoskeleton, order their chromosomes in space, and localize structural and regulatory proteins. They depend critically on this surprisingly sophisticated cellular organization. This realization—mostly within the past few years—has revolutionized the microbiology field and has resulted in a recent surge of interest in bacterial cell biology. This emerging discipline is still in its infancy and much remains to be discovered. How cellular organization is achieved in bacteria remains largely elusive. The acquisition and propagation of spatial information are often tightly coordinated with the cell cycle to ensure the maintenance of this information within the cell population. The mechanisms by which this temporal control occurs are also poorly understood.

Figure 1: Cell cycle schematic...

My laboratory primarily uses the Gram-negative bacterium Caulobacter crescentus as a model to (1) understand the control mechanisms underlying bacterial multiplication; (2) decipher the molecular mechanisms by which bacterial cellular organization is achieved, maintained, and replicated in time and space; and (3) establish how this spatiotemporal information controls the physiology, morphogenesis, and behavior of the cell. Given the dramatic impact of bacteria on human health and activities, such a fundamental understanding of bacterial biology is critical. This understanding will provide a basis for the rational design of novel antibacterial strategies and for the innovative use of bacteria in medicine, industry, and the environment. For our studies, we use a wide array of imaging techniques and an arsenal of genetic, biochemical, and computational tools.

Cell Cycle Regulation and Cell Polarization
Bacteria are infamous for their rapid replication rates, which are in part possible because of robust cell cycle mechanisms ensuring that every division produces a pair of cells with a full complement of genetic material. We seek to understand the mechanisms involved in DNA partitioning and cell cycle control. C. crescentus has a unique set of strengths for studying cell cycle events in time and space. In this bacterium, distinct cell cycle phases are readily distinguishable by morphological differences, as cell cycle progression is associated with a developmental program (Figure). Additionally, we can easily use a simple separation technique to isolate C. crescentus cells in G1 phase, providing an efficient means for synchronizing cell populations for cell cycle studies.

We and others have shown that chromosomal segregation in C. crescentus is driven by the ParA/ParB/parS system that is widely distributed across bacterial phyla. The partitioning protein ParB binds to the parS sequence near the origin of replication to form the partition complex (Movie 1). ParA forms an asymmetric DNA-bound cloud-like structure whose retraction drives the segregation of the ParB/parS partition complex. The active motion of the partition complex (Movie 1) relies on ParA ATPase activity, while the speed and directionality of DNA segregation is dependent on the polarity factor TipN, which localizes to the tip of the "new" cell pole (i.e., the pole formed by the most recent division). Once segregation is completed, the pole-organizing protein PopZ binds to the ParB/parS complex at the pole and thereby anchors the chromosome. This polar anchoring not only ensures that each daughter cell inherits a copy of the chromosome but also couples segregation with cell division by dictating when and where the cytokinetic ring forms.

Movie 1: Segregation of the chromosome partition complex ParB/parS in a Caulobacter crescentus cell. Movie: Whitman Schofield, in the Jacobs-Wagner lab.

Many factors, including signaling proteins, localize at the cell poles to regulate cell cycle events during the cell cycle. How proteins recognize the poles is therefore a fundamental question in the field. We have shown that bacteria use multifunctional "hubs" to recruit multiple proteins to the same location. For example, the chromosome-anchoring protein PopZ recruits multiple proteins to the poles, where it couples their activity to DNA segregation. Several lines of evidence suggest that PopZ localizes to cell poles through a self-organizing mechanism that relies on PopZ self-assembly in chromosome-free regions. TipN is also required for the localization of proteins and structures (e.g., the flagellum) at the new pole. TipN "recognizes" the new pole by tracking the most recent cell division event; by doing so, it reestablishes the polarity axis of the cell after each division.

Cell Morphogenesis and the Cytoskeleton
Bacteria exhibit specific shapes and sizes that are important for cell function and survival. As in eukaryotes, the cytoskeleton plays a central role in cell morphogenesis in bacteria. We discovered that intermediate filaments, which constitute one of the three major cytoskeletal systems in animal cells, are found in bacteria, contrary to previous belief. In C. crescentus, the intermediate filament protein crescentin forms a filamentous structure that mediates the characteristic curvature of this organism by mechanically affecting cell wall growth.

We also study how other cytoskeletal elements affect cell morphogenesis, combining functional studies and quantitative analyses of the localization and dynamic properties of cell wall enzymes during the cell cycle. We examine the cell wall growth pattern in relation to the function and localization of each cytoskeletal protein. Genetic screens and bioinformatic approaches are employed to identify new players. Using this strategy, we have, for example, identified RodZ, a conserved component of the core morphogenetic machinery that interacts with the actin homolog MreB to regulate cell wall growth.

Spatial Organization of mRNA Processes
By visualizing specific transcripts in C. crescentus, we recently showed that protein synthesis largely occurs from a localized source dictated by the chromosome organization. We also provided evidence that mRNA degradation is spatially regulated. Our findings revealed that bacteria spatially organize the flow of genetic information, despite lacking membrane-bound organelles such as a nucleus.

In Vivo Biochemistry
Most of our knowledge on biochemical processes inside cells comes from in vitro studies using purified components or cell extracts, even though test-tube solutions poorly recapitulate the cytoplasmic environment. Unlike test-tube solutions, the cytoplasm is heterogeneous, spatially structured, highly crowded with macromolecules, and far from thermodynamic equilibrium; all of these characteristics can affect biochemical rates. Hence, methods that allow measurements of binding kinetics and molecular mobility in live cells are critical for our understanding of any biochemical process.

To address this need, we have developed a new, broadly applicable methodology for performing in vivo biochemistry in bacterial cells. This method is based on fluorescence recovery after photobleaching microscopy and reaction-diffusion modeling. As a demonstration, we implemented our method to gain new quantitative insight into multiple phases (initiation, elongation, and ribosome recycling) of the bacterial translation cycle by determining, for the first time in live bacterial cells, binding rate constants and diffusion coefficients of free and active ribosomes.

This work is partially supported by grants from the National Institutes of Health.

As of December 12, 2012

Scientist Profile

Investigator
Yale University
Cell Biology, Microbiology