A bacteriophage tubulin harnesses dynamic instability to center DNA in infected cells.

Dynamic instability, polarity, and spatiotemporal organization are hallmarks of the microtubule cytoskeleton that allow formation of complex structures such as the eukaryotic spindle. No similar structure has been identified in prokaryotes. The bacteriophage-encoded tubulin PhuZ is required to position DNA at mid-cell, without which infectivity is compromised. Here, we show that PhuZ filaments, like microtubules, stochastically switch from growing in a distinctly polar manner to catastrophic depolymerization (dynamic instability) both in vitro and in vivo. One end of each PhuZ filament is stably anchored near the cell pole to form a spindle-like array that orients the growing ends toward the phage nucleoid so as to position it near mid-cell. Our results demonstrate how a bacteriophage can harness the properties of a tubulin-like cytoskeleton for efficient propagation. This represents the first identification of a prokaryotic tubulin with the dynamic instability of microtubules and the ability to form a simplified bipolar spindle

Shaping the Future of Research: a perspective from junior scientists

The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 nd and 3 rd, 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers’ synthesis of the outcomes

Best practices for fluorescence microscopy of the cyanobacterial circadian clock.

This chapter deals with methods of monitoring the subcellular localization of proteins in single cells in the circadian model system Synechococcus elongatus PCC 7942. While genetic, biochemical, and structural insights into the cyanobacterial circadian oscillator have flourished, difficulties in achieving informative subcellular imaging in cyanobacterial cells have delayed progress of the cell biology aspects of the clock. Here, we describe best practices for using fluorescent protein tags to monitor localization. Specifically, we address how to vet fusion proteins and overcome challenges in microscopic imaging of very small autofluorescent cells

Chapter Eleven Best Practices for Fluorescence Microscopy of the Cyanobacterial Circadian Clock

This chapter deals with methods of monitoring the subcellular localization of proteins in single cells in the circadian model system Synechococcus elongatus PCC 7942. While genetic, biochemical, and structural insights into the cyanobacterial circadian oscillator have flourished, difficulties in achieving informative subcellular imaging in cyanobacterial cells have delayed progress of the cell biology aspects of the clock. Here, we describe best practices for using fluorescent protein tags to monitor localization. Specifically, we address how to vet fusion proteins and overcome challenges in microscopic imaging of very small autofluorescent cells

The Phage Nucleus and Tubulin Spindle Are Conserved among Large Pseudomonas Phages

We recently demonstrated that the large Pseudomonas chlororaphis bacteriophage 201φ2-1 assembles a nucleus-like structure that encloses phage DNA and segregates proteins according to function, with DNA processing proteins inside and metabolic enzymes and ribosomes outside the nucleus. Here, we investigate the replication pathway of the Pseudomonas aeruginosa bacteriophages φKZ and φPA3. Bacteriophages φKZ and φPA3 encode a proteinaceous shell that assembles a nucleus-like structure that compartmentalizes proteins and DNA during viral infection. We show that the tubulin-like protein PhuZ encoded by each phage assembles a bipolar spindle that displays dynamic instability and positions the nucleus at midcell. Our results suggest that the phage spindle and nucleus play the same functional role in all three phages, 201φ2-1, φKZ, and φPA3, demonstrating that these key structures are conserved among large Pseudomonas phages

Dynamic localization of the cyanobacterial circadian clock proteins.

BackgroundThe cyanobacterial circadian clock system has been extensively studied, and the structures, interactions, and biochemical activities of the central oscillator proteins (KaiA, KaiB, and KaiC) have been well elucidated. Despite this rich repository of information, little is known about the distribution of these proteins within the cell.ResultsHere we report that KaiA and KaiC localize as discrete foci near a single pole of cells in a clock-dependent fashion, with enhanced polar localization observed at night. KaiA localization is dependent on KaiC; consistent with this notion, KaiA and KaiC colocalize with each other, as well as with CikA, a key input and output factor previously reported to display unipolar localization. The molecular mechanism that localizes KaiC to the poles is conserved in Escherichia coli, another Gram-negative rod-shaped bacterium, suggesting that KaiC localization is not dependent on other clock- or cyanobacterial-specific factors. Moreover, expression of CikA mutant variants that distribute diffusely results in the striking delocalization of KaiC.ConclusionsThis work shows that the cyanobacterial circadian system undergoes a circadian orchestration of subcellular organization. We propose that the observed spatiotemporal localization pattern represents a novel layer of regulation that contributes to the robustness of the clock by facilitating protein complex formation and synchronizing the clock with environmental stimuli

Article Dynamic Localization of the Cyanobacterial Circadian Clock Proteins

Summary Background: The cyanobacterial circadian clock system has been extensively studied, and the structures, interactions, and biochemical activities of the central oscillator proteins (KaiA, KaiB, and KaiC) have been well elucidated. Despite this rich repository of information, little is known about the distribution of these proteins within the cell. Results: Here we report that KaiA and KaiC localize as discrete foci near a single pole of cells in a clock-dependent fashion, with enhanced polar localization observed at night. KaiA localization is dependent on KaiC; consistent with this notion, KaiA and KaiC colocalize with each other, as well as with CikA, a key input and output factor previously reported to display unipolar localization. The molecular mechanism that localizes KaiC to the poles is conserved in Escherichia coli, another Gram-negative rod-shaped bacterium, suggesting that KaiC localization is not dependent on other clock-or cyanobacterial-specific factors. Moreover, expression of CikA mutant variants that distribute diffusely results in the striking delocalization of KaiC. Conclusions: This work shows that the cyanobacterial circadian system undergoes a circadian orchestration of subcellular organization. We propose that the observed spatiotemporal localization pattern represents a novel layer of regulation that contributes to the robustness of the clock by facilitating protein complex formation and synchronizing the clock with environmental stimuli

The Structure and Assembly Mechanism of a Novel Three-Stranded Tubulin Filament that Centers Phage DNA

Tubulins are a universally conserved protein superfamily that carry out diverse biological roles by assembling filaments with very different architectures. The underlying basis of this structural diversity is poorly understood. Here, we determine a 7.1 Å cryo-electron microscopy reconstruction of the bacteriophage-encoded PhuZ filament and provide molecular-level insight into its cooperative assembly mechanism. The PhuZ family of tubulins is required to actively center the phage within infected host cells, facilitating efficient phage replication. Our reconstruction and derived model reveal the first example of a three-stranded tubulin filament. We show that the elongated C-terminal tail simultaneously stabilizes both longitudinal and lateral interactions, which in turn define filament architecture. Identified interaction surfaces are conserved within the PhuZ family, and their mutagenesis compromises polymerization in vitro and in vivo. Combining kinetic modeling of PhuZ filament assembly and structural data, we suggest a common filament structure and assembly mechanism for the PhuZ family of tubulins

OmpA and OmpC are critical host factors for bacteriophage Sf6 entry in Shigella.

Despite being essential for successful infection, the molecular cues involved in host recognition and genome transfer of viruses are not completely understood. Bacterial outer membrane proteins A and C co-purify in lipid vesicles with bacteriophage Sf6, implicating both outer membrane proteins as potential host receptors. We determined that outer membrane proteins A and C mediate Sf6 infection by dramatically increasing its rate and efficiency. We performed a combination of in vivo studies with three omp null mutants of Shigella flexneri, including classic phage plaque assays and time-lapse fluorescence microscopy to monitor genome ejection at the single virion level. Cryo-electron tomography of phage 'infecting' outer membrane vesicles shows the tail needle contacting and indenting the outer membrane. Lastly, in vitro ejection studies reveal that lipopolysaccharide and outer membrane proteins are both required for Sf6 genome release. We conclude that Sf6 phage entry utilizes either outer membrane proteins A or C, with outer membrane protein A being the preferred receptor