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

The SINS/zC-SINF survey of z~2 galaxy kinematics: Outflow properties

Based on SINFONI Ha, [NII] and [SII] AO data of 30 z \sim 2 star-forming galaxies (SFGs) from the SINS and zcSINF surveys, we find a strong correlation of the Ha broad flux fraction with the star formation surface density of the galaxy, with an apparent threshold for strong outflows occurring at 1 Msun yr^-1 kpc^-2. Above this threshold, we find that SFGs with logm_\ast>10 have similar or perhaps greater wind mass loading factors (eta = Mdotout/SFR) and faster outflow velocities than lower mass SFGs. This trend suggests that the majority of outflowing gas at z \sim 2 may derive from high-mass SFGs, and that the z \sim 2 mass-metallicity relation is driven more by dilution of enriched gas in the galaxy gas reservoir than by the efficiency of outflows. The mass loading factor is also correlated with the SFR and inclination, such that more star-forming and face-on galaxies launch more powerful outflows. For galaxies that have evidence for strong outflows, we find that the broad emission is spatially extended to at least the half-light radius (\sim a few kpc). We propose that the observed threshold for strong outflows and the observed mass loading of these winds can be explained by a simple model wherein break-out of winds is governed by pressure balance in the disk. Using the ratio of the [SII] doublet in a broad and narrow component, we find that outflowing gas has a density of \sim10-100 cm^-3, significantly less than that of the star forming gas (600 cm^-3).Comment: 7 pages, 3 figures, accepted by Ap

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

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

Characterization of the First Bacteriophage Tubulin Homolog PhuZ

Interactions between viruses and their host's cytoskeletal proteins have proven to be essential components of viral infections. Newly sequenced bacteriophages have genes encoding their own putative cytoskeletal proteins, but nothing was known about what role these viral cytoskeletal proteins might play in infection. Here I characterize the first bacteriophage tubulin homolog, PhuZ, and demonstrate that it has an important and conserved role during lytic growth. It is the first prokaryotic tubulin shown to be dynamically unstable in vivo and actively position a mass of phage DNA in the center of the infected cell. This positioning is important for encapsidation of phage DNA and subsequent production of progeny phage. PhuZ polymerizes by a unique mechanism that requires its extended C-terminal tail and potentially forms a three- stranded filament, a reduced prokaryotic version of the eukaryotic microtubule. Using mass spectrometry, we have determined that PhuZ is among the proteins encoded in the phage genome that are only expressed inside the infected cell and are not found in the mature virio

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