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

Abstract

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.

Keywords: PhuZ; compartmentalization; nucleus; nucleus origin; nucleus positioning; phage; spindle; tubulin; viral eukaryogenesis.

Figure 1. 201Φ2-1, ΦKZ, and ΦPA3 assemble a nucleus-like structure that compartmentalizes proteins and DNA during viral replication

Cells were grown on an agarose pad and the fusion proteins were induced with arabinose at the indicated concentrations. P. chlororaphis was infected with 201Φ2-1 and P. aeruginosa with phages ΦKZ or ΦPA3, and imaged at approximately 60 mpi (A,B,D) or 90 mpi (C,E). Phage DNA is stained with DAPI (blue). These proteins do not assemble in uninfected cells. All scale bars equal 0.5 micron. See also Figure S1. A) Top row: Fluorescence micrographs of infected P. chlororaphis and infected P. aeruginosa cells expressing fluorescent proteins (GFP, green) or (mCherry, red) alone from the arabinose promoter. Second row: Fluorescence micrographs of infected P. chlororaphis and infected P. aeruginosa cells expressing fluorescent protein fusions to the conserved nuclear shell proteins gp105 of 201Φ2-1 (0.2% arabinose), gp054 of ΦKZ (0.025% arabinose), and gp053 of ΦPA3 (0.025% arabinose). In rows two through four, the square panel on the left is an enlarged image of fluorescent proteins shown within cells whose border is indicated by a dotted line. Third row: Fluorescence micrographs of infected P. chlororaphis and infected P. aeruginosa cells expressing fluorescent protein fusions to the major capsid proteins gp200 of 201Φ2-1 (0.2% arabinose), gp120 of ΦKZ (0.05% arabinose), and gp136 of ΦPA3 (0.05% arabinose). Fourth row: Fluorescence micrographs of infected P. chlororaphis and infected P. aeruginosa cells expressing fluorescent protein fusions to the RecA-like protein gp237 of 201Φ2-1 (0.2% arabinose), gp152 of ΦKZ (0.025% arabinose), and gp175 of ΦPA3 (0.025% arabinose). B) Co-localization of RecA-related proteins ΦKZ-gp152-mCherry (red) and ΦPA3-gp175-GFP (green) in the 201Φ2-1 nucleus (stained blue by DAPI or gray) during phage 201Φ2-1 infection. P. chlororaphis expressing ΦKZ-gp152-mCherry and ΦPA3-gp175-GFP (0.1% arabinose) was infected with 201Φ2-1 and visualized at 60 mpi. The region indicated by a dashed box is magnified and shown on the right. Scale bar equals 0.5 micron. C) Time-lapse imaging (seconds) of ΦKZ-gp152-mCherry (red) and ΦPA3-gp175-GFP (green) in the 201Φ2-1 nucleus showing both RecA-related proteins form foci which move together as the 201Φ2-1 nucleus rotates over the course of 58 seconds. The position of the focus (asterisk) was tracked for the duration of the time-lapse. Scale bar equals 0.5 micron. See also Movie S1. D) Graph showing percentage of 201Φ2-1 nuclei of infected P. chlororaphis cells containing no proteins inside (none), either ΦKZ-gp152 or ΦPA3-gp175 inside, or both RecA-related proteins inside. Data were collected from the infected cells at 60 mpi from at least 3 different fields and are represented as mean ± standard error of the mean (n = 263). E) SIM images of infected P. chlororaphis and infected P. aeruginosa at 90 mpi showing encapsidated phage clusters localized at midcell as determined by DAPI staining (blue or gray). GFP-PhuZ filaments (green) extend from both cell poles toward the viral nucleus at midcell. 3D-SIM and rotation of the phage nucleus around Y-axis show that the nucleus is surrounded with encapsidated phages (degree of rotation is indicated below each subset). Scale bar equals 0.5 micron.

Figure 2. PhuZ requires a critical threshold concentration to polymerize in P. aeruginosa

Cells were grown on an agarose pad and the fusion proteins were induced at the indicated arabinose concentrations. Cell membranes were stained with FM4-64 (red). All scale bars equal 1 micron. See also Figures S2 and S3. A) Fluorescence images of cells expressing wild-type GFP-PhuZ_ΦKZ at the indicated arabinose concentrations. B) P. aeruginosa cells expressing the GTPase mutant GFP-PhuZ_ΦKZD204A at the indicated arabinose concentrations. C) Graph showing the percentage of cells containing either wild-type or mutant PhuZ_ΦKZ (D201A or D204A) filaments when fusion proteins are expressed at increasing levels of arabinose ranging from 0% to 1%. The graph below replots the same data showing the range of arabinose concentration from 0% to 0.2%. Data represented as mean values were collected from the induced cells at indicated arabinose concentration from at least 3 different fields with at least 200 cells per field. D) Fluorescence images of cells expressing wild-type GFP-PhuZ_ΦPA3 at the indicated arabinose concentrations. E) P. aeruginosa cells expressing the GTPase mutant GFP-PhuZ_ΦPA3D190A at the indicated arabinose concentrations. F) Graph showing the percentage of cells containing either wild-type or mutant PhuZ_ΦPA3 (D187A or D190A) filaments when fusion proteins are expressed at increasing levels of arabinose ranging from 0% to 1%. The graph below replots the same data showing the range of arabinose concentration from 0% to 0.2%. Data represented as mean values were collected from the induced cells at indicated arabinose concentration from at least 3 different fields with at least 200 cells per field.

Figure 3. PhuZ_ΦKZ and PhuZ_ΦPA3 show dynamic instability and center the phage nucleus in the cell, while the GTPase mutants fail to position the nucleus at midcell

Cells were grown on agarose pads and the fusion proteins were induced with 0.05% arabinose (below the critical threshold for filament assembly) for infected cells and with 0.1% arabinose (to study the dynamic instability of filaments) for uninfected cells. Cell membranes were stained red by FM4-64 and phage DNA was stained blue by DAPI. All scale bars equal 1 micron. See also Figures S3 and S4. (A and C) Fluorescence micrographs of uninfected P. aeruginosa cells induced at 0.1% arabinose to express wild-type GFP-PhuZ_ΦKZ (A) and wild-type GFP-PhuZ_ΦPA3 (C). The filament in the dashed box is magnified and shown on the right. Time-lapse imaging and kymograph for one representative filament for each protein (GFP-PhuZ_ΦKZ (A) and GFP-PhuZ_ΦPA3 (C)) and a graph showing length changes for five representative filaments for each protein show that these filaments display dynamic instability. (B and D) Fluorescence micrographs of uninfected P. aeruginosa cells induced at 0.1% arabinose to express mutant GFP-PhuZ_ΦKZ (B) and mutant GFP-PhuZ_ΦPA3 (D). Time-lapse imaging and kymograph for one representative filament. The graph shows length changes of five representative mutant filaments. (E and G) Fluorescence micrographs of ΦKZ-infected (E) and ΦPA3-infected (G) P. aeruginosa cells induced at 0.05% arabinose to express wild-type GFP-PhuZ_ΦKZ (E) or wild-type GFP-PhuZ_ΦPA3 (G). The dynamic GFP-PhuZ spindle (green) and phage nucleus (blue) was centered in the infected cell at 60 mpi. (F and H) Fluorescence micrographs of ΦKZ-infected (F) and ΦPA3-infected (H) P. aeruginosa cells induced at 0.05% arabinose to express mutant GFP-PhuZ_ΦKZ (F) and mutant GFP-PhuZ_ΦPA3 (H). The mutant PhuZ proteins (green) assembled a single static filament instead of a bipolar spindle resulting in mispositioning of the phage nucleus (blue) at 60 mpi. (I) Graph showing the position of the phage nucleus in P. aeruginosa cells, expressed as a fraction of cell length. Cells expressed either wild-type GFP-PhuZ_ΦKZ (blue circle), wild-type GFP-PhuZ_ΦPA3 (green diamond), GTPase mutant GFP-PhuZ_ΦKZ (orange circle) and GTPase mutant GFP-PhuZ_ΦPA3 (red diamond). One hundred cells were counted for each group. (J) Histogram showing position of the phage nucleus in which the data from Figure I is replotted as percentage of total cells. Cells expressed either wild-type GFP-PhuZ_ΦKZ (blue), wild-type GFP-PhuZ_ΦPA3 (green), GTPase mutant GFP-PhuZ_ΦKZ (orange) and GTPase mutant GFP-PhuZ_ΦPA3 (red). One hundred cells were counted for each group.

Figure 4. Model of phage nucleus assembly and its role in the viral life cycle in large Pseudomonas phages

As soon as the phage injects DNA into its host, the nuclear shell protein assembles a compartment to protect its genomic DNA from host defenses and to provide a compartment for DNA replication. Dynamically unstable PhuZ filaments set up a spindle with one end anchored at the cell pole while the other end of the spindle pushes the compartment toward midcell. The spindle might rely upon a yet to be discovered factor that organizes its assembly. As DNA replication proceeds inside the compartment, the nucleus grows in size as it is pushed toward the cell center. Late during infection, capsids dock on the surface of the phage nucleus for DNA encapsidation. The mature phage particles are assembled in the cytoplasm and the host cell lyses.