A phage tubulin assembles dynamic filaments by an atypical mechanism to center viral DNA within the host cell

Abstract

Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.

Figure 1. Phylogenetic relationship and conserved sequences of PhuZ and other distantly related tubulins

A. Phylogenetic tree showing the relationship of divergent tubulins encoded by Pseudomonas phage (PhuZ), Clostridial sequences from chromosomes (Cb,Ck,Cl,CA), plasmid (pCL2), and phage (Cst, Cbc), TubZ (pBtoxis/B.thuringiensis and pH308197/B. cereus) and representative bacterial FtsZ sequences. PhuZ sequences (green): GP59 (P.chlororaphis phage 201ϕ2-1); GP39 (P.aeruginosa phage ϕKZ), and GP16 (P.aeruginosa phage EL). FtsZ sequences: Bs, B.subtilis; Ba, B.anthracis; Sa, S.aureus; Cb, C.botulinum; Mt, M.tuberculosis, Ec, E.coli; Vc, V.cholerae, Pa, P.aeruginosa; Hi, H.influenzae. Clostridial sequences (blue): CA, CAC3459 Clostridium acetobutylicum ATCC824; Cb, CBY3413 C.butyricum 5521; Ck, CKL0570 C. kluyveri DSM555; Cl, Clocel4294 C.cellulovorans 743B; pCL2, pCLG2A0045 C.botulinum str.1873; Cst, Cst189 C.botulinum phage C-st, Cbc, CBCA1765 C.botulinum C str. Eklund; eukaryotic α/β tubulin sequences (cyan): Dd, Dictyostelium discoideum, Sc, Saccharomyces cerevisiae, Ss, Sus scrofa. Sequences were aligned using Tcoffee and the tree was generated using ClustalW. Bootstrap values are given for selected branches. B. Alignment of L1 and G box motifs for PhuZ, TubZ, α/β tubulin and representative bacterial FtsZ sequences. Conserved residues are in red. PhuZ GP59 (phage 201ϕ2-1) GP39 (phage ϕKZ), and GP16 (phage EL), TubZ from pBtoxis, FtsZ sequences (Bs, B.subtilis; Mt, M.tuberculosis; Ec, E.coli). α/β-tubulin of Ss, Sus scrofa C. Alignment of the last 13 amino acids of PhuZ (GP59) that make up the acidic knuckle with PhuZ related proteins encoded by phage ϕKZ (GP39) and EL (GP16). Conserved acidic residues are in red.

Figure 2. PhuZ polymer assembly in P.chlororaphis

A. Fluorescent micrographs of P.chlororaphis cells expressing wild type GFP-PhuZ grown at 30°C and induced with the indicated amount (%) of arabinose. Scale bar equals 1 micron. B and C. The catalytic point mutant GFP-PhuZD190A forms filaments that become trapped in septa. Membranes are stained red with FM4–64. D. Photobleaching of GFP-PhuZD190A. The bleached zone generated at t0 seconds (red bracket) does not move or recover after 112s, indicating that the filaments are static. E. Graphs showing the percentage of cells containing filaments when fusion proteins are expressed at increasing levels. Cells were grown at 30°C for wild type and catalytic point mutants. See also Figure S4, Movies S1, S2, S3.

Figure 3. In vitro polymerization of PhuZ

A. Right angle light scattering traces of PhuZ polymerization at 5 (red), 6.25 (blue), 7.5 (green) µM upon addition of 1 mM GTP. Black trace is of 15 µM PhuZ with 1 mM GDP. B. Right angle light scattering traces of PhuZ mutants at 20 µM (ΔI302, black; R298A, green; D311A; blue) show no detectable polymer formation. 6.25 µM wild-type trace shown for comparison. C. Negative stain EM of 7 µM PhuZ polymerized in the presence of 1 mM GMPCPP at 36000×. Two boxed segments of filaments collected at 52000× are shown at right to show detailed filament. See also Figure S2.

Figure 4. Structure and nucleotide binding of PhuZ

A. Cartoon representation of the PhuZ structure with the N-terminal domain shown in orange, the interdomain in yellow, the C-terminal domain in slate, helix H11 in pink, and the C-terminal tail in cyan. The bound GDP is shown as spheres. B. Top-down view of the nucleotide-binding pocket. 2F_o-F_c prior to addition of Mg-GDP to model shown as mesh at 2 σ See also Figure S3, Tables S1, S2.

Figure 5. Crystal lattice contains filament-like contacts with the C-terminal tail providing most of the contact surface

A. Cartoon representation of PhuZ (wheat with hot pink tail) with five symmetry mates (grey50), nucleotide shown as spheres, reveals two-stranded filament within the crystal lattice and extensive contacts by the C-terminal tail. B. Electrostatic surface of PhuZ shown interacting with the C-terminal tail. The tail buries 1226 Ų of surface area per monomer. Residues R298, I302, and D311 are highlighted. C. Average of 500 segments of PhuZ polymers observed by negative-stain EM. Spacing between longitudinal monomers is ~47 Å. D–E. Fluorescent micrographs of P.chlororaphis cells expressing the C-terminal tail mutants D311A (D) and R298A (E) grown at room temperature and induced with the indicated amount (%) of arabinose. Both fail to assemble polymers except at the highest expression levels (1% and 2%). Scale bar equals 1 micron. F. Graph showing the percentage of cells containing filaments when fusion proteins are expressed at increasing levels at 25°C. See also Figure S1.

Figure 6. A single cell assay for phage infection reveals that PhuZ assembles filaments in vivo during infection of the host cell with 201ϕ2-1

A. In cells grown with 0.15% arabinose (below critical threshold inducer concentration), filaments first appeared 56 minutes after phage addition and the cell lysed after 175 minutes, as revealed by FM4–64 staining. Staining with DAPI/DNAseI indicates phage release. B. In cells grown with 0.25% arabinose, filaments first appeared 39 minutes after phage addition and polymers underwent cycles of assembly and disassembly until the cell lysed after 140 minutes. Scale bar equals 1 micron. C. Two examples of infected cells stained with FM4–64 (red) and DAPI (blue) at 90 minutes post infection showing a large mass of DNA in the center of the cell. D. Six examples of infected cells showing filaments of GFP-PhuZ on either side of a centrally located DAPI stained nucleoid. E–G. Cells were fixed and treated with DNAse I to degrade all DNA except that encapsidated by phage. E. An example of GFP-PhuZ filaments surrounding DAPI foci at midcell. F. Two examples of rosette structures formed during infection and visualized after DNAaseI digestion. G. A series of optical sections through a DNAseI digested nucleoid showing phage encapsidated DNA occurs in a circular pattern. Numbers indicate distance in nanometers from the first optical section. On the far right, a 3-D fluorescence intensity graph of DAPI fluorescence corresponding to the 900nm optical section showing the rosette pattern of foci localization. H. Four panels showing DAPI stained cells expressing the GTPase mutant GFP-PhuZD190A after 90 minutes of phage infection. The phage nucleoid is frequently positioned at the pole of the cell. Some cells (far left) contain two or three nucleoids. I. Histogram showing the percentage of cells with the phage nucleoid located near the center (50% cell length) of the cell for wild type GFP-PhuZ (red) or mutant GFP-PhuZD190A (blue). J. Graph showing the position of the phage nucleoid as a fraction of cell length versus cell length for wild type GFP-PhuZ (red) or mutant GFP-PhuZD190A (blue). K. Histogram showing the percentage of infected cells expressing either wild type GFP-PhuZ (red) or mutant GFP-PhuZD190A (blue) containing one, two or three phage nucleoids. See also Figure S5, Movies S4, S5, S6.

Figure 7

After 201ϕ2-1 infects a cell, the host chromosome is degraded and short PhuZ filaments appear that eventually extend from the poles of the cell to the phage nucleoid in the center. The PhuZ spindle positions the phage DNA in the center of the cell to allow 201ϕ2-1 genomes to be efficiently replicated and/or packaged into the capsids. After the completion of phage assembly, the cell lyses, expelling mature phage into the environment.