Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis

Abstract

Recent advances in cryo-electron tomography (cryo-ET) have allowed direct visualization of the initial interactions between bacteriophages and their hosts. Previous studies focused on phage infection in Gram-negative bacteria but it is of particular interest how phages penetrate the thick, highly cross-linked Gram-positive cell wall. Here we detail structural intermediates of phage Φ29 during infection of Bacillus subtilis. Use of a minicell-producing strain facilitated in situ tomographic reconstructions of infecting phage particles. Φ29 initially contacts the cell wall at an angle through a subset of the twelve appendages, which are attached to the collar at the head proximal portion of the tail knob. The appendages are flexible and switch between extended and downward conformations during this stage of reversible adsorption; appendages enzymatically hydrolyze wall teichoic acids to bring the phage closer to the cell. A cell wall-degrading enzyme at the distal tip of the tail knob locally digests peptidoglycan, facilitating penetration of the tail further into the cell wall, and the phage particle reorients so that the tail becomes perpendicular to the cell surface. All twelve appendages attain the same “down” conformation during this stage of adsorption. Once the tail has become totally embedded in the cell wall, the tip can fuse with the cytoplasmic membrane. The membrane bulges out, presumably to facilitate genome ejection into the cytoplasm, and the deformation remains after complete ejection. This study provides the first visualization of the structural changes occurring in a phage particle during adsorption and genome transfer into a Gram-positive bacterium.

Bacteriophages: Their Structural Organisation and Function

“…The structure is comparable to cryo-EM structures of mature φ29 [56,133,135]. Tomographic reconstructions demonstrated the different stages of infection [134]. In the adsorption stage, the phage is tilted to the cell wall, and both the appendages and the tail seem to contact the cell surface.…”

“…The C-terminal domain D4 acts as a chaperone for trimer assembly and is cleaved by autocatalysis. The φ29 structure attached to the lipid bilayer has been obtained by cryo-ET (34 Å) [134]. The structure is comparable to cryo-EM structures of mature φ29 [56,133,135].…”

Dual host specificity of phage SP6 is facilitated by tailspike rotation

“…It appears that each TSP can move ~30° due to rotation of the adaptor protein on its connection to the tail. By analogy with the long tail fibers of T7 and T4 (Hu et al, 2013, 2015a), and the appendages of ø29 (Farley et al, 2017), the three orientations are likely at energy minima, switching due to Brownian motion.…”

“…Recent advances in cryo-electron tomography (cryo-ET) have allowed detailed observations of some phage-host interactions at the initiation of infection (Chang et al, 2010; Farley et al, 2017; Guerrero-Ferreira et al, 2011; Hu et al, 2013, 2015a; Liu et al, 2011; Liu et al, 2010; Peralta et al, 2013; Sun et al, 2014). Two of the earliest studies capitalized on naturally small host bacteria, but the use of bacterial minicells has made it possible to determine high-resolution structures of intermediates during phage infection of other bacteria.…”

Cryo-EM Elucidation of the Structure of Bacteriophage P22 Virions after Genome Release

“…The resulting empty particles-which lack gp26 and DNA and are sometimes called ''empty heads''-contain coat protein, portal protein, tailspike protein, and all three ejection proteins (13,43). Low-resolution tomographic structures of Escherichia coli phages P1 (2) and T4 (44), cyanophage Syn5 (45), and Bacillus subtilis phage ø29 (46) bound to their target bacteria have been determined; however, there are no published high-resolution cryo-EM structures of bacteriophages that have ejected their DNA. Here, we use cryo-EM to study the structural differences between intact P22 virions and particles that have lost their DNA.…”