title: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA.
authors.forename: Madeline M
authors.initials: MM
authors.lastname: Farley
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA.
authors.forename: Jiagang
authors.initials: J
authors.lastname: Tu
authors.affiliation: Department of Biology, Indiana University, 1001 East Third Street, Bloomington, IN 47405, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Kearns
authors.affiliation: Center for Infectious Disease, Department of Molecular Biosciences, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA. Electronic address: molineux@austin.utexas.edu.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Molineux
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA. Electronic address: Jun.Liu.1@uth.tmc.edu.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
mesh_terms: D001412:Bacillus subtilis / Q000648:ultrastructure* / Q000821:virology; D001435:Bacteriophages / Q000472:pathogenicity / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods; D015999:Multivariate Analysis; D010457:Peptidoglycan / Q000648:ultrastructure
chemical_list: D010457:Peptidoglycan
keywords: Bacteriophage infection; Cryo-electron tomography; Gram-positive bacterium; Molecular machine