Entry into the host bacterial cell is one of the least understood steps in the life cycle of bacteriophages. The different envelopes of Gram-negative and Gram-positive bacteria, with a fluid outer membrane and exposing a thick peptidoglycan wall to the environment respectively, impose distinct challenges for bacteriophage binding and (re)distribution on the bacterial surface. Here, infection of the Gram-positive rod-shaped bacterium Bacillus subtilis by bacteriophage SPP1 was monitored in space and time. We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB, which is encoded by a putative type VII secretion system gene cluster. YueB was found to concentrate at the cell poles and to display a punctate peripheral distribution along the sidewalls of B. subtilis cells. The kinetics of SPP1 DNA entry and replication were visualized during infection. Most of the infecting phages DNA entered and initiated replication near the cell poles. Altogether, our results reveal that the preferentially polar topology of SPP1 receptors on the surface of the host cell determines the site of phage DNA entry and subsequent replication, which occurs in discrete foci.Bacterial viruses (phages or bacteriophages) are the most abundant biological entities in the biosphere (25). The vast majority (96% of the phages currently described [2]) use a tail device for specific recognition of the host, binding to its surface, and delivery of their genome from their icosahedral capsid to the bacterial cytoplasm. The first contact with the bacterial surface usually leads to reversible binding that is not saturable. It allows for dissociation of viable phages from the bacterium. In a second step, phages attach irreversibly to a specific cell envelope receptor committing to infection of the host. This process is normally saturable due to a limited number of active receptors accessible for the irreversible interaction at the cell surface. Reversible and irreversible adsorption can target the same receptor or involve different surface components (see reference 48 and references therein). The two strategies correlate with distinct phage adsorption machineries whose complexity can vary from a single receptor-binding protein (RBP) to complex baseplates with several RBPs (42, 47). Different phages of Gram-negative bacteria were shown to bind preferentially to cell poles at low multiplicities of infection (18). In case of phage lambda, this localization correlates with the polar topology of ManY, an inner membrane protein required for lambda DNA entry in the bacterium. The subsequent position of phage DNA replication appears to occur also at cytoplasmic positions close to the pole (18). However, the receptor for irreversible binding at the bacterial surface, LamB, is an abundant protein distributed throughout the Escherichia coli outer membrane following a helical pattern (20). It is thus likely that the preferential positioning of pha...

The mechanism of genome transfer from the virion to the host cytoplasm is critical to understand and control the beginning of viral infection. The initial steps of bacteriophage SPP1 infection of the Gram-positive bacterium Bacillus subtilis were monitored by following changes in permeability of the cytoplasmic membrane (CM). SPP1 leads to a distinctively faster CM depolarization than the one caused by podovirus ϕ29 or myovirus SP01 during B. subtilis infection. Depolarization requires interaction of SPP1 infective virion to its receptor protein YueB. The amplitude of depolarization depends on phage input and concentration of YueB at the cell surface. Sub-millimolar concentrations of Ca(2+) are necessary and sufficient for SPP1 reversible binding to the host envelope and thus to trigger depolarization while DNA delivery to the cytoplasm depends on millimolar concentrations of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.