Bacillus subtilis Operon Encoding a Membrane Receptor for Bacteriophage SPP1

The interaction of a bacteriophage with the bacterial surface (adsorption) is the first step in the infection process. This step involves recognition of and binding to one or more cell envelope constituents and leads to ejection of the phage DNA from the capsid. It has long been appreciated that adsorption can proceed in two steps, one reversible step followed by irreversible commitment (1). Irreversible adsorption and ejection of DNA are possibly different descriptions of the same phenomenon, but this has not really been clarified. Similarly, DNA ejection from the capsid and injection into the cytoplasm are at best difficult to separate in vivo, except in those cases where injection itself proceeds in distinct phases, as in T5 (13). In the gram-negative world, several phage receptors have been identified, and in a number of cases (e.g., T4, T5, and T7 of Escherichia coli), the adsorption and injection processes are now beginning to be understood at a detailed molecular level (14, 18, 26). Much less is known about the first steps of infection for phages preying on gram-positive bacteria. Early work has established that glucosylated polyglycerol phosphate, the major and essential teichoic acid in Bacillus subtilis, serves as a receptor for several phages, including φ29, φ25, and SP01 (24, 29, 34, 36). Mutations leading to a lack of glucosylation of this polymer block the phage adsorption process and plaque-forming ability. More recently, selection for B. subtilis mutants resistant to bacteriophage φ3T led to the identification of another type of cell wall receptor, a minor cell wall anionic polymer, poly-(glucosyl-N-acetylgalactosamine 1-phosphate) (6). Interestingly, no membrane protein has yet been implicated in phage binding to B. subtilis, in contrast to the evidence from gram-negative systems. Nevertheless, phage infection of B. subtilis protoplasts from both wild-type and phage-resistant strains has been reported, suggesting the presence of membrane-linked receptors (9, 10). In Lactococcus lactis, Geller et al. have established that phages of the c2 group adsorb first reversibly to a cell wall polymer and then irreversibly to a membrane protein, which was designated Pip (for phage infection protein) (7). To our knowledge, this is the only instance where a membrane receptor for a phage infecting a gram-positive bacterium has been documented. Twenty years ago, a B. subtilis phage resistance mutation (pha-2) blocking irreversible binding of phage SPP1 was described (29). pha-2 mutants retained the capacity to adsorb phages at a wild-type rate, but phage-host complexes could be readily separated by dilution, showing the process to be wholly reversible. The mutation was found to be specific for SPP1 and related phages and was mapped, by PBS1-mediated transduction, in the vicinity of the ald-1 marker (29, 30). Based on the map position of the pha-2 locus and the availability of the complete sequence of the B. subtilis genome, we have now attempted to identify the gene required for SPP1 irreversible adsorption. The experiments described here identify yueB, an orthologue of pip, as the key gene involved in irreversible adsorption of SPP1. yueB and upstream genes are organized as an operon, the products of which may contribute additionally to efficient SPP1 infection. Nonetheless, only YueB appears to be required for irreversible binding of the phage. This is the first demonstration of a membrane-bound protein acting as a phage receptor in B. subtilis. Furthermore, it strengthens the notion that YueB/Pip-like proteins, which are widely distributed among gram-positive species, may be generally used as bacteriophage receptors.