Bacterial strains and plasmids
All strains, plasmids, oligonucleotides and phages used in this study are listed in Supplementary Tables 1–4. Genetic modifications were performed as described previously6. Chromosomal deletions were performed using the λ-Red recombination method; PCR products containing the kanamycin cassette were integrated into bacteria containing pKD46, as described previously42. All deletions were then transduced using phage P2243 lysates and appropriate selection. Curing of phage P22 after transduction was verified by purifying the strains on EBU (Evans-blue uranine) plates to indicate lysis and PCR at the mutation site43. The kanamycin cassette was then removed through recombination at frt (flippase recognition target) sites using FLP (Flippase) recombinase encoded on pCP2042. Unmarked deletions and point mutations were generated through allelic exchange with pTOX2 or pTOX344. In brief, a 2–3 kb fragment including the target mutation was cloned into pTOX plasmids44 at the SwaI site by Gibson Assembly (New England Biolabs). This plasmid was transformed into the auxotrophic MFDλpir strain for conjugation into S. Typhimurium45. Selection for MFDλpir transformants was performed with chloramphenicol and 300 μM meso-diaminopimelic acid. S. Typhimurium was selected on chloramphenicol after conjugation and counter-selection was performed on lysogeny broth plates with 2% rhamnose. All insertions, deletions and substitutions were verified by colony PCR with OneTaq (New England Biolabs) using primers flanking the cassette or by localized sequencing at the mutation site. All other plasmids were constructed using restriction cloning or Gibson Assembly with 2x Gibson Assembly Master mix (New England Biolabs; construction details for each plasmid noted in Supplementary Table 2). All plasmids were verified by sequencing of the insertion (Quintara Biosciences) or the entire plasmid (SNPsaurus). Plasmids that were not transformed into another strain background were maintained in E. coli DH5α.
Prophage curing
Prophages were cured using counter selection with MqsR from pTOX2. In brief, λ-Red recombination was used to insert a portion of the pTOX2 vector including the chloramphenicol resistance gene and rhamnose-inducible MqsR toxin into each prophage. This cassette was transduced into the desired strain using P22 with selection on chloramphenicol. Following curing of P22, the counter selection with 2% rhamnose was performed after brief exposure to 2 mM H 2 O 2 (shown to induce prophages in 14,028 s). Prophage curing was verified by PCR at the att site and whole genome sequencing (SeqCenter).
Prophage lysogenization
To construct lysogens of the temperate prophage ES1846 in strain 14028, concentrated stocks of each phage were spotted onto a lawn of the recipient strain embedded in 0.5% lysogeny broth agar in 12-well culture plates. Plates were incubated at 37 °C for 12–16 h. Bacteria from the turbid centre of the region of lysis were purified onto lysogeny broth twice and screened by PCR for the presence of the temperate phage to which they had been exposed.
Quantification of phage plaquing and defence
Plaque assays were performed on lysogeny broth with 0.5% top agar as described. Tenfold dilutions of phage stocks were plated on lawns of indicator strains of interest. All plaque assays were performed with stationary phase cultures inoculated from a single colony (16 h). Plaque assays were incubated overnight (12–16 h) before imaging with a BioRad Chemidoc. Where defences prevented the formation of single plaques on some strains, plaquing of phage was scored on the basis of clearance across the tenfold dilution series and the size of plaques. Thus, phage plaquing is reported as the lowest phage tenfold dilution at which clearance was observed and the relative plaque size at the next dilution in 0.25-fold increments. This is a score of plaquing on a log scale. The fold defence, as calculated in Figs. 1e and 2a, is the difference between the score of plaquing in the presence and absence of the hepS gene.
Phage stocks and phage propagation
All phages used in this study are reported in Supplementary Table 4. Lytic phages were propagated on prophage-cured strains of S. Typhimurium. Lysates were collected from phage-infected cultures and treated with chloroform. Temperate phage lysates were generated by inducing prophages from the appropriate lysogens with MMC. The supernatants of MMC-treated cultures were collected 2–4 h after treatment and treated with chloroform. Phage stocks were stored at 4 °C until use. When higher concentrations of phage were needed, lysates were concentrated using Amicon Ultra Centrifugal Filter with a 30 kDa molecular weight cutoff (MWCO).
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