(c) Endolysin forward and reverse primers yield a 750-bp PCR product of the parent phage P954 and 2400-bp product of the recombinant phage P954. (d) The holin forward primer and endolysin reverse primer yield a 1000-bp PCR product with parent phage P954 and 2650-bp product of the recombinant phage P954. Both PCR panels include lane 1: PCR buffer (negative control); Pifithrin-�� concentration lane 2: parent phage P954 lysogen B7, lane 3: molecular weight marker (λ/HindIII-EcoRI); lane 4: recombinant phage P954 lysogen H10. Mitomycin C induction of parent and

endolysin-deficient phage P954 We examined the prophage induction pattern and phage progeny release from TSA HDAC in vitro parent and endolysin-deficient phage P954 lysogens. Absorbance and extracellular phage titers

were monitored every hour until the end of induction. Induction of the parent phage P954 lysogen (B7) resulted in cell lysis and gave a phage titer of 1 × 109 PFU/ml. In contrast, the endolysin-deficient phage P954 lysogen did not lyse and gave a phage titer of about 103 PFU/ml (Figure 2). Figure 2 Mitomycin C induction of parent and endolysin-deficient phage P954 lysogens. (a) Growth profiles of the parent (B7) and endolysin-deficient (H10) phage P954 lysogens after Mitomycin C induction showing absorbance of cultures at 600 nm. The graph is representative of two experiments. The error bars represent mean plus standard deviation (n = 3) (b) Phage release into the culture medium from parent (B7) and endolysin-deficient (H10) phage P954 lysogens after Mitomycin C induction. The graph is representative of 2 experiments. Endolysin complementation for buy Sirolimus phage enrichment and enumeration Endolysin-deficient phage P954 could be enriched to titers of up to 5 × 1010 PFU/ml in S. aureus RN4220 that constitutively expressed phage P926 endolysin. This strain was used also to determine titers of the endolysin-deficient phage preparations. When preparations of the endolysin-deficient phage were spotted on a non-complementing host, a zone of lysis

characteristic of “”lysis from without”" was observed at lower dilutions, and no plaques were discernible (Figure 3a). The recombinant phage formed plaques only on the endolysin-complementing host (Figure 3b, c, d). Figure 3 Complementation with heterologous endolysin gene for enrichment of endolysin-deficient phage P954. Ten-fold serial dilutions of endolysin-deficient phage P954 (5 × 1010 PFU/ml) spotted on (a) S. aureus RN4220 lawn and (b) complementing host pGMB540/S. aureus RN4220, which expresses a heterologous endolysin. Plaque assay of enriched endolysin-deficient phage P954 on (c) non-complementing host S. aureus RN4220 and (d) complementing host pGMB540/S. aureus RN4220.