entomophila L48 prophage1 – PSEEN4129 through PSEEN4186; P. check details aeruginosa PAO1 prophage1 – PA0610 through PA0648; P. aeruginosa PA14 prophage1 – PA14_07950 through PA14_08330; P. aeruginosa PA7 prophage1 – PSPA7_0754 through PSPA7_0789; P. aeruginosa PA7 prophage2 – PSPA7_2366 through PSPA7_2431. The homologous prophage elements from Pf-5 and Q8r1-96 have simple overall organization, lack integrase and head morphogenesis genes, and carry conserved regulatory, lytic and lambda-like tail morphogenesis genes also found in phage SfV of Shigella flexneri (Fig.

1). Taken together, the results of sequence analyses suggest that these regions are not simple prophage remnants but rather, are similar to F-type pyocins. F-type pyocins were first discovered in P. aeruginosa and represent a class GSK458 of high molecular weight protease-

and nuclease-resistant bacteriocins that resemble flexible and non-contractile tails of bacteriophages [18, 19]. This notion is further supported by the fact that the putative lytic genes found within Pf-5 prophage 01 (Fig. 3) and Q8r1-96 (data not shown) seem to be fully functional. In non-filamentous bacteriophages buy LY294002 and bacteriophage tail-like bacteriocins, the lytic activity is provided by the combined action of the small membrane protein holin and a cytoplamic muralytic enzyme, endolysin [19, 20]. During phage-mediated cell lysis, holin permeabilizes the cytoplasmic membrane and allows endolysin, which lacks a secretory signal sequence, to gain access to peptidoglycan. To confirm that the prophage 01-like loci indeed encode functional holin and endolysin, we cloned genes PFL_1211 and PFL_1227 from Pf-5 and their counterparts

from Q8r1-96 (Fig. 1) in Escherichia coli under the control of an inducible T7 promotor. As shown in Fig. 3, induction of both of the putative holin and endolysin genes by IPTG had a strong lethal effect on the host, resulting in rapid cell lysis. In accordance with the current model of action of holin and endolysin, the lethal effect of the endolysin encoded by PFL_1227 was not apparent unless the cytoplasmic membrane was destabilized by addition of small amount of chloroform to the induced E. coli culture (Fig. 3B). Gene induction experiments carried out with putative holin and endolysin genes from the ssh6 locus of Q8r1-96 had a similar lytic effect on E. coli (data not shown). Figure 3 Thiamine-diphosphate kinase Lytic activity associated with the prophage 01 of P. fluorescens Pf-5. Putative holin (PFL_1211) (A) and endolysin (PFL_1227) (B) genes encoded by prophage 01 from P. fluorescens Pf-5 were cloned in the plasmid vector pCR-Blunt (Invitrogen) under the control of the IPTG-inducible T7 promoter. Broth cultures of E. coli Rosetta/pLysS bearing the cloned holin and endolysin genes were induced with 3 mM IPTG and incubated with shaking for 5 hours while monitoring cell growth by measuring OD600. The arrow indicates the time of addition of chloroform to the endolysin-expressing culture.