​pseudomonas.​com). buy Sapanisertib However, the role of these proteins during phage infection is unclear and is currently under investigation in our laboratory. The gene PD173074 mw PA0654 encodes the SpeD protein, an S-adenosylmethionine decarboxylase, which is an essential part of the spermidine

biosynthesis pathway in P. aeruginosa [43]. These results suggest that the infection process of phage JG004 is dependent on spermidine. As pointed out earlier, JG004 also possesses a probable homospermidine synthase, which uses spermidine and purtrescine to synthesize homospermidine. Spermidine itself or homospermidine could be important substances essential for compact packaging of phage DNA by balancing the negative charge of the DNA [35]. The analysis of P. aeruginosa transposon mutants resistant buy Alvocidib to phage infection confirmed that phage JG004 recognizes LPS as receptor. Moreover,

this approach revealed details of phage JG004 biology, e.g. its dependance on spermidine. Conclusions We characterized a P. aeruginosa specific broad-host-range phage which is a member of the Myoviridae phage family. JG004 has a contractile sheath and a central tube with a length of 115 nm and an isometric head structure with a diameter of 67 nm. JG004 uses LPS as receptor and has a burst size of 13 phage particles. Genome analysis revealed that this phage shares 87% identity to phage PAK-P1. Despite its morphological similarity to other phages, no significant identity to other phage genomes was detected. We used a transposon

mutagenesis approach of the host to identify genes important for phage infection. This approach indicated a dependance of JG004 on spermidine production of the host bacterium and confirmed LPS as host receptor. In addition to the characterization of host-phage biology, this approach could be an interesting tool to perform host receptor studies or to investigate genes of unknown function such as e.g. P. aeruginosa genes involved in LPS biosynthesis. Methods Bacterial strains The bacterial strains used in this study are listed in Table 1. P. aeruginosa strains were routinely grown in Luria Bertani (LB) broth medium aerobically at 37°C. Transposon mutagenesis Transposon mutagenesis was performed with the mariner transposon as previously described [44] pheromone with the following modifications. After incubation of the mating mixture, the cells were scraped and resuspended in 1 ml LB. For selection of P. aeruginosa strains resistant to phage infection, the cells were incubated with a ten fold excess of the phage JG004 for 30 min at 37°C. The cells were plated on LB medium containing 200 μg/ml gentamicin and 10 μg/ml chloramphenicol for the inhibition of the E. coli S17λpir strain. The insertion of the transposon was identified by arbitrary PCR and sequencing as described previously [45].