Different P. aeruginosa mutant strains which lack flagella (ΔfliM), pili (ΔpilM) or a complete LPS (ΔalgC ) were used to investigate the ability of JG004 to infect these mutant strains. The gene algC encodes an enzyme with phosphoglucomutase and phosphomannomutase activity and is required for the biosynthesis of the complete P. aeruginosa LPS core [16]. The phage JG004 is able to lyse flagella and pili mutants but not the algC mutant defect in LPS biosynthesis, which indicates that LPS is the receptor of JG004. In order to determine the host range of JG004, we used a set of 19 clinical isolates to investigate the ability of JG004 to infect Luminespib chemical structure these strains (Table 1). JG004 is able to infect around

50% of the tested clinical isolates (Table 1), suggesting that JG004 belongs to the broad-host-range phages. Additionally, JG004 is even capable of infecting a P. aeruginosa mucA mutant, which produces large amounts of exopolysaccharides and displayes 10058-F4 a mucoid phenotype [17]. Mucoid P. aeruginosa strains are frequently isolated from patients suffering from cystic fibrosis and are correlated with a poor prognosis [18]. Table 1 Strains and phages used in this study. Bacterial strain or phage Phenotype or genotype Reference PAO1* Wild type [54] PA14 Wild type [55] PAO1 ΔmucA* PAO1 mucA::aacC1-gfp GmR Sabrina Thoma, this laboratory, unpublished PAO1 ΔpilA* pilA inactivated by allelic displacement;

tagged with eGFP, TcR, GmR [56] PAO1 ΔfliM * fliM inactivated by allelic displacement; tagged with eGFP, TcR, GmR [56] PAO1 ΔalgC algC::aacC1-gfp GmR Julia Garbe, this laboratory, unpublished BT2, BT72, BT73, RN3,

RN43, RN45*, NN84 Clinical CF isolates Medical Highschool Hannover, www.selleckchem.com/products/AG-014699.html Germany PACF15, PACF21*, PAKL1, Clinical CF isolates Gerd Döring, PAKL4*, PACF60*, PACF61*, PACF62, PACF63*   Tübingen, Germany Nr. 18*, 19*, 26*, 29 Urinary tract infection isolate Michael Hogardt, München, Germany JG004 Wild type PAO1 LPS-specific lytic bacteriophage This study * = strains infected by phage JG004 in the host range and receptor studies. Abbreviations: GmR, resistant to gentamicin; TcR, resistant to tetracyclin; eGFP, enhanced green fluorescent protein; IKBKE LPS, lipopolysaccharide. Growth characteristics Figure 2 shows the one step growth curve of phage JG004. The burst size, which describes the average number of phages liberated per bacterial cell as well as the latent phase were calculated as described in Methods. JG004 is able to produce approximately 13 progeny phages per cell and has a latent phase of 31 min. Figure 2 Growth of JG004. One step growth curve of phage JG004. A representative growth experiment of three independent experiments is shown. Within 34 min, the phage is able to produce about 13 phage progeny per infected cell. Genome properties and organization The entire genome sequence of phage JG004 was determined as described in Methods and revealed a genome with a size of 93,017 bp.