A total of approximately 30000 transposon mutants were screened and 14 phage resistant mutants were isolated and analyzed. Since two mutants, TM20 and TM22 are defect in the same gene, rmlB, a total of 13 genes was identified, which are essential for phage infection. The transposon find more screen revealed genes important for LPS biosynthesis (see Table 4 for details) like the gene algC which is needed for a complete LPS core in P. aeruginosa . It also revealed the genes rmlA and rmlB, which are involved in the biosynthesis of the LPS core sugars [39, 40]. These findings confirm that the phage JG004 uses LPS as receptor.
Other identified genes involved in LPS biosynthesis are wzz2, PARP activation waaL, migA, PA5000 and PA5001 (Table 4) . Since nine out of 13 identified genes encoded proteins involved in LPS biosynthesis, we additionally isolated LPS from all mutant strains and analyzed it by electrophoresis (see Materials and Methods). Figure 4 shows the LPS profiles of the transposon mutants. The lipid A, which migrates furthest due to its size, is seen as a dark grey spot at the end of the gel. The migration depends on changes in the LPS composition, mostly in the core polysaccharide which
is adjacent to the lipid A . Not all LPS biosynthesis genes cause changes in the LPS which are visible by electrophorsis e.g. migA , which appears as wild type LPS. The black line in Figure 4 indicates the migration level of the wild type lipid A. Dramatic changes in the LPS profile which differs clearly from the P. aeruginosa wild type LPS can be seen for the algC, the wzz2 and the PA5001 mutant. Further analysis of the LPS for example Western blot analysis with antibodies specific to the different components of the LPS could provide a better understanding
of the mutants, not but was not involved in this phage characterization study. Figure 4 LPS profile of transposon mutants. Silver stained SDS-PAGE illustrating the isolated LPS of the wild type PAO1 and the transposon mutants. Only the gene, interrupted by the transposon of the respective mutant is indicated on top of the lanes, PAO1 is the P. aeruginosa wild type. The arrow points to the black line in the lower part of the gel. This line indicates the migration of wild type lipid A and core sugars of the LPS . As indicated, the LPS of the speD, PA0534, PA0421, PA2555 and migA mutant strains appears similar to wild type LPS. The LPS profile of the remaining mutant strains is different and indicates an altered LPS structure. Interestingly, the selleck chemicals llc biochemical analysis of LPS indicates that gene PA2200 might be involved in biosynthesis or modification of P. aeruginosa LPS due to altered migration. We also identified genes essential for phage infection, which encode proteins of unknown function.