The data shown is the
mean of at least 2 independent experiments (with n = 10 insects/experiment). For clarity the standard deviations are not shown but these values were within expected limits (0-35%). Role of iron uptake in symbiosis In this study we wanted to determine the affect of the iron homeostasis mutations in Pl TT01 on nematode growth and development. Therefore lipid agar plates were inoculated with the PFT�� in vitro strains to be tested (Pl TT01, ΔexbD, Δyfe, Δfeo, ΔexbD Δyfe Δfeo) and, 4 days later, the bacterial biomass was seeded with 40 surface-sterilised H. bacteriophora IJs. We observed that all of the Pl TT01 mutants, even the ΔexbD Δyfe Δfeo triple mutant, were as competent as, if not better than, the WT in their Ricolinostat ability to support the growth and development of their nematode partner as measured by Galunisertib in vivo the IJ yield i.e. total number of IJs collected/number of IJs inoculated (see Figure 5). This is in sharp contrast to what we had previously observed with Pt K122 exbD::Km where we reported that H. downesi nematodes failed to reproduce on the mutant bacteria . Figure 5 The IJ yield after growth on P. luminescens. The different bacterial strains were inoculated onto lipid agar plates, incubated for 3-4 days at 30°C and
40 surface-sterilised H. bacteriophora IJs were added to the biomass. The plates were incubated for 21 days at 25°C and the IJ yields were determined (i.e. total number of IJs/50). For each experiment 5 plates were analyzed for each strain and the experiment was repeated 3 times. Therefore the data shown is the mean ± standard deviation of n = 15 plates for each strain. Statistical significance was determined using a T-test and IJ yields significantly different (P < 0.01) to those obtained using TT01 are indicated with an asterisk. Nematode development culminates
in the formation of a new generation of IJs that are colonized by the bacteria on which the nematodes have been Adenosine cultured. Therefore, in order to ensure the symbiotic cycle had been completed, the IJs recovered from these symbiosis assays were surface sterilised, crushed individually and the lysate was spread onto LB agar. In this way it was determined that there were, on average, 42 CFU of Pl TT01 present in the gut of each IJ (Figure 6A). Moreover the ΔexbD and Δfeo mutant strains were able to colonize the IJ as well as the WT (Figure 6A). However the Δyfe and ΔexbD Δyfe Δfeo mutants appeared to colonize the nematodes at a level that was significantly lower than WT (P < 0.0001) suggesting that the yfeABCD locus may be important during colonization of the IJ (Figure 6A). Figure 6 Colonization of IJ nematodes with TT01 and mutant derivatives. A) Individual IJ nematodes (n = 10), grown on the different bacterial strains (as indicated), were crushed and the lysate was plated on LB agar to enumerate the CFU within the nematode. The data is shown as a boxplot where the horizontal line within the box represents the median value.