Figure 3 JIB04 cell line Attachment to abiotic surface by P. luminescens. Photorhabdus strains (as indicated) were grown overnight at 30°C in LB broth (+ Km). The OD600 of the culture was adjusted to 0.05 and 200 μl was added to the well of a 96-well Costar® PP microtitre plate. The plates were incubated for 72 h at 30°C before staining with crystal violet to quantify bacterial attachment. Relative BTK inhibitor biofilm formation was determined by calculating the OD595 (mutant):OD595 (TT01gfp) ratio and the results shown are the mean ± SD of 3 experiments. Virulence of mutants to insect larvae Photorhabdus is highly

virulent to insect larvae and previous work had shown that mutants affected in their ability to colonize IJs were also affected in their virulence to insects [5]. Therefore 200 cfu of each of the mutants was injected into 10 final instar larva of the Greater Wax Moth (Galleria mellonella)

and insect death was assessed by gently prodding the insects at different DMXAA in vitro time points post-infection. As expected the LT50 of TT01gfp was observed to be approximately 45-46 h (see Figure 4). This was similar to the LT50′s of the proQ, hdfR and asmA mutants suggesting that these genes are not important during virulence. We had previously shown that a mutation in the pbgPE operon was avirulent and this has now been confirmed in this study (see Figure 4). In addition the galE and galU mutants appeared to be completely avirulent under the conditions tested here implying an important role for polysaccharide production during virulence (see Figure 4). Figure 4 Virulence of P. luminescens to insect larvae. TT01gfp

and mutant strains were grown overnight in LB broth at 30°C and diluted in PBS so that approximately 200 cfu were injected into each of 10 final-instar G. mellonella larvae. The insects were incubated at 25°C and insect death was monitored over the next 72 hours. In each graph the virulence of the mutant (■) is compared to TT01gfp (□). The results shown are of a representative experiment that was independently repeated at least 3 times. Sensitivity of mutants to polymyxin B Insects have a sophisticated innate immune system that includes the production of CAMPs [18]. One mechanism employed by bacteria to adapt PJ34 HCl to, and resist, the presence of CAMPs is to reduce the net negative charge associated with the LPS present in their outer membrane. This can be achieved by, amongst other means, replacing a negatively charged phosphate group on the lipid A moiety of the LPS with a positively charged L-aminoarabinose. In Salmonella and E. coli this modification is carried out by the products of the arnBCADTFE operon (formerly the pmrHFIJKLM operon) [7]. In P. luminescens the closest homologue to the arnBCADTFE operon is annotated as the pbgPE operon and we have previously shown that a mutation in this operon is hyper-sensitive to the presence of the CAMP, polymyxin B [5].