The pbgE1 mutant did produce altered LPS compared to the wildtype implicating LPS structure as PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28724915 a nematode Ing DC (left panel) and the grey value analysis along the colonization factor in Photorhabdus . In this study we screened a library of Photorhabdus mutants with the aim of extending our understanding of the transmission process by identifying genes important in the colonization of the H. bacteriophora IJ nematode by P. luminescens TT01.ResultsConstruction of a GFP-tagged strain of P. luminescens TTIn order to facilitate a relatively rapid screening method for the identification of transmission mutants we decided to tag TT01 with a constitutively expressed gfp using a mini-Tn7 transposon (see Methods). This would enable the translucent IJs to be viewed beneath a fluorescent microscope and be scored qualitatively for the presence/absence of bacteria. Colonies of the gfp-tagged strain (called TT01gfp) were initially checked for fluorescence using a UV light box before overnight cultures were checked for gfp expression using a fluorescent microscope. This confirmed that the vast majority of cells in an overnight population of TT01gfp were expressing gfp (see Figure 1A). Phenotypic comparisons of TT01 and TT01gfp confirmed that there was no difference in growth rate, bioluminescence, pigmentation or virulence to insect larvae. Furthermore we also verified that TT01gfp was able to colonize IJ nematodes (see Figure 1B) with a transmission frequency identical to TT01 (between 80-85 ). As has been previously shown, the TT01gfp bacteria were confirmed to occupy the proximal region of the nematode gut extending from just below the pharynx of the IJ (see Figure 1C).Identification of TT01gfp mutants affected in colonization of the IJIn this study we were using a qualitative screen that was designed to identify mutants that were affected in transmission frequency i.e. we were looking for mutants that colonized significantly fewer IJs than the 80 level observed with TT01gfp. Ull list of author info is readily available in the finish of Therefore PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27663262 TT01gfp was subjected to transposon mutagenesis using the Tn5 interposon delivered by plasmid pUT-Km2 and individual mutants were arrayed into 96 well plates and frozen. From this arrayed library 3271 mutants were screened for a defect in transmission frequency by growing the mutant on a lipid agar plate and inoculating the biomass with 30 surface-sterilized H. bacteriophora IJs. After 21 days incubation the new generation of IJs were collected and checked for colonization using a fluorescent microscope. In this way 40 mutants were identified as having a qualitative defect in transmission frequency i.e.