The pbgPE operon is predicted to be involved in modification of the lipid A moiety of LPS with L-aminoarabinose. Interestingly we also identifed a mutation in the downstream gene, pbgE3, confirming a key role for this operon in IJ colonization. From this group of 3 mutants we used
mutant #28 F4 for all further analysis. Mutants #6 D10 and #6 E10 were identified as interuptions of galE and galU respectively. Selleck Olaparib The galE gene is predicted to encode UDP-glucose 4-epimerase and galU is predicted to encode glucose-1-phosphate uridyltransferase. Both of these activities are important in the production of polysaccharides including O-antigen [9–11]. Mutant #36 F4 was identified as an interuption of a gene with homology to
the asmA gene in E. coli. The AsmA protein is localised to the outer INCB018424 mouse membrane of E. coli and mutations in this gene resulted in significantly lower levels of LPS [12, 13]. Mutant #22 G12 was identified as an interuption of a gene with homology to hdfR in E. coli. The hdfR gene has been shown to repress flhDC expression, and thus motility, in E. coli . Finally mutant selleck screening library #2 D6 was shown to be an interuption of gene with homolgy to proQ from E. coli. In E. coli proQ encodes a protein that modifies the activity of ProP, a MFS transporter involved in the adaptation of the cell to osmotic stress [15, 16]. However we could not identify a ProP homologue on the genome of TT01 suggesting a different role for ProQ in this bacterium. Figure 2 The genetic loci important for colonization
of the IJ. The position of the transposon HA-1077 in each mutant was identified by sequencing and subsequent BLAST analysis using PhotoList http://genolist.pasteur.fr/PhotoList. Table 1 Colonization mutants identified in this study. Mutant ID Gene Transmission frequency #2 D6 proQ 27% #6 D10 galE 31% #6 E10 galU 23% #12 E12 pbgE2 27% #22 G12 hdfR 26% #26 F7 nd 10% #28 F4 pbgE2 30% #30 F4 pbgE3 10% #32 H12 nd 10% #36 F4 asmA 20% Attachment of mutants to abiotic surfaces Previous transmisson electron microscopy of Photorhabdus within the gut of the IJ had revealed features of the bacterial population that resembled growth as a biofilm i.e. the bacteria were seen to be in close association with the epithelial cells of the gut and encased in a matrix of unidentified composition . Therefore we wanted to determine if any of the mutants defective in transmission to the IJ were affected in biofilm formation, as measured by attachment to an abiotic surface. The mutants were grown in the wells of a polypropylene (PP) microtitre plate for 72 h and the attached biomass was measured using crystal violet (see Figure 3). As can be seen only 2 mutants were affected in their ability to attach to PP, proQ and galU (20% and 45% of wild-type levels, respectively).