CpxRA Regulates Mutualism and Pathogenesis in Xenorhabdus nematophila

ΔcpxR1 mutant is attenuated in virulence for M. sexta insects and mutualistic nematode colonization. (A) Fourth-instar M. sexta larvae were injected with log-phase X. nematophila cells, and insect survival was monitored over time. The strains included in this analysis were the WT (Tn7) (□), WT (Tn7/cpxRA) (⋄), ΔcpxR1 (Tn7) (○), and ΔcpxR1 (Tn7/cpxRA) (•) strains. The symbols indicate the averages of six to eight experiments (10 insects per experiment). The error bars indicate standard errors. The letters b and c indicate values significantly different from the WT (Tn7) strain value (a). (B) X. nematophila bacteria were isolated from colonized S. carpocapsae nematodes, and the average number of CFU/infective juvenile (IJ) for each strain was normalized to wild-type values, which were defined as 100%. Nematodes were colonized with the strains indicated at the bottom. The values are averages of three or more experiments. An asterisk indicates the number of CFU/infective juvenile is statistically different from the wild-type value (WT) (P < 0.01).

ΔcpxR1 mutant has altered morphology and phenotypic properties. (A) Wild-type X. nematophila (WT) and ΔcpxR1 mutant colony and cell morphologies. Images were obtained using phase-contrast microscopy at magnifications of ×100 (colony morphology) and ×1,000 (cell morphology). Cell and colony morphologies were analyzed using X. nematophila strains grown in plates containing liquid LB and LB with 0.1% pyruvate, respectively. The scale bars indicate 5 and 100 μm in the cell and colony morphology images, respectively. (B) Phenotypes inhibited (antibiotics, hemolysin, and protease) and activated (lipase and motility) by CpxR. The values under the antibiotic, hemolysin, and protease images indicate the halo area/colony area (averages ± standard errors). The values under the lipase images indicate the precipitate density, and the values under the motility images are growth diameters. An asterisk indicates a value that is significantly different (P < 0.01) from the WT (Tn7) strain value.

CpxR positively regulates nilA, nilB, and nilC expression. lacZ reporter fusions were used to measure expression of nilA (A), nilB (B), and nilC (C) in the ΔcpxR1 mutant by the β-galactosidase assay. Assays were performed using stationary-phase cultures (averages of three replicates are indicated by the bars). The error bars indicate standard errors. An asterisk indicates a value significantly different from the wild-type value (WT) (P < 0.001).