FIGURE 9.

VgrG4b can deliver PldA when fused to its C-terminus. (A) The STOP codon of vgrG4b, the intergenic region and START-codon of pldA were deleted resulting in a single chimeric gene product. (B) Representative figure of a western blot from a secretion assay using PAO1ΔrsmA with an active or inactive H2-T6SS (ΔtssB2) encoding VgrG4b-PldA (ΔTAA^vgrG4b-ATG^pldA). As a positive control for VgrG4b secretion, PAO1ΔrsmA (lane 1) was included, while the negative control lacked vgrG4b (lane 2). Antibodies used (from top to bottom) are against VgrG4b^C, RpoB, Hcp2 and LasB, a T2SS substrate (Olson and Ohman, 1992) acting as a loading control for the supernatant, as indicated on the right. (C) Bacterial competition represented by plots of recovered cfu of prey strain PAO1ΔrsmAΔpldAtli5a:: lacZ after contact with the attacker strain encoding WT PldA, WT VgrG4b or the fusion VgrG4b-PldA (ΔTAA^vgrG4b-ATG^pldA) as indicated. As a positive control for PldA-mediated killing, PAO1ΔrsmA was included (lane 2) while the negative controls lacked pldAtli5a (lane 3) or vgrG4b (lane 4). Spots were incubated for 24 h at 25 ^∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on the data set obtained from the recovered prey on its own with ^∗p < 0.05 and ^∗∗p < 0.01.