FIG 5.

Impact of expression of hcp genes in trans on T6SS function in vitro. (A) Coincubation assay with ES114 and the indicated FQ-A001-derived strains. ES114 is labeled with CFP via pYS112. The FQ-A001-derived strains are FQ-A001 (WT) and NPW58 (Δhcp Δhcp1), which harbors the vector control pVSV105 (unlabeled), pNPW16 (hcp1⁺), or pNPW17 (hcp⁺). Images show the CFP fluorescence of the bacterial growth resulting from the 24-h incubation of cellular mixtures spotted onto agar. Approximately 5 × 10⁷ total CFU were introduced into each spot. Dotted lines indicate the border of bacterial growth within each spot. Bar, 1 mm. (B) Ratio of Erm^r CFU to Cam^r CFU resulting from a 5-h coincubation of the Erm^r ES114-derived strain TIM313 labeled with Cam^r by plasmid pYS112 with the FQ-A001-derived strains described in the legend to panel A. Each point represents the ratio associated with an individual spot at 0 h (open symbols) or 5 h (filled symbols), and each horizontal line represents the geometric mean (n = 4). Approximately 5 × 10⁷ total CFU were introduced into each spot. Two-way analysis of variance revealed significant differences among the means of log-transformed ratios due to time (F_1,24 = 299.5; P < 0.0001), genotype (F_3,24 = 37.46; P < 0.0001), and their interaction (F_3,24 = 42.95; P < 0.0001). A Sidak post hoc test was performed to compare the means statistically, with P values adjusted for multiple comparisons. Groups with different letters have significantly different medians (P < 0.0001), and groups with the same letter do not have significantly different medians (P ≥ 0.05).