FIG 2.

Calcium promotes cell-cell contact and sheath assembly in liquid media. (A and C) Representative fluorescence microscopy images of cocultures of ES401 WT or T6⁻ (magenta) incubated with ES114 (yellow) (A) or monocultures of each strain in LBS plus 10 mM CaCl₂ for 12 h (C). Each experiment was performed three times with one biological replicate and four fields of view (n = 12). (B) Results from mixed-strain coaggregation assays, displayed as the percentage of each strain (ES114, yellow; ES401, magenta) within aggregates (top) or in the single-cell fraction (bottom). (D) Representative GFP-labeled images of ES401 harboring a VipA_2-GFP expression vector incubated in LBS with 10 mM CaCl₂ and supplemented with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 3 h. Percentages are shown for VipA_2-GFP-expressing cells that contained at least one sheath after being incubated in LBS with or without CaCl₂ and supplemented with IPTG for 3 h. The asterisk indicates a significantly different percentage of cells with sheaths between media types (Student's t test, P < 0.0001). Each experiment was performed twice with two biological replicates and five fields of view (n = 20). (E) Conceptual model for the conditional regulation of the V. fischeri T6SS2 in liquid media displayed as a logic gate. The V. fischeri T6SS2 facilitates interbacterial killing both on surfaces and in liquid environments when specific conditions are met. Either calcium or high viscosity combined with neutral or acidic pH promote T6SS activity in a liquid environment.