Functional verification of the RelA-YFP and SpoT-GFP reporter fusions using swarming.

Since the fluorescence fusions were in-frame at the C-terminal end of the gene of interest, a fluorescent signal would indicate a correctly folded fusion protein. However, overexpression of fusion proteins can lead to misfolding and subsequent sequestering into insoluble inclusion bodies (83). To verify that both fusion proteins were correctly folded and functional, we complemented the ΔrelA ΔspoT double mutant with either RelA-yellow fluorescent protein (YFP) or SpoT-green fluorescent protein (GFP) and tested the ability of the strain to swarm on semisolid agar plates (BM2 plates containing 0.4% agar). The PAO1 stringent response double mutant is unable to swarm (Fig. S7) (37, 84). The PAO1 wild-type strain as well as the ΔrelA ΔspoT mutant were transformed with an empty pHERD20T vector control and RelA-YFP and SpoT-GFP fusions. All strains were scraped from overnight-grown plates and suspended in sterile demineralized water to an OD₆₀₀ of 0.025. Ten microliters of a bacterial cell suspension was applied onto a swarming agar plate and incubated at 37°C for 18 h. Experiments were repeated at least three times. The motility complementation further verified an intact fusion protein (Fig. S6).

Verification of functional RelA-YFP and SpoT-GFP in the PAO1 ΔrelA ΔspoT stringent stress response mutant. Shown is swarming on BM2 agar plates at 37°C for 24 h. Representative images of the swarming-deficient stringent stress response mutant (left) and the complementation with RelA-YFP (middle) or SpoT-GFP (right) are shown. Experiments were repeated three times. Download FIG S7, EPS file, 0.8 MB.