Figure 2. CrvA forms a periplasmic filament that does not measurably alter sacculus composition.

(A) Diagram of predicted CrvA-GFP domains showing signal sequence (sig seq, residues 2-36), coiled-coil domain 1 (cc1, residues 44-110), coiled-coil domain 2 (cc2, residues 143-182), PEGA-like CT domain (residues 310-386), and monomeric superfolder GFP fusion. See Figure S4 for alignments of CrvA amino acid sequence to proteins with predicted similar features.

(B) Phase images overlaid with GFP fluorescence images; scale bar = 3 μm. The top panel is the parental CrvA-GFP, included for comparison. The second panel is a signal sequence deletion (CrvA-GFPΔ(2-37)), yielding straight cells with diffuse cytoplasmic signal. The third panel is a cc2 domain deletion (CrvA-GFPΔ(143-182)), yielding straight cells with polar foci. Panels four and five are deletions within the CT domain of CrvA-GFP (CrvA-GFPΔ(324-327) and CrvA-GFPΔ(332-336)), yielding cells with drastically reduced curvature and shorter filaments. See Video S3 for time-lapse of CT domain mutants.

(C) TEM reveals that CrvA forms filaments in vitro. The protein was purified and allowed to polymerize at 37°C, and then negatively stained with uranyl acetate and lead citrate. See Method Details for more information, and Figure S5A–D for more on CrvA in vitro purification and characterization. See Video S2 for time-lapse of CrvA-GFP structures during cell lysis. Scale bar = 200 nm.

(D) Fluorescence images of OMVs purified from stationary phase cultures of CrvA-GFP cells. See Figure S5E–F for further characterization of OMVs. Scale bar = 3 μm.

(E–F) UPLC analysis of V. cholerae wild-type and ΔcrvA sacculi. See Table S1 for UPLC data.

(E) UPLC shows no significant difference between PG species of wild-type and ΔcrvA sacculi, at OD₆₀₀ 0.8 and 2.0. Means of three runs ± SEM.

(F) Overlay of representative chromatograms of WT and ΔcrvA at OD 0.8 and OD 2.0 with baselines shifted for ease of visualization.