Journal: Nature Communications

Article Title: Temporal gene regulation enables controlled expression of gas vesicles and preserves bacterial viability

doi: 10.1038/s41467-025-67667-8

Figure Lengend Snippet: a Amino acid sequence and cryo-EM structure (PDB 7R1C) of the major gas vesicle shell protein GvpA2. Structural segments are annotated as N-terminal (Nt), first α-helix (α1), first β-sheet (β1), second β-sheet (β2), second α-helix (α2), and C-terminal (Ct) regions. Hydrophobic residues are depicted in yellow, while hydrophilic residues are shown in light blue. b Structural organization of GvpA2 within the GV shell (PDB 7R1C). Hydrophobic amino acid residues of GvpA2 are oriented toward the GV’s interior, establishing a gas-facing surface, while hydrophilic residues are exposed on the exterior, interfacing with the aqueous environment. c Growth profiles of E. coli with mean ± s.d. for n = 6 biologically independent samples for the GvpA2-expressing cultures and MBP-expressing cultures. Absorbance ( y -axis) is reported in absorbance units (AU). d CFU/mL of E. coli expressing GvpA2 or MBP under 20 µM IPTG, quantified over 24 h (mean ± s.d., n = 3 biological replicates). The light-purple inset shows representative GvpA2 plates at 4 h (1:10 7 for 0 and 20 µM IPTG; 1:10 5 for 20 µM), illustrating reduced viable counts. Additional representative plates are shown in Supplementary Fig. . e Cell viability of E. coli expressing GvpA2 or MBP under 20 µM IPTG, quantified by SYTO9/PI flow cytometry (mean ± s.d., n = 3 biological replicates). Two-tailed paired Student’s t test: NS at 0, 12, and 24 h; P = 0.0229 at 4 h and <0.0001 ( P = 0.00006) at 8 h. Insets show representative GvpA2 plots at 4 and 8 h with red gates marking PI-positive cells. Additional representative plots are shown in Supplementary Fig. . f Fluorescence microscopy images of E. coli cells expressing mCherry, mCherry::GvpA2, and GvpA2::mCherry. g Fluorescence microscopy images of E. coli cells expressing mCherry with its C-terminus fused to defined segments of GvpA2. Structural annotations on the right corners of micrographs correspond to those shown in ( a ). Scale bars, 20 mm in ( d ) and 2 µm in ( f , g ).

Article Snippet: The monomeric Cherry red fluorescent protein (mCherry) gene was obtained from Addgene (plasmid #29747), and the superfolder green fluorescent protein (sfGFP) gene was acquired from Addgene (plasmid #85492).

Techniques: Sequencing, Cryo-EM Sample Prep, Expressing, Flow Cytometry, Two Tailed Test, Fluorescence, Microscopy

Journal: Nature Communications

Article Title: Temporal gene regulation enables controlled expression of gas vesicles and preserves bacterial viability

doi: 10.1038/s41467-025-67667-8

Figure Lengend Snippet: Schematic representation of the genetic constructs of the dual-inducer system ( a ) and two single‑reporter constructs ( b ) for fluorescent reporter proteins expression. c , d Fluorescence fold change of dual‑inducer vs. single‑reporter across inducer concentrations. c mCherry-only vs. mCherry-dual: NS at 0 µM IPTG; P = 0.0003, <0.0001 ( P = 0.00000058), and <0.0001 ( P = 0.000003) at 20, 200, and 400 µM. d sfGFP-only vs. sfGFP-dual: NS for all. e , f Fluorescence fold change of the dual-inducer vs. single-reporter without the corresponding inducer. e mCherry-dual vs. mCherry-only. P = 0.00461, 0.0165, <0.0001 ( P = 0.00002) at 0, 50, 500 ng/mL aTc. NS at 1000 ng/mL. f sfGFP-dual vs. sfGFP-only. NS at 0, 20, 200 µM IPTG; P = 0.00461 at 400 μM. Schematic representation of the dual-inducer system induced with varying concentrations of IPTG at fixed aTc concentration ( g ) or with varying concentrations of aTc at fixed IPTG concentration ( j ). Fold change in mCherry ( h ) and sfGFP ( k ). mCherry: 0 vs. 500 ng/mL aTc across IPTG concentrations: NS at 0, 20 µM; P = 0.0238, 0.0044 at 200, 400 µM. sfGFP: 0 vs. 200 µM IPTG across aTc concentrations: NS at 0, 50 ng/mL; P = 0.0089, 0.0012 at 500, 1000 ng/mL. Fold change in sfGFP ( i ) and mCherry ( l ). i sfGFP-dual cultures at 500 ng/mL aTc without IPTG vs. 0–400 µM IPTG: NS at 0 µM; P = 0.0009, 0.0007, 0.0010 at 20, 200, 400 µM. l mCherry-dual cultures at 200 µM IPTG without aTc vs. 0–1000 ng/mL aTc: NS at 0 ng/mL; P = 0.0478, 0.0013, 0.0145 at 50, 500, 1000 ng/mL. Fold change in c – f , h , i , k , l is plotted in arbitrary units (AU, y -axis), data representing mean ± s.d. ( n = 6 biologically independent samples). Statistics: c , d , h , k by two-tailed unpaired Welch t-test; e , f , i , l vs. grey controls by Brown–Forsythe and Welch one-way ANOVA with Dunnett T3 correction.

Article Snippet: The monomeric Cherry red fluorescent protein (mCherry) gene was obtained from Addgene (plasmid #29747), and the superfolder green fluorescent protein (sfGFP) gene was acquired from Addgene (plasmid #85492).

Techniques: Construct, Expressing, Fluorescence, Concentration Assay, Two Tailed Test