Extended Data Fig. 9: Fluorescent protein-based reporters for c-di-GMP level and for Vibriophage N4 infection in V. cholerae cells.
From: Bacteria use exogenous peptidoglycan as a danger signal to trigger biofilm formation
a, Calibration results for the c-di-GMP reporter. The reporter is based on the triple-tandem riboswitch Bc3-569, which permits the transcription of the sfGFP-LAA gene, coding for an unstable superfolder-GFP with the LAA degradation tag53. The bc3-5-sfGFP-LAA fragment was cloned into a low copy-number plasmid (pSC101*) harboured in V. cholerae. The bar graph shows the quantification of the unstable-sfGFP fluorescent intensity levels in microscopy images, normalized by the mean of the WT level, for three different strains that are known to have different levels of c-di-GMP70, which were grown in liquid shaking culture until OD600 = 0.4. The ∆4DGC strain lacks four diguanylate cyclases (∆cdgD∆cdgK∆cdgH∆cdgL), which are proteins that can produce c-di-GMP. The ∆2PDE strain lacks two phosophodiesterases (∆rocS∆cdgJ), which are proteins that can degrade c-di-GMP. The cellular c-di-GMP levels were expected to be intermediate for the WT, low for the ∆4DCG mutant, and high for the ∆2PDE mutant70,71. The fluorescence levels of the unstable sfGFP correspond qualitatively to the expected c-di-GMP levels. Bars are mean values with points denoting n = 3 biological replicates and error bars indicate the standard deviation. Statistical significances were calculated using a one-way ANOVA with Bonferroni’s correction, yielding p-values that are indicated in brackets in the graph underneath the * symbol. b, To construct the fluorescent protein-based reporter for Vibriophage N4 infection in V. cholerae cells, the promoter of the gene VN4_32 (encoding the major capsid protein) was identified on the Vibriophage N4 genome using PHIRE72. c, Schematic drawing of the phage infection reporter system: The phage promoter PVN4_32 was fused to mNeonGreen (cyan) and inserted at the lacZ locus on the V. cholerae chromosome. Constitutive fluorescence was achieved by engineering a Ptac-TagRFP-T (red) construct on a low-copy plasmid (pSC101*). d, Confocal microscopy image time series showing the production of mNeonGreen (cyan) during phage infection, followed by cell lysis. All cells produce TagRFP-T (red).
