Fig. 2: TCSs 13 and 17 are important for comG promoter activity and natural transformation in CS2 static (biofilm) growth conditions.

a The percentage of cells expressing P_comG-gfp reporter in Nef and its derivatives, Nef-H (overexpressing SigH), Δ13, Δ17, and ΔH (sigH-null mutant). Cells were statically grown in CS2 medium. The mean of at least n = 3 independent experiments is shown with error bars indicating SD. ND: none detected. b Chromosomal complementation of the mutants restores the percentage of cells expressing GFP at day 3 in the biofilm-growth conditions. Bars represent the mean of at least n = 7 independent experiments with error bars indicating SD. Gray dots represent independent experiments. ND: none detected. Chromosomally complemented strains were used for consistency with the transformation experiments (Fig. 2f). Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. Nef-GFP vs. Δ13-GFP ^*P = 0.0439, Nef-GFP vs. Δ17-GFP ^*P = 0.0223, ^**P = 0.0093, Δ17-GFP vs. Δ17(TCS17)-GFP ^*P = 0.0466. c–f Transformation assays. Data points represent independent experiments. The dotted lines represent detection limits. The mean of at least n = 2 independent experiments is shown. Error bars indicate SD. c Transformation frequencies of Nef (red), Nef-H (blue), and ΔH (black) at day 3 in the planktonic or biofilm-growth conditions. Transformants were selected by tetracycline. d Nef was statically grown with bacitracin, vancomycin, or nisin in CS2 medium. Transformants were selected by tetracycline after 3 days. e Time-course development of natural transformation in the biofilm for 4 days. Nef and its derivatives, Nef-H, Δ13, Δ17, ΔH, NefΔcomG (comG operon-null mutant), and NefΔcomE (comE operon-null mutant) were statically grown in CS2 medium. Natural transformation frequencies were determined every 24 h. f Complementation of the TCS13 and TCS17 restores the transformation frequencies at day 3 in the biofilm. Chromosomally complemented strains were used because plasmid-based complemented strains are tetracycline resistant. Statistical significance was determined by one-way ANOVA with Tukey’s multiple-comparison test. Δ13 vs. Δ13(TCS13) ^*P = 0.0272, Δ17 vs. Δ17(TCS17) ^*P = 0.0386, ^**P = 0.0031, ^****P < 0.0001. Source data are provided as a Source Data file.