Fig. 5: Fitness impact of integrating an additional defense system or island into the chromosome of S. thermophilus DGCC7710.

A Schematic illustration of the insertion of a defense system into the chromosome of S. thermophilus through natural competence. A recombination template, consisting of the defense system flanked by 1 kb regions that are homologous to the upstream and downstream sequences in the recipient strain (e.g., DGCC7710), was transformed. Phages were used to select bacteria that integrated the defense system. B, C Assessment of the fitness cost (bacterial growth and milk acidification monitoring) following chromosomal integration of PD-Lambda-1 (B) or a defense island comprising Hachiman and RM II (C) at their native locus in DGCC7710. For bacterial growth graphs, each line represents the mean of biological replicates (n = 3) with shaded areas indicating confidence intervals. In milk acidification assays with phages, a phage cocktail was used at an initial MOI 0.0005. Each line shows the mean values, with shaded areas representing confidence intervals calculated from either three technical replicates (for PD-Lambda-1, Hachiman clone 2.4, and Hachiman clone 2.1 knockout conditions) or two biological replicates, each with three technical replicates (for Hachiman clone 2.1 and DGCC7710 WT). D Genomic context of selected defense systems integrated at non-native loci in DGCC7710. Thoeris originates from a short contig lacking conserved neighboring genes, preventing its precise localization within the DGCC7710 genome. E Evaluation of defense efficiency (EOP) and fitness cost (bacterial growth monitoring) for systems integrated at non-native loci. All experiments were performed in biological replicates (n = 3). Bars and curves indicate mean values, with standard deviations shown as error bars and confidence intervals as shaded areas. In the EOP plots, filled circles represent conditions with countable plaques, while hollow red circles indicate lysis zones without distinct plaques. EOP Efficiency of Plaquing, WT Wild Type, OD Optical Density, RM Restriction-modification system, R-S-M Restriction-Specificity-Methylase subunits of RM type I system. Source data are provided as a Source Data file.