(A) Schematic representation of the triple-labeled strain VL3126 harboring three reporter cassettes: hlpA-mScarlet-I, spv_1159-msfYFP, and ftsZ-mTurquoise2 at the hlpA, CEP, and ftsZ loci, respectively. Strain VL3127 contains stop codon mutations in the linker between each of the fluorescent fusion proteins. Gray arrows indicate the direction of the DNA replication fork relative to the reporter cassette. (B) Microscope image of strain VL3127 treated with CSP and transformed with the tDNAs of hlpA-mScarlet-I, spv_1159-sfGFP, and ftsZ-mTurquioise2 (3.2 nM each) amplified from VL3126. Merge image of phase contrast, cyan (FtsZ-mTurquoise2), yellow (spv_1159-msYFP) and red (HlpA-mScarlet-I) fluorescence is shown. Scale bar: 10 μm. (C and D) Proportion of transformed phenotypes. Population of each transformed phenotype was quantified from microscope images. Representative images for each phenotype are shown. Scale bar: 2 μm. (D) Stacked bars represent proportion of single transformed (cyan, yellow or red), double transformed [green (cyan+yellow), blue (cyan+red), orange (yellow+red)], triple transformed (white) and non-transformed (gray) cells. Bars represent mean ± SD of three independent replicates. Analyzed data of the positive control strain VL3126 (ftsZ-mTurquoise2, spv_1159-msfYFP, hlpA-mScarlet-I) and negative control non-transformed strain VL3127 (ftsZ-stop-mTurquoise2, spv_1159-stop-msfYFP, hlpA-stop-mScarlet-I) are also shown, demonstrating the accuracy of the threshold used to score positive transformants. (E) Competition effect of unrelated DNA on transformation frequency. CSP-treated VL1803 was transformed with 7 kb hlpA-mScarlet-I tDNA at the final concentration of 0.32 nM in the absence or the presence of an unrelated DNA fragment (0.32 nM or 3.2 nM) amplified from E. coli. After incubation of 4 hr post transformation, cells were separated and analyzed by flow-cytometry. Red vertical line indicates the threshold of positive cells in mScarlet-I signal expression. The proportion of positive cells (%) is depicted in the plots. (F). Fragmented tDNA recombination model. The fluorescence-based reporters used in this study rely on replacement of the stop codon SNP (gray star) by intact (amino acid coding) SNP (yellow star) that is located in the middle of the tDNA fragment (orange line). All prepared tDNA molecules have obviously intact SNP, but, integration into host chromosome may take place outside the SNP, which is never distinguished from true untransformed cells by the fluorescence-based system and effectively acting as competing DNA for tDNAs that transform the SNP.