Fig. 6. Self-association of ComEA is required for transformation.

All mutations were introduced into the chromosome of BD9007, which carries a competence-specific fusion of a sequence encoding cyan fluorescent protein (CFP) expressed from the promoter of comG, placed at the ectopic amyE locus to enable the identification of competence-expressing cells by epifluorescence microscopy. Transformation experiments were performed as biological triplicates and average frequencies, normalized to the wild-type values are plotted as bar graphs in panel A with the data points included. The larger number of data points for the wild-type control reflects the inclusion of this strain in all three biological replicates. The mean normalized values with standard deviations for the mutants were 0.0012 ± 0.00047 (ΔOD), 0.0.0005 ± 0.00024 (A106Y), 0.11 ± 0.03 (D111N). The p values were determined using two-sided t-tests. Panel B shows Western blots for the corresponding wild-type and mutant extracts using anti-GFP antiserum. ΔcomEA extracts were included as controls for the identity of the ComEA signals. Panel C shows typical epifluorescent images from the wild-type (BD9007) and its isogenic A106Y mutant equivalent, after transformation with rhodamine-labeled lambda bacteriophage DNA for 45-min. Competence-expressing cells were identified by CFP fluorescence (pseudocolored cyan) and detectable cell-associated rhodamine-tDNA signals are circled. For the wild type, 13 out of 42 cells exhibited at least one red dot, while for the 23 mutant cells only one barely detectable cell-associated dot was observed. The large red blotches in the lower panel are not associated with cells and are due to contaminating fluorescent material of unknown origin. The scale bar on the lower left of the image corresponds to 1 micron. The microscopy and Western blotting experiments in panels B and C were each repeated three times with closely similar results. Source data are provided as a Source Data file.