| Summary: | <i>Bacillus subtilis</i> conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor Rco<sub>pLS20</sub>, the anti-repressor Rap<sub>pLS20</sub>, and the signaling peptide Phr*<sub>pLS20</sub>. In previous studies, artificial overexpression of <i>rap<sub>pLS20</sub></i> in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of <i>rap<sub>pLS20</sub></i> led to various phenotypic traits, including cell aggregation and death, which might have affected the correct determination of the conjugation efficiency when determined by colony formation assay. In the current study, conjugation efficiencies were determined under different conditions using a two-color fluorescence-activated flow cytometry method and measuring a single-round of pLS20-mediated transfer of a mobilizable plasmid. Under standard conditions, the conjugation efficiency obtained by fluorescence-activated flow cytometry was 23-fold higher than that obtained by colony formation. Furthermore, the efficiency difference increased to 45-fold when <i>rap<sub>pLS20</sub></i> was overexpressed.
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