Abstract
Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In previous studies, artificial overexpression of rappLS20 in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of rappLS20 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 rappLS20 was overexpressed.
Highlights
The conjugation efficiency of transferring pLS20 itself may be misestimated because a generated transconjugant can serve as a donor that transfers the plasmid in the second round of conjugation
In the case of the colony formation assay, the efficiency panels A and B of Figure 6). This result is consistent with the Cell Aggregate Rate (CAR) value being almost was elevated 3-fold (0.14%/0.045% = 3.1) higher upon the artificial overexpression of equal to 1 when cells were grown in the absence of IPTG (Figure 2A), suggesting that conjugation genes, which was half of the elevation of 6-fold calculated as above from the excessive cell–cell aggregation does not occur under native mating conditions
On the other hand, when the conjugation genes were overexpressed, several lines of evidence suggested that overexpression of the conjugation genes resulted the formation
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conjugation requires direct contact between two cells, as the DNA element is transferred from a donor cell to a recipient cell through a channel that connects them. The selection of a suitable recipient cell involves proteins playing a role in a process called exclusion that inhibits the transfer of the conjugative element between two donor cells. Recently an alternative methodology has been described in which the conjugation efficiency is determined by labeling the conjugative element and the recipient cells with genes encoding different fluorescent proteins and analyzing the cells with fluorescence-activated flow cytometry and microscopy [25,26,27,28]. A mobilizable plasmid and the recipient cell labeled with a different gene for fluorescent protein were subjected to fluorescence-activated flow cytometry to investigate pLS20-mediated conjugation. We re-evaluate the efficiency of pLS20 conjugation upon the enhanced production of RappLS20 , and visualize possible effects of the artificial overexpression of the conjugation genes on cell aggregation
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