Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20

Abstract

Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.

Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

“…Unlike the P c and P r promoters, the activities of P rap and P phr were not affected by the presence of pLS20 in the cell, indicating that Rco pLS20 does not regulate these promoters. The absence of Rco pLS20 binding motifs in the vicinity of the P rap and P phr promoters supports this conclusion (Singh et al, 2020). In summary, 1) the conjugation genes are under the control of the strong P c promoter, 2) the rco pLS20 and rap pLS20 genes, driving expression of the proteins regulating the activity of the P c promoter, are preceded by very weak promoters, 3) expression of phr is controlled by P rap and additionally by the two-fold stronger promoter P phr , presumably to accommodate for the dilution effect upon secretion, and 4) Rco pLS20 represses P c and regulates the P r promoter, but not promoters P rap and P phr .…”

“…Furthermore, AUC analyses provided conclusive evidence that Rap pLS20 forms predominantly dimers in solution. Sedimentation velocity experiments of mixtures of Rap pLS20 and Rco pLS20 resulted in the appearance of a novel species whose deduced molecular weight was compatible with a complex of one Rap pLS20 dimer and one Rco pLS20 tetramer (Singh et al, 2020). This conclusion FIGURE 3 | Relative strength of promoters P c , P r , P rap and P phr determined by FACS analysis of strains containing transcriptional gfp fusions.…”

“…A homogeneous pattern of P c promoter activity was also found for cells containing a derivative of pLS20cat in which a copy of the gfp gene was placed on pLS20 behind gene 28, the first gene of the conjugation operon. Together, these results strongly indicate that the multi-levels of regulation results in a sensitive genetic switch that activates the P c promoter in a coordinated manner in most or all pLS20-containing cells (Singh et al, 2020). This does not automatically imply that activation of the P c promoter guarantees successful plasmid transfer or even the initiation of the conjugation process in all cells.…”

“…At low expression levels Rco pLS20 stimulates the activity of P r but at high expression level it represses its own promoter ( Ramachandran et al, 2014 ). It is important to mention that the maximum strength of the P r promoter is about 50–75-fold lower than that of the P c promoter ( Singh et al, 2020 ). In summary, Rco pLS20 represses the strong promoter P c and simultaneously regulates the activity of its own promoter P r ( Ramachandran et al, 2014 ).…”

“…Light and dark colored bars reflect strains lacking or containing pLS20spec, respectively. The figure is adapted from (Singh et al, 2020 was reinforced by multi-signal sedimentation velocity (MSSV) analysis from which it was determined that the heterocomplex was composed of a stoichiometry of 2.1 mol of Rco pLS20 per mol of Rap pLS20 . More importantly, besides demonstrating that Rap pLS20 interacts with free Rco pLS20 , AUC analyses also showed that Rap pLS20 is able to interact with DNA-bound Rco pLS20 , and that this results in the release of Rco pLS20 from the DNA and the concomitant formation of Rap pLS20 /Rco pLS20 complexes.…”