Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression

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.

Constitutive expression of the global regulator AbrB restores the growth defect of a genome-reduced Bacillus subtilis strain and improves its metabolite production

“…Flow cytometric analysis was carried out as described previously with the following modifications. 27 Cells cultured in MMOPS medium in a microplate as described above were fixed with 4% (v/v) formaldehyde at 25°C for 30 min, washed once in a buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 200 mM KCl, and 5% [v/v] glycerol) suspended in phosphate-buffered saline (1 mM KH 2 PO 4 , 3 mM Na 2 HPO 4 , and 155 mM NaCl), and analyzed by flow cytometry using CytoFlexS (Beckman Coulter, Brea, CA, USA) without sonication. Data analysis was performed using the CytoExpert software bundle.…”

A conjugative plasmid exploits flagella rotation as a cue to facilitate its transfer

“…To study the nature of the link between flagellum functionality and conjugation events, we assessed whether the necessity for flagella within donor cells could be bypassed by enhancing the transcription of conjugative genes. To achieve this, the anti-Rco conjugation repressor, RapApLS20 (herein RapA) 23,26 , was ectopically overexpressed in flagellar mutants and assayed for conjugation. Indeed, ectopic expression of RapA effectively bypassed the conjugation defect in all tested flagellar mutants (Figure 3A), hinting that flagella might play a role in augmenting the transcription of conjugation genes.…”

“…In parallel, an anti-repressor, termed Rap, is held inactive through binding to a short quorum-sensing signaling peptide, Phr, encoded by the plasmid. The transition to an "ON" state is facilitated by a decrease in Phr concentration, and the consequent liberation of Rap, which counteracts Rco, thus promoting conjugation [23][24][25][26] .…”

Phenotypic plasticity: The role of a phosphatase family Rap in the genetic regulation ofBacilli

“…This ability, known as horizontal gene transfer (HGT), is an efficient mechanism for individual organisms to acquire genes, regardless of functionality (Ochman et al, 2000; Soucy et al, 2015). HGT of Rap‐Phr cassettes is heightened due to the fact that many rap‐phr genes are encoded within mobile genetic elements (Bongiorni et al, 2006; Butala & Dragoš, 2022; Meijer et al, 2021; Mori et al, 2021; Parashar, Konkol, et al, 2013; Rösch et al, 2014; Singh et al, 2013). Importantly, Rap‐Phr can also control mobile elements, as RapI‐PhrI sensory system activates gene expression within the ICEBs1 mobile genetic element, therefore contributing to excision and transfer (Auchtung et al, 2005).…”

“…Even‐Tov et al estimated that up to 75% of Rap‐Phr cassettes may be mobile (i.e., coded on plasmids, prophages, or integrative and conjugative elements), based on a GC‐content comparison with their host strain (Even‐Tov, Omer Bendori, Pollak, et al, 2016). Furthermore, some Rap‐Phr cassettes are able to regulate the mobility of the genetic element that contains them, be them plasmids (Meijer et al, 2021; Mori et al, 2021; Singh et al, 2013), or transposons (Auchtung et al, 2005, 2007). These features could then favor a rapid expansion of Rap‐Phr cassettes through HGT among Bacilli .…”