The alternative sigma factor σ^Xmediates competence shut-off at the cell pole inStreptococcus pneumoniae

Abstract

SummaryBacterial competence for genetic transformation is a well-known species-specific differentiation program driving genome plasticity, antibiotic resistance and virulence in many pathogens. How competence regulation is spatiotemporally integrated in the cell is ill-defined. Here, we unraveled the localization dynamics of the key regulators that master the two intertwined transcription waves controlling competence in Streptococcus pneumoniae. The first wave relies on a stress-inducible phosphorelay system, made up of the ComD and ComE proteins, and the second is directed by an alternative sigma factor, σX, which includes in its regulon the DprA protein that turns off competence through interaction with phosphorylated ComE. Remarkably, we found that ComD, σX and DprA stably co-localize at a single cell pole over the competence period. In contrast, ComE assembles into dynamic patches in the cell periphery, colocalizing temporarily with DprA and ComD at the pole. Furthermore, we provide evidence that σX directly conveys DprA polar anchoring. Through this protein targeting function, σX is shown to be actively involved in the timely shut-off of the competence cycle, hence preserving cell fitness. Altogether, this study unveils an unprecedented role for a bacterial transcription σ factor in spatially coordinating the negative feedback loop of its own genetic circuit.

The RecA-directed recombination pathway of natural transformation initiates at chromosomal replication forks in Streptococcus pneumoniae

“…We took advantage of this strain and these conditions to visualise DprA-GFP during transformation as described above, with 48% of cells possessing DprA-GFP foci, mostly at midcell (Figures 2AB). By contrast, only 7% of cells possessed foci in the absence of tDNA, in agreement with previous results 38 . Most cells present DprA-GFP foci at midcell, while late dividing cells present foci at the ¼ and ¾ positions, future sites of midcell in daughter cells (Figure 2B).…”

“…By contrast, only 7% of cells possessed foci in the absence of tDNA, in agreement with previous results 38 . Most cells present DprA-GFP foci at midcell, while late dividing cells present foci at the ¼ and ¾ positions, future sites of midcell in daughter cells (Figure 2B).…”

“…We showed previously using a strain expressing dprA under the control of an IPTG-inducible P lac promoter ( CEP lac -dprA ) and lacking native dprA that reducing cellular levels of DprA in competent cells prevented competence shut-off from occurring whilst maintaining optimal transformation efficiency 39 . Using a CEP lac -dprA-gfp fusion, we also showed that in similar conditions (6 µM IPTG), no polar foci of DprA-GFP were observed 38 . We took advantage of this strain and these conditions to visualise DprA-GFP during transformation as described above, with 48% of cells possessing DprA-GFP foci, mostly at midcell (Figures 2AB).…”

“…To observe the early DprA-mediated HR steps of natural transformation in individual living competent pneumococcal cells, we tracked the localisation of a fluorescent DprA-GFP fusion proven to be fully functional in transformation assays 38 . Purified DprA-GFP was as efficient as DprA in assisting RecA-directed HR in an in vitro D-loop assay (Extended Figure 1), validating use of this fusion for analysing DprA localisation dynamics during transformation.…”

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

“…R304 ( smR; rifR; novR ) ( Chastanet et al, 2001 ) strain was transformed with genomic DNA from R4574 ( ∆comCDE::trmpR ) ( Johnston et al, 2020 ) and was selected by trimethoprim, obtaining R4692.…”