Natural transformation and cell division delay in competent Staphylococcus aureus

ABSTRACT Genetic competence for natural transformation, considered one of the three main mechanisms leading to horizontal gene transfer in bacteria, is able to promote evolution, through genomic plasticity, and foster antibiotic resistance and virulence factors spreading. Conserved machinery and actors required to perform natural transformation have been shown to accumulate at different cellular localizations depending on the model organism considered. In this study, we investigate the transformation apparatus composition, localization, and dynamics in the human pathogen Staphylococcus aureus. We particularly show that most of the natural transformation actors co-localize in clusters. We also reveal that the localization of natural transformation proteins is dynamic, following the cell cycle. Ultimately, the natural transformation apparatus is preferentially established in the vicinity of the division septum. All these results demonstrate that DNA binding, uptake, and recombination are spatially and temporally coordinated to ensure S. aureus natural transformation. Finally, we hypothesize that S. aureus competent cells would initiate and then block cell division to ensure the success of natural transformation before the final constriction of the cytokinetic ring. IMPORTANCE Natural transformation, considered one of the three main mechanisms leading to horizontal gene transfer in bacteria, is able to promote genomic plasticity and foster antibiotic resistance spreading. Conserved machinery and actors required to perform natural transformation have been shown to accumulate at different cellular localizations depending on the model organism considered. Here, we show in the human pathogen Staphylococcus aureus that DNA binding, uptake, and recombination are spatially and temporally coordinated to ensure S. aureus natural transformation. We also reveal that localization of natural transformation proteins occurs in the vicinity of the division septum allowing S. aureus competent cells to block cell division to ensure the success of natural transformation before the final constriction of the cytokinetic ring.

(b) Examples of the three ComGA-EGFP (pRIT-P comGA -comGA-egfp) cellular localizations observed using the St113 strain grown in CS2 medium (from left to right: cytoplasmic, associated to the inner face of the membrane and accumulation in foci near the membrane).
For each localization pattern, we provide 3D reconstructions (top row) as well as 360 degrees videos.Bar = 4 µm (c) Dynamic of ComGA-EGFP localization patterns in S. aureus competent cells from diluted cultures (10 -3 top panel; 10 -4 , middle panel; 10 -5 , bottom panel).In addition to the evolution of the percentage of ComGA-EGFP-expressing cells (10 -3 in green, 10 -4 in blue and 10 -5 in red), histograms represent the percentage of each ComGA-EGFP localizing pattern : in the cytoplasm (light grey), associated to the membrane (dark grey) or as single foci (black).At least 1500 cells were counted for each time point in each culture.This table completes the results presented in Fig. 5. Strains St29 (pRIT-PcomGA-egfp), St113 (pRIT-PcomGA-comGA-egfp) and St243 (pRIT-PcomGA-comGCflag) were grown to competence for 23h in CS2 medium (10 -5 dilution).After exposition to fluorescently labelled DNA, the percentage of competent cells, of cells binding DNA were evaluated.In addition, the percentage of cells binding DNA that were also competent as well as competent cells that were also binding DNA was calculated (two last columns).

Supplementary
This table completes the results presented in Fig. 6.
At least 400 cells were counted for each strain.

. 1 .
Spatial and temporal dynamic of ComGA-EGFP localization in competent S. aureus cells.

Table 3 . ComGC-FLAG localization in S. aureus competent cells.
PcomGA-comGA-mCherry / pRIT-PcomGA-comGC-FLAG) was grown to competence for 25h in CS2 medium (10 -5 dilution).The percentage of ComGA-mCherry-expressing cells, ComGC-FLAG-expressing cells as well as the percentage of ComGA-mCherry-expressing cells that also express ComGC-FLAG and ComGC-FLAG-expressing cells that also express ComGA-mCherry were evaluated by microscopy.Pearson's correlation coefficients were calculated based on 10 individual cells as explained in the Material and methods.This table completes the results presented in Fig.4.At least 400 cells were counted for each strain.