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• W3105441053 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Competence is a widespread bacterial differentiation program driving antibiotic resistance and virulence in many pathogens. Here, we studied the spatiotemporal localization dynamics of the key regulators that master the two intertwined and transient transcription waves defining competence in Streptococcus pneumoniae. The first wave relies on the stress-inducible phosphorelay between ComD and ComE proteins, and the second on the alternative sigma factor σX, which directs the expression of the DprA protein that turns off competence through interaction with phosphorylated ComE. We found that ComD, σX and DprA stably co-localize at one pole in competent cells, with σX physically conveying DprA next to ComD. Through this polar DprA targeting function, σX mediates the timely shut-off of the pneumococcal competence cycle, preserving cell fitness. Altogether, this study unveils an unprecedented role for a transcription σ factor in spatially coordinating the negative feedback loop of its own genetic circuit. Introduction In bacteria, sigma (σ) factors are essential transcription effectors that direct the RNA polymerase to and activate RNA synthesis at specific genes promoters. All bacterial species encode a single, highly conserved σ factor that drives the expression of house-keeping genes essential for vegetative growth and cell homeostasis. In addition, many bacteria encode a variable set of alternative σ factors that control specific regulons, providing appropriate properties to the cells in response to various stimuli. These alternative σ factors play pivotal roles in the multifaceted lifestyles of bacteria. They trigger specific developmental programs, such as sporulation or biofilm formation, as well as adapted responses to multiple types of stress and virulence in some pathogenic species (Kazmierczak et al., 2005). How these alternative σ factors are activated in the cell has been extensively studied, revealing multiple mechanisms underlying their finely tuned regulation (Österberg et al., 2011). However, how these mechanisms are orchestrated spatiotemporally within the cell remains poorly understood. The human pathogen Streptococcus pneumoniae (the pneumococcus) possesses a unique alternative σ factor σX (Lee and Morrison, 1999). It is key to the regulatory circuit controlling the transient differentiation state of competence. Pneumococcal competence is induced in response to multiple types of stresses, such as antibiotic exposure (Prudhomme et al., 2006; Slager et al., 2014). This induction modifies the transcriptional expression of up to 17% of genes (Aprianto et al., 2018; Dagkessamanskaia et al., 2004; Peterson et al., 2004; Slager et al., 2019). Competence is a key feature in the lifestyle of pneumococci as it promotes natural transformation, a horizontal gene transfer process widespread in bacteria that facilitates adaptation by acquisition of new genetic traits (Johnston et al., 2014). In addition, pneumococcal competence development provides the cells with the ability to attack non-competent cells, a scavenging property defined as fratricide (Claverys and Håvarstein, 2007), is involved in biofilm formation (Aggarwal et al., 2018; Vidal et al., 2013) and virulence (Johnston et al., 2018; Lin et al., 2016; Lin and Lau, 2019; Zhu et al., 2015). Pneumococcal competence induction is primarily regulated by a positive feedback loop involving the genes encoded by the comAB and comCDE operons (Figure 1A). The comC gene codes for a peptide pheromone coordinating competence development within the growing cell population. This peptide, accordingly named CSP (Competence Stimulating Peptide), is secreted by the dedicated ComAB transporter (Hui et al., 1995). After export, it promotes autophosphorylation of the membrane-bound two-component system histidine kinase ComD, which in turn phosphorylates its cognate intracellular response regulator ComE (Figure 1A). Phosphorylated ComE (ComE~P) specifically induces the expression of 25 genes, which include the comAB and comCDE operons, generating a positive feedback loop that controls competence development. Conversely, unphosphorylated ComE acts as repressor of its own regulon, the expression of which is thus modulated by the ComE/ComE~P ratio (Martin et al., 2013). The ComE regulon includes two identical genes encoding σX, named comX1 and comX2 (Lee and Morrison, 1999). The σX regulon comprises ~60 genes, with ~20 involved in natural transformation (Claverys et al., 2006; Peterson et al., 2004), five in fratricide (Claverys and Håvarstein, 2007) but the majority having undefined roles. The reason why the σX-encoding gene is duplicated is unknown, the inactivation of one of them having no impact on transformation (Lee and Morrison, 1999). To fully activate transcription, σX needs to be assisted by ComW, another protein whose production is controlled by ComE~P (Luo et al., 2004). ComW is proposed to help σX association with the RNA polymerase at promoter sequences presenting the consensual 8 bp cin box motif (Peterson et al., 2004; Sung and Morrison, 2005). Altogether, ComE~P and σX trigger two successive waves of competence (com) gene transcription, commonly referred to as early and late, respectively. Importantly, competence shut-off is mediated by the late com protein DprA (Mirouze et al., 2013; Weng et al., 2013), which directly interacts with ComE~P to turn-off ComE~P-dependent transcription (Mirouze et al., 2013). In addition to defining the negative feedback loop of the pneumococcal competence regulatory circuit, DprA also plays a crucial, conserved role in transformation by mediating RecA polymerization onto transforming ssDNA to facilitate homologous recombination (Mortier-Barrière et al., 2007; Quevillon-Cheruel et al., 2012; Figure 1A). Over 8000 molecules of DprA are produced per competent cell (Mirouze et al., 2013). Although only ~300–600 molecules are required for optimal transformation, full expression of dprA is required for optimal competence shut-off (Johnston et al., 2018). Uncontrolled competence induction in cells lacking DprA results in a large in vitro growth defect, and high cellular levels of DprA thus maintain the fitness of the competent population and of resulting transformants (Johnston et al., 2018). In addition, inactivation of dprA was shown to be highly detrimental for development of pneumococcal infection, dependent on the ability of cells to develop competence (Lin and Lau, 2019; Zhu et al., 2015). Together, these studies showed that the DprA-mediated shut-off of pneumococcal competence is key for pneumococcal cell fitness. Figure 1 with 1 supplement see all Download asset Open asset DprA: Localization and roles in competence and transformation. (A) (1) Pre-CSP is exported and matured by the ComAB transporter, and then promotes phosphorylation of the histidine kinase ComD. (2) ComD transphosphorylates ComE, which then stimulates the expression of 17 early com genes, including two copies of comX. (3) These encode an alternative sigma factor σX, which controls late com genes including dprA. (4) DprA dimers load RecA onto ssDNA to mediate transformation and interact with ComE~P to shut-off competence. (5) Transforming DNA is internalized in single strand form and is protected from degradation by DprA and RecA (6), which then mediate transformation (7). Orange arrows, early com promoters; purple arrows, late com promoters. (B) Western blot tracking cellular levels of DprA-GFP after competence induction in strain R3728. α-DprA antibody used. (C) Sample fluorescence microscopy images of R3728 strain producing DprA-GFP 15 min after competence induction. Scale bars, 1 µm. (D) Schematic representation of focus density maps with half cells represented as vertical lines in ascending size order and localization of foci represented along the length axis of each half cell. Black half-cells represent those presented, and grey those not presented. (E) DprA-GFP accumulates at the cell poles during competence. 1290 cells and 1128 foci analyzed. (F) Sample immunofluorescence microscopy images of a strain producing wild-type DprA (R1502; wildtype) and a strain lacking DprA (R2018; dprA-) fixed 15 min after competence induction and probed using anti-DprA antibodies. Scale bars, 1 µm. A hallmark of pneumococcal competence is its tight temporal window, which lasts less than 30 minutes in actively dividing cells (Alloing et al., 1998; Håvarstein et al., 1995). How this regulation is coordinated within the cell remains unknown. Here, we studied the choreography of pneumococcal competence induction and shut-off at the single cell level by tracking the spatiotemporal localization of the main effectors of these processes, DprA, σX, ComW, ComD, ComE and exogenous CSP. Remarkably, DprA, σX, ComD, CSP and to some extent ComE were found to colocalize at a single-cell pole during competence. This study revealed that the entire pneumococcal competence cycle occurs at cell pole, from its induction triggered by ComD, ComE, and CSP to its shut-off mediated by DprA and assisted by σX. In this regulatory mechanism, σX is found to exert an unprecedented role for a σ factor. In addition to directing the transcription of the dprA gene, σX associates with and anchors DprA at the same cellular pole where ComD and CSP are located, allowing this repressor to interact with newly activated ComE~P and promoting timely extinction of the whole transcriptional regulatory circuit of competence. Results DprA displays a polar localization in competent cells, which correlates with competence shut-off To investigate the localization of DprA during competence in live cells, we used a fluorescent fusion protein DprA-GFP, produced from the native dprA locus (Figure 1—figure supplement 1A). DprA-GFP was synthesized during competence and remained stable up to 90 min after induction (Figure 1B), similarly to wild-type DprA (Mirouze et al., 2013), without degradation (Figure 1—figure supplement 1B). A strain possessing DprA-GFP was almost fully functional in transformation (Figure 1—figure supplement 1C), but partially altered in competence shut-off (Figure 1—figure supplement 1D). In pneumococcal cells induced with exogenous CSP, the peak of competence induction occurs 15–20 min after induction. 15 min after competence induction, DprA-GFP showed a diffuse cytoplasmic localization, punctuated by discrete foci of varying intensity (Figure 1C). The distribution and localization of DprA-GFP foci was analyzed by MicrobeJ (Ducret et al., 2016), with results presented as focus density maps ordered by cell length. Spots represent the localization of a DprA-GFP focus on a representative half pneumococcal cell, while spot color represents density of foci at a particular cellular location. DprA-GFP foci were present in 70% of cells, predominantly at a single cell pole (Figure 1DE). To ascertain whether the polar localization of DprA-GFP was due to the GFP tag or represented functional localization, we carried out immunofluorescence microscopy with competent cells possessing wild-type DprA using α–DprA antibodies. Results showed that native DprA exhibited a similar accumulation pattern as the DprA-GFP foci upon competence induction (Figure 1F). Next, to explore the relation between these foci and the dual role of DprA in transformation and competence regulation, we investigated focus formation in cells possessing a previously published mutation in DprA impairing its dimerization (DprAAR) (Quevillon-Cheruel et al., 2012). This mutant strongly affected both transformation and competence shut-off (Mirouze et al., 2013; Quevillon-Cheruel et al., 2012). Results showed that 15 min after competence induction, DprAAR-GFP did not form foci, despite being produced at wild-type levels (Figure 1—figure supplement 1E). DprA thus accumulates at the cell pole during competence, dependent on its ability to dimerize. To explore whether the polar foci of DprA were involved in its role in transformation, or competence shut-off, we began by investigating how a dprAQNQ mutation, specifically abrogating the interaction between DprA and RecA and thus affecting transformation (Quevillon-Cheruel et al., 2012), affected the localization of DprA-GFP. Fifteen minutes after competence induction, the DprAQNQ-GFP mutant formed polar foci at wild-type levels (Figure 1—figure supplement 1F). In addition, the inactivation of comEC, encoding for an essential protein of the transmembrane DNA entry pore (Pestova and Morrison, 1998), or recA, encoding the recombinase with which DprA interacts during transformation (Mortier-Barrière et al., 2007), did not alter the frequency or localization of DprA-GFP foci (Figure 1—figure supplement 1GH). Altogether, these results suggested that the polar foci of DprA-GFP may not be related to the DNA entry or recombination steps of transformation but could be linked to competence shut-off. We recently reported that optimal competence shut-off relies on the maximal cellular concentration of DprA (~8000 molecules), while wild-type transformation required only around 1/10th of these molecules (Johnston et al., 2018). This conclusion was obtained by expressing dprA under the control of the IPTG-inducible Plac promoter (CEPlac-dprA), which enables the modulation of the cellular concentration of DprA by varying IPTG concentration in the growth medium. Here, we reproduced these experiments with the DprA-GFP fusion, to test whether its concentration correlates with the formation of polar foci in competent cells. The expression, transformation, and competence profiles of a dprA- mutant strain harboring the ectopic CEPlac-dprA-gfp construct in varying concentrations of IPTG (Figure 2—figure supplement 1) were equivalent to those reported previously for CEPlac-dprA (Johnston et al., 2018). Notably, a steady decrease in DprA-GFP foci was observed as IPTG was reduced (Figure 2AB). When comparing the cellular localization of DprA-GFP foci, a sharp reduction in the proportion of polar foci was observed as IPTG was reduced, with most of the remaining foci observed at midcell and appearing weaker in intensity (Figure 2AC). This shift correlated with a progressive loss of competence shut-off (Figure 2—figure supplement 1C), presenting a strong link between the presence of polar DprA-GFP foci and the shut-off of pneumococcal competence. Altogether, these results strongly support the notion that the polar foci of DprA-GFP represent the subcellular site where DprA mediates competence shut-off. Figure 2 with 1 supplement see all Download asset Open asset Reducing cellular DprA-GFP levels results in loss of polar accumulation and competence shut-off. (A) Focus density maps of DprA-GFP foci at different cellular levels during competence. CEPlac-dprA-gfp from strain R4262. Cellular DprA-GFP levels were controlled by growing cells in a gradient of IPTG. 1.5 µM IPTG, 11267 cells and 791 foci analyzed; 3 µM IPTG, 10623 cells and 748 foci analyzed; 6 µM IPTG, 10603 cells and 743 foci analyzed; 12.5 µM IPTG, 6985 cells and 1010 foci analyzed; 25 µM IPTG, 2945 cells and 1345 foci analyzed; 50 µM IPTG, 3678 cells and 1964 foci analyzed. Sample microscopy images of strain R4262 in varying IPTG concentrations. Scale bars, 1 µm. (B) Reducing cellular levels of DprA-GFP reduces the number of cells with foci. Error bars represent triplicate repeats. (C) Reducing cellular levels of DprA-GFP results specifically in loss of polar foci. Error bars represent triplicate repeats. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Timelapse move of DprA-GFP after competence induction by CSP addition. Images taken every 5 min. Left panel, fluorescence; right panel, phase contrast; middle panel, overlay. Finally, to further explore the temporal dynamics of these DprA-GFP foci, their distribution within competent cells was analyzed over the competence period and beyond. The results are presented in Figure 3A as focus density maps ordered by cell length. The number of cells with foci, as well as their intensity, was found to increase gradually to reach a maximum of 74% at 30 min after competence induction (Figure 3A), with the majority of cells possessing a single focus that persisted long after induction (Figure 3B). Notably, the DprA-GFP foci localization pattern rapidly evolved from a central position to a single cell pole (Figure 3C). DprA-GFP foci were not observed in a particular cell type, with polar foci found in small, large or constricted cells throughout competence (Figure 3C). Finally, tracking DprA-GFP foci formed in the cells after 10 min of competence induction by time-lapse microscopy showed that once generated, they remained static over 20 min (Figure 1 and Figure 1—figure supplement 1IJ, Video 1). In conclusion, DprA-GFP forms discrete and static polar foci during competence, with most cells possessing a single focus. This polar localization of DprA correlates with its regulatory role in competent shut-off. Figure 3 Download asset Open asset Analysis of the cellular localization of DprA-GFP. (A) DprA-GFP foci persist at the cell pole after competence shut-off. Data at different time points after CSP addition represented as in Figure 1E. 4 min, 8336 cells and 1383 foci analyzed; 6 min, 2871 cells and 1674 foci analyzed; 10 min, 2614 cells and 1502 foci analyzed; 15 min, 1290 cells and 1128 foci analyzed; 20 min, 2110 cells and 1900 foci analyzed; 30 min, 789 cells and 831 foci analyzed; 60 min, 842 cells and 839 foci analyzed. Sample microscopy images of strain R3728 at varying times after competence induction. Scale bars, 1 µm. (B) Most competent cells possess a single polar focus of DprA-GFP. Error bars represent triplicate repeats. (C) Most cells possess polar DprA-GFP foci. Along the length of a cell of arbitrary length 1, polar foci are found between positions 0–0.15 and 0.85–1, midcell foci are found between 0.35 and 0.65, and anything in between is localized as betwixt. Error bars represent triplicate repeats. The polar localization of DprA-GFP requires induction of the late com genes Transcriptional expression of dprA is only detected during competence (Aprianto et al., 2018; Dagkessamanskaia et al., 2004; Peterson et al., 2004). To explore whether a competence-specific factor was required for the formation of polar DprA-GFP foci during competence, dprA-gfp was ectopically expressed from a promoter inducible by the BIP peptide in dprA- cells. This BIP-derived induction mimics rapid, strong induction by CSP during competence (Johnston et al., 2016). These cells were found to produce stable DprA-GFP after exposure to BIP, and upon addition of CSP to the growth medium, to transform at wild-type levels and to partially shut-off competence (Figure 4—figure supplement 1). However, BIP-induced production of DprA-GFP in the absence of CSP resulted in the formation of weak, barely detectable polar foci in only 10% of non-competent cells (Figure 4A, B). In comparison, 47% of cells producing DprA-GFP during competence formed bright polar foci (Figure 4A, B). This stark increase in DprA-GFP foci showed that a competence-specific factor was crucial for their formation and anchoring at the cell pole. To explore whether the competence-specific factor needed for the polar localization of DprA-GFP in competent cells was part of the early or late com regulons, we generated two constructs allowing us to artificially control DprA-GFP expression and either one or the other of these two connected regulons (Figure 4C, D and Materials and methods). Observation of DprA-GFP in conditions where only late com genes were induced revealed the presence of polar foci at wildtype levels (Figure 4E, F). Conversely, no foci were observed when DprA-GFP was ectopically expressed with only the early com genes (Figure 4E), showing that late com regulon expression was required for the polar accumulation of DprA-GFP. Figure 4 with 1 supplement see all Download asset Open asset Polar accumulation of DprA-GFP appears to depend on late com regulon expression. (A) Sample fluorescence microscopy images of strain R4060 producing DprA-GFP 15 min after induction with BIP or BIP and CSP. Scale bars, 1 µm. (B) Competence induction is required for optimal accumulation of DprA-GFP at the cell poles. Focus density maps as in Figure 1E. BIP+, 7845 cells and 790 foci analyzed; BIP+ CSP+, 2707 cells and 1478 foci analyzed. (C) Genetic context strain R4107 expressing dprA-gfp and only the late com regulon. PBIP and PX as in panel A, PE represents early com promoter controlled by ComE. Light blue circle, ComW; light green oval, RNA polymerase; purple hexagon, σX. (D) Genetic context of strain R4140 expressing CEPR-dprA-gfp and only the early com regulon. PBIP as in panel A, PE as in panel D. (E) Sample fluorescence microscopy images of strains producing DprA-GFP with only late (R4107) or only early (R4140) com operons 15 min after competence induction. Scale bars, 2 µm. (F) Induction of the late com regulon is required for accumulation of DprA-GFP at the cell poles. Focus density maps as in Figure 1E. 1988 cells and 1824 foci analyzed. (G) Focus density maps produced as in Figure 1E from images where DprA-GFP was produced outside of competence in presence or absence of σX. DprA-GFP alone, 7845 cells and 790 foci analyzed; DprA-GPF + σX, 3545 cells and 1355 foci analyzed. Strains used: DprA-GFP alone, R4060; DprA-GFP and σX, R4489. The polar localization of DprA-GFP depends on the alternative sigma factor σX The late com regulon is comprised of 62 genes, organized in 18 operons (Claverys et al., 2006; Dagkessamanskaia et al., 2004; Peterson et al., 2004). To identify the hypothetical late com gene product needed for DprA localization at the cell pole, the cin boxes that define the late com promoters were individually mutated, generating a panel of 18 mutant strains, each lacking the ability to induce a specific late com operon. The inactivation was validated by comparing transformation efficiency in three strains, where cin box inactivation mirrored gene knockout levels (Supplementary file 1). Visualization of the red fluorescent fusion DprA-mKate2 showed that in all 18 mutants, DprA-mKate2 formed foci at levels and localization comparable to wildtype (Supplementary file 2). This result contrasted with our previous result (Figure 4E), which suggested that expression of the late com regulon was required for formation of polar DprA-GFP foci, causing us to revisit our interpretation of Figure 4C–F. In fact, to express only the early com regulon, we inactivated comX1, comX2, and comW (Figure 4C), so this strain produced only early com proteins, except σX and ComW, and lacked DprA-GFP foci (Figure 4E). Conversely, to express only the late com regulon, we ectopically expressed comX and comW (Figure 4D), so this strain produced the late com regulon but also the early com proteins σX and ComW and displayed polar DprA-GFP foci at wild-type levels (Figure 4E and F). This led us to consider that the only proteins whose presence correlated directly with the presence of polar DprA-GFP foci were thus σX and ComW. To first investigate whether ComW played a role in the formation of polar DprA-GFP foci, the comW gene was inactivated in a strain possessing a rpoDA171V mutation, enabling σX-RNA polymerase interaction and resulting in late com regulon expression in the absence of ComW (Tovpeko et al., 2016). DprA-GFP expressed from the native locus still formed polar foci in this strain at levels comparable to the wildtype strain (Figure 5—figure supplement 1A). ComW was thus not required for the formation of DprA-GFP foci. In light of this, the only remaining candidate whose presence in competent cells correlated directly with formation of polar DprA-GFP foci was σX. Thus, to determine if σX alone was necessary and sufficient to localize DprA-GFP to the cell poles, both comX and dprA-gfp or dprA-gfp alone were expressed in non-competent rpoDwt cells. Western blot analysis using α-SsbB antibodies indicated that the late com regulon was weakly induced when σX was ectopically produced in the absence of comW (Figure 5—figure supplement 1B). Cells producing DprA-GFP alone showed polar DprA-GFP foci in 10% of cells (Figure 4G). In contrast, DprA-GFP foci were formed in 37% of cells when σX was also produced in non-competent cells (Figure 4G). Importantly, induction of competence in both of these strains resulted in similar foci numbers (Figure 5—figure supplement 1D). Altogether, this result suggested that σX alone was sufficient to stimulate polar foci of DprA-GFP, highlighting an unexpected role for this early competence σ factor only known to act in concert with ComW to induce late competence gene transcription. σX mediates the localization of DprA at the cell pole of competent cells To investigate how σX could be involved in the polar localization of DprA-GFP, we explored how it localized in competent cells. To this end, we generated a comX1-gfp construct at the native comX1 locus combined with a wild-type comX2 gene. In the absence of comX2, the second gene producing σX, σX-GFP induced the late com regulon poorly and was thus weakly transformable (Figure 5—figure supplement 2). As a result, a strain possessing comX1-gfp and comX2 was used to determine the localization of σX -GFP. Remarkably, σX-GFP formed bright polar foci 15 min after competence induction (Figure 5A), reminiscent of those formed by DprA-GFP (Figure 1C). Immunofluorescence microscopy on wild-type cells using anti-σX antibodies confirmed the polar localization of σX in competent cells (Figure 5—figure supplement 2). A time-course experiment after competence induction showed that σX-GFP localized to the cell pole as soon as 4 min after CSP addition (Figure 5B), when DprA-GFP forms weak foci at midcell (Figure 3A). In contrast, a ComW-GFP fusion protein displayed a diffuse cytoplasmic localization in the majority of competent cells, with only 7% of cells possessing weak foci at the cell poles (Figure 5—figure supplement 2A and Supplementary results). Thus, σX-GFP localizes to the cell pole without its partner in transcriptional activation ComW, despite the fact that σX is an alternative sigma factor directing RNA polymerase to specific promoters on the chromosome. Figure 5 with 2 supplements see all Download asset Open asset σX-GFP interacts directly with DprA at the cell pole during competence. (A) Sample fluorescence microscopy images of strain R4451 producing σX-GFP from comX1 and wild-type σX from comX2 15 min after competence induction. Scale bars, 1 µm. (B) σX-GFP accumulates at the cell pole during competence. Focus density maps presented as in Figure 1E. 4 min, 7544 cells and 489 foci analyzed; 6 min, 5442 cells and 1711 foci analyzed; 10 min, 4358 cells and 2691 foci analyzed; 15 min, 3746 cells and 2144 foci analyzed; 20 min, 4211 cells and 1754 foci analyzed; 30 min, 4695 cells and 1920 foci analyzed; 60 min, 5713 cells and 1016 foci analyzed. (C) Most cells have a single σX-GFP focus. Data from the time-course experiment presented in panel B showing the number of foci per cell at each time point. Error bars represent triplicate repeats. (D) DprA-GFP foci persist in cells longer than σX-GFP foci. Comparison of cells with foci at different timepoints from timecourse experiments. DprA-GFP from Figure 3A, σX-GFP from panel B. Error bars represent triplicate repeats. (E) Accumulation of σX at the cell poles does not depend on DprA. Sample microscopy images of a comX1-gfp, dprA- strain (R4469). Focus density maps generated from cells visualized 15 min after competence induction presented as in Figure 1E. 1104 cells and 638 foci analyzed. (F) σX and DprA colocalize at the cell pole. Colocalization of σX-YFP and DprA-mTurquoise in R4473 cells visualized 15 min after competence induction. 7460 cells and 3504 DprA-mTurquoise foci analyzed. Scale bars, 1 µm. (G) DprA is copurified with σX-GFP while DprAAR is not. Western blot of pull-down experiment carried out on strains producing σX-GFP and either DprA (R4451) or DprAAR (R4514) 10 min after competence induction. WCE, whole cell extract; FT1, flow through; E, eluate. Analysis of σX-GFP foci distribution showed that they are detected in up to 51% of cells 10 min after competence induction, with most cells possessing a single focus (Figure 5B and C). The number of cells with foci decreased steadily after this point (Figure 5B, C, D), contrasting with polar DprA-GFP which remained stable over 60 min after induction. Importantly, σX-GFP continued to form polar foci in a strain lacking dprA (Figure 5E), showing that σX does not depend on DprA for its localization. Together, these results strongly supported the notion that σX promotes the targeting and assembly of DprA-GFP foci at the cell pole. To further explore this hypothesis, we co-expressed DprA-mTurquoise and σX-YFP fluorescent fusions in the same cells and found that 86% of DprA-mTurquoise foci colocalized with σX-YFP foci (Figure 5F). DprA and σX are thus present at the same pole of the cell at the same time and the polar accumulation of DprA molecules in competent cells depends on σX. These results suggested that σX could interact with DprA to anchor it to the pole of competent cells. The potential interaction between σX and DprA was tested in live competent pneumococcal cells in pull-down experiments. To achieve" @default.
• W3105441053 created "2020-11-23" @default.
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• W3105441053 date "2020-10-29" @default.
• W3105441053 modified "2023-09-30" @default.
• W3105441053 title "Author response: The alternative sigma factor σX mediates competence shut-off at the cell pole in Streptococcus pneumoniae" @default.
• W3105441053 doi "https://doi.org/10.7554/elife.62907.sa2" @default.
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