Fine‐tuning cellular levels of DprA ensures transformant fitness in the human pathogen Streptococcus pneumoniae

Abstract

Natural genetic transformation is a widespread mechanism of horizontal gene transfer. It involves the internalization of exogenous DNA as single strands and chromosomal integration via homologous recombination, promoting acquisition of new genetic traits. Transformation occurs during a distinct physiological state called competence. In Streptococcus pneumoniae, competence is controlled by ComDE, a two-component system induced by an exported peptide pheromone. DprA is universal among transformable species, strongly induced during pneumococcal competence, and crucial for pneumococcal transformation. Pneumococcal DprA plays three crucial roles in transformation and competence. Firstly, DprA protects internalized DNA from degradation. Secondly, DprA loads the homologous recombinase RecA onto transforming DNA to promote transformation. Finally, DprA interacts with the response regulator ComE to shut-off competence. Here, we explored the effect of altering the cellular levels of DprA on these three roles. High cellular levels of DprA were not required for the primary role of DprA as a transformation-dedicated recombinase loader or for protection of transforming DNA. In contrast, full expression of dprA was required for optimal competence shut-off and transformant fitness. High cellular levels of DprA thus ensure the fitness of pneumococcal transformants by mediating competence shut-off. This promotes survival and propagation of transformants, maximizing pneumococcal adaptive potential.

The alternative sigma factor σXmediates competence shut-off at the cell pole inStreptococcus pneumoniae

“…Here, we reproduced these experiments with the DprA-GFP fusion, to test whether 162 its concentration correlates with the formation of polar foci in competent cells. The expression, 163 transformation and competence profiles of a dprAmutant strain harbouring the ectopic CEPlac-dprA-164 gfp construct in varying concentrations of IPTG ( Figure S2ABC) were equivalent to those reported 165 previously for CEPlac-dprA (Johnston et al, 2018). Notably, a steady decrease in DprA-GFP foci was 166 observed as IPTG was reduced ( Figure 2AB).…”

“…DprA and neosynthesized ComE~P to promote efficient shut-off ( Figure 7H). Importantly, we 368 previously reported that DprA-mediated competence shut-off is crucial to the fitness of competent 369 cells (Johnston et al, 2018;Mirouze et al, 2013), and we revealed here that this vital role of DprA in actively limiting the competence window is orchestrated at the cell pole, which defines a coordination 371 hub of competence regulation. Unexpectedly, DprA is targeted to this hub by σ X , describing an 372 unprecedented role for an alternative sigma factor.…”

“…Inactivation of either comX gene not only slightly reduced the peak of late com gene expression 325 but also markedly delayed the rate of competence shut-off ( Figure 7B), revealing that reducing the 326 cellular level of σ X impacts competence shut-off efficiency, presumably because less σ X is present to 327 promote accumulation of DprA at the cell poles. In addition, this unregulated competence shut-off of 328 single comX mutants is accompanied by a reduced growth rate of the cell population, consistent with 329 an alteration of cell fitness linked to altered competence shut-off (Johnston et al, 2018). In conclusion, 330 this finding suggests that pneumococci possess two copies of comX to optimize DprA-mediated 331 competence shut-off and maintain the fitness of competent cells.…”

“…We thus propose that 420 two copies of comX are maintained within the genome to optimize the shut-off of competence. Since 421 unregulated competence is toxic for the cell (Johnston et al, 2018), two copies of comX provide a fail-422 safe in case of loss or inactivation of one comX gene by transformation or spontaneous mutation, 423 allowing the cell to nonetheless exit the competent state. In light of the second role of σ X uncovered 424 here, two copies of comX provide a simple yet elegant means for S. pneumoniae and close relatives to 425 ensure they are always equipped to survive competence, allowing cells to reap the potential benefits 426 of competence without the fitness cost associated with unregulated competence.…”

“…Altogether, these results suggested that the 154 polar foci of DprA-GFP were not related to the DNA entry or recombination steps of transformation 155 but could be linked to competence shut-off. 156 We recently reported that optimal competence shut-off relies on the maximal cellular 157 concentration of DprA (~8000 molecules), but this level could be reduced by 10 fold while still 158 maintaining wildtype frequency of transformation (Johnston et al, 2018). This conclusion was 159 obtained by expressing dprA under the control of the IPTG-inducible Plac promoter (CEPlac-dprA), which 160 enables the modulation of the cellular concentration of DprA by varying IPTG concentration in the 161 growth medium.…”