The pneumococcal σX activator, ComW, is a DNA-binding protein critical for natural transformation.

Natural genetic transformation via horizontal gene transfer enables rapid adaptation to dynamic environments and contributes to both antibiotic resistance and vaccine evasion among bacterial populations. In Streptococcus pneumoniae (pneumococcus), transformation occurs when cells enter competence, a transient state in which cells express the competence master regulator, SigX (σΧ), an alternative σ factor (σ), and a competence co-regulator, ComW. Together, ComW and σX facilitate expression of the genes required for DNA uptake and genetic recombination. SigX activity depends on ComW, as ΔcomW cells transcribe late genes and transform at levels 10- and 10,000-fold below that of WT cells, respectively. Previous findings suggest that ComW functions during assembly of the RNA polymerase-σX holoenzyme to help promote transcription from σX-targeted promoters. However, it remains unknown how ComW facilitates holoenzyme assembly. As ComW seems to be unique to Gram-positive cocci and has no sequence similarity with known transcriptional activators, here we used Rosetta to generate an ab initio model of pneumococcal ComW's 3D-structure. Using this model as a basis for further biochemical, biophysical, and genetic investigations into the molecular features important for its function, we report that ComW is a predicted globular protein and that it interacts with DNA, independently of DNA sequence. We also identified conserved motifs in ComW and show that key residues in these motifs contribute to DNA binding. Lastly, we provide evidence that ComW's DNA-binding activity is important for transformation in pneumococcus. Our findings begin to fill the gaps in understanding how ComW regulates σΧ activity during bacterial natural transformation.