New PALM-compatible integration vectors for use in the Gram-positive model bacterium <i>Bacillus subtilis</i>

Super-resolution microscopy techniques allow localization of proteins and cellular components in prokaryotic and eukaryotic cells with unprecedented spatial resolution. Plasmids remain a powerful approach to clone fluorescent protein fusions in bacterial cells. In the current work, we expanded the toolbox of vectors available to engineer the Gram-positive model organism <i>Bacillus subtilis</i> for PALM studies. Four integrative vectors in total, two carrying the gene encoding the photoswitchable green fluorescent protein dronPA and two carrying the gene encoding the photoactivatable red fluorescent protein PAmCherry1, were constructed and tested by generating translational fusions to the LacI repressor. The LacI fluorescent fusions successfully reported the subcellular localization of viral DNA in infected <i>B. subtilis</i> cells, either live or upon fixation, by PALM. Our dronPA and PAmCherry1 integration vectors expand the genetic toolbox for single-molecule localization microscopy studies in <i>B. subtilis</i>.