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

Improvements in super-resolution and single-molecule techniques together with the development of new fluorescent proteins and labelling methods have allowed super-resolution microscopy to be applied to bacterial cells. Cloning vectors remain important tools for researchers to perform efficient labelling. Here, we describe the creation of four PhotoActivated Localization Microscopy (PALM)-compatible integration plasmids for the Gram-positive model organism Bacillus subtilis. These plasmids carry either the photoswitchable green fluorescent protein dronPA or the photoactivatable red fluorescent protein PAmCherry1, codon-optimized or not for B. subtilis. For fast and interchangeable cloning, we have inserted multi cloning sites at both the C-terminal and the N-terminal end of the fluorophores. The plasmids replicate in Escherichia coli and allow integration at the ectopic amyE or thrC loci of B. subtilis via double homologous recombination, for stable chromosomal insertions of dronPA and PAmCherry1 protein fusions respectively. Dual color imaging is accessible with the simultaneous use of the two vectors. Insertion of the gene encoding the LacI repressor under control of a constitutive promoter in each of the four plasmids yielded four derivative vectors that, combined with an array of lacO operator sites, allow Fluorescent Repressor-Operator System (FROS) localization studies. We demonstrated the successful photoactivation of the LacI-dronPA or PAmCherry1 fusions, and used them to report with nanoscale precision the subcellular localization of bacteriophage SPP1 DNA in infected B. subtilis cells as proof of concept. Our PALM-compatible integration vectors expand the genetic toolbox for single molecule localization microscopy studies in B. subtilis.