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

Improvements in super-resolution and single-molecule techniques, along with the development of new fluorescent proteins and labeling methods, have allowed super-resolution imaging of bacterial cells. Cloning vectors are important tools for engineering fluorescent fusions and perform efficient labeling. Here, we report the construction of four photoactivated localization microscopy (PALM)-compatible integration plasmids for the Gram-positive model organism Bacillus subtilis. These plasmids carry genes encoding either the photoswitchable green fluorescent protein dronPA or the photoactivatable red fluorescent protein PAmCherry1, codon-optimized or not for expression in B. subtilis. For fast and flexible cloning, multiple cloning sites were added at both the C-terminal and the N-terminal ends of the fluorescent protein genes. 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 single copy number dronPA and PAmCherry1 fusions, respectively. Two-color imaging is accessible with the simultaneous use of both vectors. Insertion of the LacI repressor gene under control of a constitutive promoter in each plasmid yielded four derivative vectors that, combined with an array of lacO operator sites, allow fluorescent repressor-operator system localization studies. We demonstrated the effective photoactivation of the LacI-dronPA and LacI-PAmCherry1 fusions, and used them to report with nanoscale precision bacteriophage SPP1 DNA within infected B. subtilis cells, both live and fixed, as proof of concept. Our integration vectors provide a convenient and versatile workflow for qualitative and quantitative, single- and dual-color PALM studies in B. subtilis.

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 Bacillus subtilis 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 B. subtilis 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 B. subtilis.