A modular genetic toolbox for precise gene regulation and multi-color imaging in streptococci

Fluorescent labeling is a powerful tool in microbiology allowing live cell imaging and providing insights into dynamic cellular processes, quantification of gene expression, and protein subcellular localization. Although multicolor imaging is widely used in <i>Streptococcus pneumoniae</i> and <i>S. mutans</i>, this is less common in other streptococcal species. To address this gap in the streptococcal molecular toolbox, we benchmarked five different fluorescent proteins. They were fused to the C-terminus of <i>S. pneumoniae</i> HlpA, a small non-specific DNA binding histone-like protein. These reporters, combined with four different antibiotic resistance genes, were engineered with various expression systems (inducible or constitutive) to form versatile cassettes. We provide methods to transfer these cassettes to different streptococcal species including <i>S. salivarius, S. thermophilus</i>, and <i>S. pyogenes</i>. As a proof of concept, we generated a triple labeled <i>S. salivarius</i> strain in which HlpA, FtsZ, and DivIVA were fused to three spectrally distinct compatible fluorescent proteins. Multiple fluorescent labeling has broad applications for deciphering a wide range of scientific problems, from cellular processes to infectious disease mechanisms. The availability of these cassettes should allow for a wider use of single-cell labeling strategies in the streptococcus clade and other closely related bacteria.