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Although multicolor imaging is widely used in Streptococcus pneumoniae and S.\u00a0mutans, 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 S.\u00a0pneumoniae 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 S.\u00a0salivarius, S.\u00a0thermophilus, and S.\u00a0pyogenes. As a proof of concept, we generated a triple labeled S. salivarius 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.<\/jats:p>","DOI":"10.1093\/femsml\/uqag006","type":"journal-article","created":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T12:47:55Z","timestamp":1772023675000},"source":"Crossref","is-referenced-by-count":0,"title":["A modular genetic toolbox for precise gene regulation and multi-color imaging in streptococci"],"prefix":"10.1093","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3721-4307","authenticated-orcid":false,"given":"Johann","family":"Mignolet","sequence":"first","affiliation":[{"name":"Faculty of Biology and Medicine, University of Lausanne Department of Fundamental Microbiology, , CH-1015 Lausanne ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Albane","family":"Schmid","sequence":"additional","affiliation":[{"name":"Faculty of Biology and Medicine, 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