Expanding the genetic toolbox for the obligate human pathogenStreptococcus pyogenes

Abstract Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes . In this study, we set out to develop a comprehensive set of plasmids, promoters and reporters for S. pyogenes . We present an expansion to the current genetic toolbox that comprises new replicative and site-specific integrative plasmids. Moreover, we established a collection of constitutive promoters with a wide variety of strengths as well as a set of novel inducible regulatory elements, including a zinc-inducible promoter, an erythromycin-inducible riboswitch and an IPTG-inducible promoter that outperform previously described inducible systems in terms of tightness and inducibility. In addition, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes . For this, we adapted a novel chemically defined medium called RPMI4Spy. This medium showed a highly reduced autofluorescence compared to other growth media and allowed efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS *, which improved the generation of scarless gene deletions. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.