Efficient and rapid one-step method to generate gene deletions in <i>Streptococcus pyogenes</i>

Group A Streptococcus (GAS) is a major human pathogen, causing diseases ranging from mild and superficial infections of the skin and pharyngeal epithelium to severe systemic and invasive diseases. Since June 2022, several European countries, the US, and Australia are facing an upsurge of invasive life-threatening GAS infections. Finding a good vaccine antigen and understanding the role of virulence factors in GAS infections have been hampered, in part, by technical difficulties to transform the many different GAS strains and generate knockout mutants. Moreover, these tools must be adapted to a large range of different strains, since GAS are divided into more than 260 emm-types (M-type). We have set up a method allowing the generation of non-polar mutants of GAS in 3 days and in diverse backgrounds, which contrasts with previously published protocols.