Genetic modification of by natural transformation.

Unlabelled: is an emerging human and animal pathogen. Functional studies of genes involved in virulence of and other pyogenic group streptococci are often hampered by limited genetic tractability. It is known that pyogenic streptococci carry genes required for competence for natural transformation; however, in contrast to other streptococcal subgroups, there is limited evidence for gene transfer by natural transformation in these bacteria. In this study, we systematically assessed the genomes of 179 . strains of both human and animal origins (subsp. and respectively) for the presence of genes required for natural transformation. While a considerable fraction of the strains contained inactive genes, the majority (64.2%) of the strains had an intact gene set. In selected strains, we examined the dynamics of competence activation after addition of competence-inducing pheromones using transcriptional reporter assays and exploratory RNA-seq. Based on these findings, we were able to establish a protocol allowing us to utilize natural transformation to construct deletion mutants by allelic exchange in several strains of both subspecies. As part of the work, we deleted putative lactose utilization genes to study their role in growth on lactose. The data presented here provide new knowledge on the potential of horizonal gene transfer by natural transformation in and, importantly, demonstrates the possibility to exploit natural transformation for genetic engineering in these bacteria.

Importance: Numerous spp. exchange genes horizontally through natural transformation, which also facilitates efficient genetic engineering in these organisms. However, for the pyogenic group of streptococci, including the emerging pathogen , there is limited experimental evidence for natural transformation. In this study, we demonstrate that natural transformation indeed is possible in strains under optimal conditions. We utilized this method to perform gene deletion through allelic exchange in several strains, thereby paving the way for more efficient gene engineering methods in pyogenic streptococci.