Genetic modification of Streptococcus dysgalactiae by natural transformation

Abstract

ABSTRACT Streptococcus dysgalactiae is an emerging human and animal pathogen. Functional studies of genes involved in virulence of S. dysgalactiae 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 S. dysgalactiae strains of both human and animal origins (subsp. equisimilis and dysgalactiae, 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 S. dysgalactiae 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 S. dysgalactiae and, importantly, demonstrates the possibility to exploit natural transformation for genetic engineering in these bacteria.IMPORTANCENumerous Streptococcus 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 Streptococcus dysgalactiae, there is limited experimental evidence for natural transformation. In this study, we demonstrate that natural transformation in vitro indeed is possible in S. dysgalactiae 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.

Keywords

• natural transformation
• pyogenic streptococci
• genetic tools
• genetic competence