Methylome-dependent transformation ofemm1 group A streptococci

Abstract

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group AStreptococcus(GAS), a lethal human pathogen currently associated with an unprecedented surge of infections world-wide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this “restriction barrier” thus presents a major technical challenge. Here we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Further, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRDAG, encoded by all sequenced strains of the dominant and upsurge-associatedemm1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol whereby the restriction barrier is overcome by addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRDAGstrains including clinical isolates representing allemm1 lineages, and will expedite critical research interrogating the genetics ofemm1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation, and the importance of defining lineage-specific mechanisms of genetic recalcitrance.