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Abstract
Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.
Highlights
As part of our research into molecular pathogenesis of infections due to group A Streptococcus (S. pyogenes or GAS), our laboratory routinely constructs mutant strains of GAS using allelic exchange mutagenesis by transformation of a wild type strain with a temperature-sensitive plasmid carrying the desired mutation
Since restriction modification systems target foreign DNA for destruction by the host cell, we hypothesized that inactivation of the Hsd system by deletion of hsdM would result in a strain with increased transformation efficiency
We introduced a 1,005 bp inframe deletion into the hsdM coding sequence of GAS strain 854, an emm1 isolate representative of the globally prevalent M1T1 clonal group that is dominant among GAS emm1 strains associated with invasive infections [4]
Summary
As part of our research into molecular pathogenesis of infections due to group A Streptococcus (S. pyogenes or GAS), our laboratory routinely constructs mutant strains of GAS using allelic exchange mutagenesis by transformation of a wild type strain with a temperature-sensitive plasmid carrying the desired mutation. Type I restriction modification systems are comprised of three subunits that work together to recognize and cleave intracellular foreign DNA at specific sites and to protect host cell DNA from cleavage by methylating it at the same recognition sites. HsdR can exert endonuclease activity at specific recognition sites, activity which is dependent on the methylation state of the target sequence. These functions of the three enzymes are tightly linked, as interaction with HsdM is necessary both for HsdS to bind to DNA and for HsdR endonuclease activity to occur [1]. The three-protein complex is able to cleave foreign DNA after it enters the cell as a type of bacterial defense system
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