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Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system.

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.

SUBMITTER: Finn MB

PROVIDER: S-EPMC8084154 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Although prokaryotic DNA methylation investigations have long focused on immunity against exogenous DNA, it has been recently recognized that DNA methylation impacts gene expression and phase variation in Streptococcus pneumoniae and Streptococcus suis. A comprehensive analysis of DNA methylation is lacking for beta-hemolytic streptococci, and thus we sought to examine DNA methylation in the major human pathogen group A Streptococcus (GAS). Using a database of 224 GAS genomes encompassing 80 emm types, we found that nearly all GAS strains encode a type I restriction modification (RM) system that lacks the hsdS' alleles responsible for impacting gene expression in S. pneumoniae and S. suis. The GAS type I system is located on the core chromosome, while sporadically present type II orphan methyltransferases were identified on prophages. By combining single-molecule real-time (SMRT) analyses of 10 distinct emm types along with phylogenomics of 224 strains, we were able to assign 13 methylation patterns to the GAS population. Inactivation of the type I RM system, occurring either naturally through phage insertion or through laboratory-induced gene deletion, abrogated DNA methylation detectable via either SMRT or MinION sequencing. Contrary to a previous report, inactivation of the type I system did not impact transcript levels of the gene (mga) encoding the key multigene activator protein (Mga) or Mga-regulated genes. Inactivation of the type I system significantly increased plasmid transformation rates. These data delineate the breadth of the core chromosomal type I RM system in the GAS population and clarify its role in immunity rather than impacting Mga regulon expression. IMPORTANCE The advent of whole-genome approaches capable of detecting DNA methylation has markedly expanded appreciation of the diverse roles of epigenetic modification in prokaryotic physiology. For example, recent studies have suggested that DNA methylation impacts gene expression in some streptococci. The data described herein are from the first systematic analysis of DNA methylation in a beta-hemolytic streptococcus and one of the few analyses to comprehensively characterize DNA methylation across hundreds of strains of the same bacterial species. We clarify that DNA methylation in group A Streptococcus (GAS) is primarily due to a type I restriction modification (RM) system present in the core genome and does not impact mga-regulated virulence gene expression, but does impact immunity against exogenous DNA. The identification of the DNA motifs recognized by each type I RM system may assist with optimizing methods for GAS genetic manipulation and help us understand how bacterial pathogens acquire exogenous DNA elements.

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Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system.

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