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

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 transformation 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 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.

24 Abstract 25 Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial 26 pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-27 infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal 28 glomerulonephritis. Genetic manipulation of this important pathogen is complicated by 29 resistance of the organism to genetic transformation. Very low transformation efficiency is 30 attributed to recognition and degradation of introduced foreign DNA by a type I restriction-31 modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten 32 GAS strains that had been previously transformed with an unrelated plasmid revealed that six of 33 the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, 34 and at least five of the six were predicted to result in loss of function of the respective hsd gene 35 product. The unexpected occurrence of such mutations in previously transformed isolates 36 suggested that the process of transformation selects for spontaneous inactivating mutations in 37 the Hsd system. We investigated the possibility of exploiting the increased transformability of 38 hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate 39 representative of the globally dominant M1T1 clonal group. Mutant strain 854hsdM exhibited a 40 5-fold increase in transformation efficiency compared to the wild type parent strain and no 41 obvious change in growth or off-target gene expression. We conclude that genetic 42 transformation of GAS selects for spontaneous mutants the hsdRSM restriction modification 43 system. We propose that use of a defined hsdM mutant as a parent strain for genetic 44 manipulation of GAS will enhance transformation efficiency and reduce the likelihood of 45 selecting spontaneous hsd mutants with uncharacterized genotypes. 46 47 48 Introduction 49 50 As part of our research into molecular pathogenesis of infections due to group A Streptococcus 51 (S. pyogenes or GAS), our laboratory routinely constructs mutant strains of GAS using allelic 52 exchange mutagenesis by transformation of a wild type strain with a temperature-sensitive 53 plasmid carrying the desired mutation. During characterization of one such mutant strain, we 54 made the incidental discovery that the mutant harbored a 694 bp deletion in hsdM, which 55 encodes a component of a type I restriction modification system. The deletion resulted in a 56 frameshift mutation at codon 123, altering the remainder of the protein sequence and 57 introducing a premature stop codon at amino acid 170. As discussed below, because 58 inactivation of this system can be associated with enhanced susceptibility to transformation with 59 foreign DNA, three questions arose concerning inactivating mutations in hsdM or in genes 60 encoding other components of the restriction modification system: (1) Does inactivation of hsdM 61 result in increased transformation efficiency? (2) Does the process of transformation select for 62 spontaneous inactivating mutations in the restriction modification system? (3) Could deliberate 63 disruption of the system be exploited to facilitate introduction of foreign DNA for genetic 64 manipulation of GAS? The current investigation was undertaken to answer these questions.

65
66 Type I restriction modification systems are comprised of three subunits that work together to 67 recognize and cleave intracellular foreign DNA at specific sites and also to protect host cell DNA 68 from cleavage by methylating it at the same recognition sites. Together, the three protein 69 subunits comprise the "host specificity of DNA" system, or Hsd. The three enzymes of the Hsd 70 system include the specificity subunit, HsdS, the restriction subunit, HsdR, and the modification 71 subunit, HsdM. HsdS requires interaction with HsdM in order to bind to DNA. Once bound, the 72 HsdS/HsdM complex can interact with HsdR. HsdR can then exert endonuclease activity at 4 74 sequence. These functions of the three enzymes are tightly linked, as interaction with HsdM is 75 necessary both for HsdS to bind to DNA and for HsdR endonuclease activity to occur [1].
76 Together, the three-protein complex is able to cleave foreign DNA after it enters the cell as a 77 type of bacterial defense system. 78 79 The Hsd system in GAS is a typical type I restriction modification system. It efficiently targets 80 and cleaves foreign DNA that may enter the cell either naturally or artificially through 81 electroporation. Accordingly, GAS is notoriously difficult to manipulate genetically in the 82 laboratory setting. Previous studies have reported that inactivation of the Hsd system in GAS is 83 associated with increased transformation efficiency [2,3]. One study done in an M28 strain 84 found that deletion of the three components of the Hsd system was associated with increased 85 transformation efficiency [2]. Another study characterized a group of emm1 clinical isolates 86 associated with invasive infections. These strains were found to harbor a spontaneous deletion 87 that included part of hsdR, the entire hsdS and hsdM gene sequences, and a portion of an 88 adjacent two-component system. Strains with the deletion were shown to have increased 89 transformation efficiency compared to contemporaneous emm1 isolates without the deletion [3].

91
In this study, we introduced a large deletion in hsdM in a GAS strain representative of the M1T1 92 clonal group widely implicated in invasive GAS disease. We found that inactivation of hsdM 93 resulted in increased transformation efficiency of the strain, and that the mutation was not 94 associated with a significant change in transcript abundance for other GAS genes including 95 those encoding key virulence factors. In addition, we observed that the process of 96 transformation selects for strains that have acquired spontaneous mutations in the Hsd system.

125 Transformation of wild type GAS selects for mutants in hsdRSM.
126 Because inactivation of HsdM resulted in a higher transformation rate, we wondered whether 127 the process of transformation of wild type GAS might select for bacterial cells that had acquired 128 a spontaneous inactivating mutation in the Hsd system. We screened for such mutations by 129 sequence analysis of the hsdM locus of ten independently derived mutant strains of GAS 854 130 that had been previously transformed during allelic exchange mutagenesis of various genes.
131 We found that four of the ten had acquired spontaneous mutations in the hsdM gene. These 132 mutations included a deletion encompassing the entire hsdM coding sequence, a 1.5kb 133 insertion, a missense point mutation, and a nonsense mutation (Table 1). Sequence analysis of 134 the entire hsdRSM locus revealed that two other strains among these ten had mutations in 135 either hsdR or hsdS (Table 1). Thus, including the mutations in hsdM, we found spontaneous 136 mutations in the hsdRSM locus in six of the ten strains that had been previously transformed.

158
159 Since each of the spontaneous mutations we observed in transformants of strain 854 is 160 different, we infer that the various mutations in the hsdRSM locus arose as independent events, 161 rather than a single ancestral mutation in the parental wild type strain. Taken together, these 162 observations strongly suggest that GAS isolates that have been successfully transformed in the 163 laboratory setting may have a mutation that inactivates the Hsd system, and that the process of 164 transformation selects for such mutants.          Table S1.
260 Triplicate assays were performed for each gene tested with 1 ng total template. Expression 261 levels of target genes were normalized to recA (M5005_Spy_1799), and then further normalized 262 to the wild type strain expression levels to obtain a final Ct value. These values were used to 263 calculate relative expression changes for the mutant strain compared to the parental wild type 264 strain. Data were reported as mean log 2 fold-change.