Sau1: a novel lineage-specific type I restriction-modification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages

Abstract

The Sau1 type I restriction-modification system is found on the chromosome of all nine sequenced strains of Staphylococcus aureus and includes a single hsdR (restriction) gene and two copies of hsdM (modification) and hsdS (sequence specificity) genes. The strain S. aureus RN4220 is a vital intermediate for laboratory S. aureus manipulation, as it can accept plasmid DNA from Escherichia coli. We show that it carries a mutation in the sau1hsdR gene and that complementation restored a nontransformable phenotype. Sau1 was also responsible for reduced conjugative transfer from enterococci, a model of vancomycin resistance transfer. This may explain why only four vancomycin-resistant S. aureus strains have been identified despite substantial selective pressure in the clinical setting. Using a multistrain S. aureus microarray, we show that the two copies of sequence specificity genes (sau1hsdS1 and sau1hsdS2) vary substantially between isolates and that the variation corresponds to the 10 dominant S. aureus lineages. Thus, RN4220 complemented with sau1hsdR was resistant to bacteriophage lysis but only if the phage was grown on S. aureus of a different lineage. Similarly, it could be transduced with DNA from its own lineage but not with the phage grown on different S. aureus lineages. Therefore, we propose that Sau1 is the major mechanism for blocking transfer of resistance genes and other mobile genetic elements into S. aureus isolates from other species, as well as for controlling the spread of resistance genes between isolates of different S. aureus lineages. Blocking Sau1 should also allow genetic manipulation of clinical strains of S. aureus.

FIG. 1.

Horizontal gene transfer assays with sau1hsdR-positive and sau1hsdR mutant strains. 8325-4 carries sau1hsdR, and RN4220 carries the sau1hsdR mutation. RN4220 pSK5632 carries the control plasmid, and RN4220 phsdR carries an intact copy of sau1hsdR. All experiments were performed in triplicate, and data are presented as means and standard deviations. There was a significant difference between sau1hsdR and sau1hsdR mutant strains for all three assays (P < 0.001). (A) Electroporation. Data are presented as the number of transformants per 140 ng of pMAD plasmid DNA selected using erythromycin. (B) Conjugation. Data are presented as the number of transconjugants carrying Tn918 per 1 × 10⁸ E. faecalis donors selected using tetracycline. (C) Transduction. Data are presented as the number of transductants carrying pT181 per 1 × 10¹¹ donor 80α bacteriophage selected using tetracycline.

FIG. 2.

Illustration of bacteriophage susceptibility experiments. Bacteriophage φ75 was grown on S. aureus 879R4RF, where we assume its DNA was modified by an R-M system. This phage could infect RN4220 pSK5632 but could not infect RN4220 phsdR as the intact restriction enzyme digested the “foreign” DNA. In the process of lysing RN4220 pSK5632, φ75 replicated and its phage genome was modified by the RN4220 R-M system. The progeny phage is now capable of infecting RN4220 phsdR because the phage DNA is modified appropriately and not recognized as “foreign.”

FIG. 3.

Variation in hsdS genes. Artemis Comparison Tool comparison of hsdS1 nucleotide sequences of (in descending order) MRSA252, 8325, N315, and MW2. The major regions marked are according to those described by Kim et al. (14) and are TRD1 and TRD2, PrCR (labeled PCR), CCR, and DCR.

FIG. 4.

Distribution of sau1hsdS TRD variant regions in 161 community isolates of S. aureus by using microarrays. Each vertical line represents an isolate of S. aureus and has been clustered into the dominant lineages (marked on the bottom row) by using core variable genes (18). The branches are colored according to the multilocus sequence typing clonal complex, and the two methods show high homology. The horizontal rows correspond to six different TRD-specific PCR product spots on the microarray. In descending order, they are N315sau1hsdS2TRD1, MW2sau1hsdS1TRD1, MW2sau1hsdS1TRD2, MRSA252sau1hsdS2TRD2, MRSA252sau1hsdS1TRD1, and MRSA252sau1hsdS1TRD2. The signal generated on the microarray is a ratio of test strain fluorescence (Cy3) to reference strain fluorescence (Cy5), with the reference strain being MRSA252. A yellow square represents a positive signal in both the test and reference strains. Blue is positive in the reference only. Red is positive in the test only. Negative signals are brown/green or gray. The data show that there is variation in carriage of sau1hsdS TRD regions between isolates, and this variation correlates with lineage.