Inactivation of the SauI type I restriction-modification system is not sufficient to generate Staphylococcus aureus strains capable of efficiently accepting foreign DNA

Abstract

Genetic manipulation of Staphylococcus aureus is limited by the availability of only a single strain, RN4220, that is capable of easily accepting foreign DNA. Inactivation of the hsdR gene of the SauI type I restriction-modification system was shown previously to be responsible for the high transformation efficiency of RN4220 (D. E. Waldron and J. A. Lindsay, J Bacteriol. 188:5578-5585, 2006). However, deletion of this gene in three different S. aureus strains was not sufficient to make them readily transformable, which would be remarkably useful for genetic studies of this pathogenic organism. These results indicate that another unknown factor(s) is required for the transformable phenotype in S. aureus.

FIG. 1.

Genetic characterization of hsdR mutants. The strains used were RN4220 (lane 1), NCTC8325-4 (lane 2), NCTC8325-4ΔHsdR (lane 3), NCTC8325-4ΔC-term HsdR (lane 4), SH1000 (lane 5), SH1000ΔHsdR (lane 6), COL (lane 7), and COLΔHsdR (lane 8). (A) Localization of primers used to construct hsdR mutants (panel I) (see Materials and Methods for details); of primers used to characterize the hsdR mutants by PCR for wild-type strains RN4220, NCTC8325-4, SH1000, and COL (panel II), for hsdR null mutants NCTC8325-4ΔHsdR, SH1000ΔHsdR, and COLΔHsdR (panel III), and for strain NCTC8325-4ΔC-term HsdR, which contains a truncated version of the hsdR gene (panel IV); and of primers HsdRP7 and HsdRP8 used to amplify the hsdR probe used for Southern blotting (panel II). (B) PCR fragments obtained by amplifying the DNA of the eight strains indicated above using primers HsdRP5 and HsdRP6, which flank the hsdR gene. (C) Results of Southern blot hybridization of SmaI-restricted genomic DNA of the eight strains indicated above with an internal probe for the hsdR gene amplified using primers HsdRP7 and HsdRP8, showing that this gene is absent from the hsdR mutant strains. Note that the probe hybridized with the DNA fragment which was deleted in strain NCTC8325-4ΔC-term HsdR.