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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Mol Microbiol. 2016 Mar 14;100(4):675–685. doi: 10.1111/mmi.13340

Fig. 1.

Fig. 1

(A) S. aureus N315, MR14, MR14EX. S. aureus strain N315 contains a 53 kb SCCmec type II element. The gene mecA encodes methicillin resistance; mecR1 and mecI encode mecA regulators; and ccrAB encode recombinases for SCCmec excision and integration. N315 was modified to construct MR14 for positive selection for excision. A promoterless tetracycline resistance gene, tetM, was inserted immediately downstream of attL, and Tn554 and 15.4 kb of DNA upstream of tetM were deleted. MR14EX is the excised tetracycline-resistant derivative of MR14 with tetM expression driven from the orfX promoter.

(B) S. aureus MR14 spontaneous excision. Serial dilutions of an overnight MR14 culture were plated on TSA and TSA with tetracycline (TET). The frequency of spontaneous excision was the excised population (TET) divided by the total population (TSA). Bars indicate the standard deviation of three independent experiments.

(C) S. aureus MR14 pWA46 inducible excision monitored by positive selection on tetracycline in the presence or absence of inducer (5μM cadmium chloride). Excision percentages were determined by plating overnight cultures on TSA and TSA with tetracycline and comparing growth on tetracycline to total growth without antibiotic selection. Bars indicate the standard deviation of three independent experiments.

(D) Diagram of the PCR approach used to determine integrated, excised, and circular SCCmec. Primer sets 1 and 2 amplify attR and attL integrated junctions, respectively. Primer set 3 amplifies excised chromosomal junction (attB). Primer set 4 amplifies excised circular SCCmec junction (attS).

(E) PCR analysis of strain MR14. Lane A, 1 kb molecular size marker; lanes 1 and 2, integrated attR and attL amplicons, respectively; lane 3, excised chromosomal junction (attB); lane 4, circular SCCmec junction (attS).

(F) PCR analysis of strain MR14EX. Same lanes as in Fig. 1E. Lane 3 shows only the excised chromosomal junction (attB) is present. Lane 4 shows the circular SCCmec intermediate (attS), following excision, has been lost.