S. aureus mutants.

Allelic recombination with the plasmid pKOR1 was used to delete the spa gene of S. aureus WU1 (87). To construct the Δspa mutant, two 1-kb DNA fragments upstream and downstream of the spa gene were amplified from the chromosome of S. aureus WU1 with primers ext1F (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCATTTAAGAAGATTGTTTCAGATTTATG-3′), ext1R (5′-ATTTGTAAAGTCATCATAATATAACGAATTATGTATTGCAATACTAAAATC-3′), ext2F (5′-CGTCGCGAACTATAATAAAAACAAACAATACACAACGATAGATATC-3′), and ext2R (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCAACGAACGCCTAAAGAAATTGTCTTTGC-3′). The two flanking regions were fused in a subsequent PCR, and the final PCR product was cloned into pKOR1 using the BP Clonase II kit (Invitrogen). The resulting plasmids were consecutively transferred into E. coli DH5α, S. aureus strain RN4220, and finally S. aureus strain WU1 and temperature shifted to 40°C, blocking replication of the plasmids and promoting their insertion into the chromosome (87). Growth at 30°C was used to promote allelic replacement. Mutations in the spa genes were verified by DNA sequencing of the PCR amplification products.