FIG. 1.

Genomic structure of mec DNA. (a) Cloning strategy of mec DNA. The mec DNA is shown in light yellow; the chromosomal region found in both the pre-MRSA strain N315 and the MSSA strain NCTC8325 is shown in green. All the hybridization probes are shown in orange. DNA fragments SJ1 and SJ2 were cloned from a N315 plasmid library by colony hybridization by using probe 1 prepared from plasmid pES1 (14). DNA fragments SJ4, SJ5, SJ7, and SJ8 were sequentially cloned by using probes 2, 3, and 4, respectively. All the fragments were cloned from a plasmid library of Sau3AI-partial digests except for SJ8, which was cloned from an N315 library of HindIII digests. LD8325 was cloned from a phage library of NCTC8325 by plaque hybridization by using the probe 11A. LN4 was cloned from a phage library of N315 by plaque hybridization by using cR as a probe. The region downstream of mecA was identified and sequenced by using the DNA fragment directly amplified by long PCR with mA3 (5′-ACAACGTTACAAGATATGAAGTGGTAAATGGTA-3′) and cR2 (5′-AAACGACATGAAAATCACCAT-3′) primers and the N315 DNA as a template. Only relevant restriction enzyme sites are indicated (H, HindIII; E, EcoRI; P, PstI), and those in overlapped DNA fragments are omitted. The nucleotide sequence accession numbers of the N315 chromosome containing the entire mec DNA (56,939 bp) starting at the first nucleotide of the leftmost HindIII site and of LE1a, the EcoRI-PstI subfragment of LD8325, are given in the Materials and Methods section. (b) Boundary of mec DNA. Nucleotide sequences around the left and right boundaries of mec DNA, attL and attR, respectively, are aligned with the sequence around the presumptive integration site, attB, on NCTC8325 chromosome. The four possible integration sites are located in the attB at the 3′ end of orfX (indicated by red arrowheads). The stop codons for orfX in NCTC8325 and modified orfX (orfX*) of N315 are indicated by red bars. Inverted complementary repeats, IR-L and IR-R, at both extremities of mec DNA are indicated by thin arrows. Direct repeats are indicated by thick arrows. Asterisks indicate identical nucleotides in the two sequences. The numbers on top of the nucleotides indicate possible terminal nucleotides of mec DNA. The entire length of mec DNA was 51,669 bp; the left-most nucleotide was inferred to be one of the four nucleotides (positions from 4685 to 4688) designated nucleotides 1 to 4 in the figure.