Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315 - PubMed (original) (raw)

Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315

T Ito et al. Antimicrob Agents Chemother. 1999 Jun.

Abstract

In methicillin-resistant Staphylococcus aureus, the methicillin resistance gene mecA is localized within a large chromosomal region which is absent in the methicillin-susceptible S. aureus chromosome. The region, designated mec DNA, is speculated to have originated from the genome of another bacterial species and become integrated into the chromosome of the S. aureus cell in the past. We report here cloning and determination of the structure of the entire mec DNA sequence from a Japanese S. aureus strain, N315. The mec DNA was found to be 51,669 bp long, including terminal inverted repeats of 27 bp and a characteristic pair of direct repeat sequences of 15 bp each: one is situated in the right extremity of mec DNA, and the other is situated outside the mec DNA and abuts the left boundary of mec DNA. The integration site of mec DNA was found to be located in an open reading frame (ORF) of unknown function, designated orfX. Clusters of antibiotic resistance genes were noted in mec DNA carried by transposon Tn554 and an integrated copy of plasmid pUB110. Both the transposon and plasmid were integrated in the proximity of the mecA gene, the latter being flanked by a pair of insertion sequence IS431 elements. Many ORFs other than those encoding antibiotic resistance were considered nonfunctional because of the acquired mutations or partial deletions found in the ORFs. Two ORFs potentially encoding novel site-specific recombinases were found in mec DNA. However, there was no ORF that might encode mec DNA-specific transposase or integrase proteins, indicating that the mec DNA is not a transposon or a bacteriophage in nature.

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Figures

FIG. 1

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 _Sau_3AI-partial digests except for SJ8, which was cloned from an N315 library of _Hin_dIII 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, _Hin_dIII; E, _Eco_RI; P, _Pst_I), 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 _Hin_dIII site and of LE1a, the _Eco_RI-_Pst_I 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, _att_L and _att_R, respectively, are aligned with the sequence around the presumptive integration site, _att_B, on NCTC8325 chromosome. The four possible integration sites are located in the _att_B 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.

FIG. 2

FIG. 2

(a) The essential structure of mec DNA. Locations of the essential genes are illustrated. Only restriction sites of _Hin_dIII are indicated. att_L and att_R (tentatively defined by the primer pairs cL1 with mL1 and mR8 with cR2) are shown by bars. orfX* is indicated by an arrow. (b) ORFs in and around mec DNA. The ORFs more than 200 bases in size in six possible reading frames are indicated by squares. Those above and those below the line are the ORFs whose transcription is directed to the right and those whose transcription is directed to the left, respectively. Colored squares indicate the ORFs whose extant gene homologues were found in the databases, though many of them were considered incomplete ORFs (underlined). These are potentially defective genes or pseudogenes containing either deletion, nonsense mutation, or frameshift mutation. The colors correspond to the inferred functional properties of the ORFs: red, drug resistance; yellow, transposases (for Tn_554, IS_431, and putative transposons); gray, plasmid replication (rep for pUB110); magenta, site-specific recombinase (N034 and N037); brown, metabolism and physiology; blue, plasmid replication (for pUB110); black, unknown function. CN051 (stippled green) corresponds to orfX*. Three ORFs, N001 to N003, located outside mec DNA have unknown function (black). (c) Regional change of G+C content in and around mec DNA. The G+C content was calculated and plotted as a line graph by using the Window program of The Wisconsin Package, with a window size of 100 bases and a shift increment of 100. The yellow bar indicates the range of G+C content of S. aureus strains, which is 32 to 36% (34). The range delimited by the orange bars is that of the genus Staphylococcus, which is 30 to 39% (34). The regions with substantially lower than average G+C content (regions 1, 3, 4, 6) and with high G+C content (regions 2, 5, 7) are indicated by red bars and green bars, respectively.

FIG. 3

FIG. 3

Nucleotide and predicted amino acid sequences of orfX genes of NCTC8325 and orfX* genes of N315. The nucleotide sequence of the orfX gene of NCTC8325 located on the plasmid pLE1a was sequenced and compared to orfX* of N315. A possible integration site of mec DNA is indicated by a dotted arrow; the nucleotide position was ambiguous but within four nucleotides. Arrows indicate the locations and directions of primers used for the PCR assay. The accession number for the sequence of pLE1a is given in the Materials and Methods section.

FIG. 4

FIG. 4

PCR for the detection of spontaneous precise excision of mec DNA. Two sets of primers were used. The primers cR2 (5′-AAACGACATGAAAATCACCAT-3′) and cL3 (5′-TGAAACTTCATTGGTATATT-3′) were used as the outer primers in (the first round of) PCR to detect generation of a DNA fragment containing _att_B, and the primers cR1 (5′-AAGAATTGAACCAACGCATGA-3′) and cL1 (5′-ATTTAATGTCCACCATTTAACA-3′) were used as the inner primers in (the second round of) PCR to generate a DNA fragment of 275 bp in _att_B. The results of the first round (lanes 2 to 12) and the second round (lane 14) of PCR are shown. Ten nanograms each of chromosomal DNA of N315, NCTC8325, and the mixtures (wt/wt) of N315 and NCTC8325 DNAs at the ratios indicated below were used as templates. Lanes: 1 and 13, 1-kb ladder (Gibco-BRL, Gaithersburg, Md.); 2, N315; 3 to 11, mixtures of N315 and NCTC8325 DNAs at ratios of 1:10−8, 1:10−7, 1:10−6, 1:10−5, 1:10−4, 1:10−3, 1:10−2, 1:10−1, and 1:0.5, respectively; 12, NCTC8325; 14, N315 (nested PCR).

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