Phim46.1, the main Streptococcus pyogenes element carrying mef(A) and tet(O) genes

Andrea Brenciani et al. Antimicrob Agents Chemother. 2010 Jan.

Abstract

Phim46.1, the recognized representative of the most common variant of mobile, prophage-associated genetic elements carrying resistance genes mef(A) (which confers efflux-mediated erythromycin resistance) and tet(O) (which confers tetracycline resistance) in Streptococcus pyogenes, was fully characterized. Sequencing of the Phim46.1 genome (55,172 bp) demonstrated a modular organization typical of tailed bacteriophages. Electron microscopic analysis of mitomycin-induced Phim46.1 revealed phage particles with the distinctive icosahedral head and tail morphology of the Siphoviridae family. The chromosome integration site was within a 23S rRNA uracil methyltransferase ren. BLASTP analysis revealed that the proteins of Phim46.1 had high levels of amino acid sequence similarity to tướng the amino acid sequences of proteins from other prophages, especially Phi10394.4 of S. pyogenes and lambdaSa04 of S. agalactiae. Phage DNA was present in the host cell both as a prophage and as không lấy phí circular DNA. The lysogeny module appears to tướng have been split due to tướng the insertion of a segment containing tet(O) (from integrated conjugative element 2096-RD.2) and mef(A) (from a Tn1207.1-like transposon) into the unintegrated phage DNA. The phage attachment sequence lies in the region between tet(O) and mef(A) in the unintegrated size. Thus, whereas in this size tet(O) is approximately 5.5 kb upstream of mef(A), in the integrated size, tet(O), which lies close to tướng the right kết thúc of the prophage, is approximately 46.3 kb downstream of mef(A), which lies close to tướng the left kết thúc of the prophage.

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Figures

FIG. 1.

Bacteriophage Φm46.1 integration site. Φm46.1 was integrated into the chromosome of S. pyogenes m46 within a 23S rRNA uracil methyltransferase ren. This ren, detected in all S. pyogenes genomes sequenced to tướng date, encodes a protein having the highest degree of amino acid similarity to tướng the amino acid sequence of a protein encoded by the corresponding ren from S. pyogenes MGAS10750 (EMBL accession no. CP000262), which, lượt thích S. pyogenes m46, belongs to tướng M type 4. Chromosomal ORF designations and aligned host genome sequences are thus from S. pyogenes MGAS10750. (A) Left junction. (Above) ORF map; chromosomal ORFs are indicated as Đen arrows, and Φm46.1 ORFs are indicated as white arrows, with mef(A) being indicated by a checkered arrow. (Below) Alignment of the amplified sequence (64 bp) at the attL region with the Φm46.1 and S. pyogenes MGAS10750 genomes. Numbers above and below the attL sequence refer to tướng base positions in the amplicon obtained with primers LYT-for and MEFA2 (4,425 bp). (B) Right junction. (Above) ORF map; chromosomal ORFs are indicated as Đen arrows and Φm46.1 ORFs are indicated as white arrows, with tet(O) being indicated by a striped arrow. (Below) Alignment of the amplified sequence (64 bp) at the attR region with the Φm46.1 and S. pyogenes MGAS10750 genomes. Numbers above and below the attR sequence refer to tướng base positions in the amplicon obtained with primers TETO1 and THIO-rev (6,573 bp). In the almost completely overlapping 18-bp sequence shared by the bacteriophage and the host bacterium genomes and representing the core site, nucleotides are indicated with capital letters.

**FIG. 2.**
ORF map and genome organization of the Φm46.1 prophage and its alignment with the genome maps of Φ10394.4 from *S. pyogenes* MGAS10394 and λSa04 from *S. agalactiae* A909. The ORFs, depicted as arrows pointing in the direction of transcription, are numbered consecutively (*orf1* to tướng *orf63* in Φm46.1, with some predicted functions being reported in Table 2; *orf1* to tướng *orf61* in Φ10394.4; and *orf1* to tướng *orf41* in λSa04). White arrows indicate ORFs likely of phage origin. Black arrows indicate ORFs likely of chromosomal origin, except for *mef*(A) (checkered arrow) in transposons Tn1207.1 and Tn1207.1-like and *tet*(O) (striped arrow) in the fragment from ICE 2096-RD.2. In Φm46.1, phage functional modules are identified by bars.

FIG. 3.

Electron microscopic analysis of phage particles purified from S. pyogenes m46.

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