INTRODUCTION
============

In *Streptococcus pyogenes,* the *mef* (A)/*msr*(D) pair of genes encoding eﬄux resistance to 14- and 15-membered macrolides is carried by a mobile genetic element originally described as a conjugative transposon which was called Tn*1207.3* ([@B18]; [@B16]). This element was found to be 52.5 kb in size, and to contain a complete copy of Tn*1207.1*, a 7,244-bp defective transposon previously found to carry *mef*(A)/*msr*(D) in *S. pneumoniae* ([@B19]). Integration of the element into the *S. pyogenes* chromosome occurred at a specific GA dinucleotide target site located into the *comEC* coding sequence, with integration producing a duplication of the GA site. Upon conjugal transfer to *S. pneumoniae*, chromosomal integration occurred in *celB*, the pneumococcal homolog of *comEC*, at the same insertion site of Tn*1207.1* ([@B18]; [@B16]). A copy of Tn*1207.1* was also found integrated in the 58,761-bp genetic element Φ10394.4, described as a prophage integrated at the same GA site within the *comEC* coding sequence of an erythromycin resistant clinical strain of *S. pyogenes* ([@B2], [@B1]).

Here we report the manually annotated DNA sequence of Tn*1207.3* which indicates that the element is in fact a prophage, identical to the right end of Φ10394.4. For this reason the element was renamed Φ1207.3.

MATERIALS AND METHODS {#s1}
=====================

*Streptococcus pyogenes* STRAINS AND GROWTH CONDITIONS
------------------------------------------------------

2812A is an erythromycin-resistant italian clinical isolate containing Φ1207.3 ([@B18]). Bacteria were routinely grown in tryptic soy broth or tryptic soy agar (Difco) supplemented with 3% horse blood and, where appropriate, in presence of 1 μg/ml erythromycin.

PCR AND SEQUENCING
------------------

Long PCR fragments were obtained with *Takara LA Taq* (Takara) following essentially the protocol suggested by the manufacturer. Briefly, the 25-μl reaction mixture was in 1X LA PCR Buffer II Taq buffer and contained: (i) 2.5 mM MgCl~2~, (ii) 200 μM dNTPs, (iii) 10 pmol of each primer, (iv) 0.25 units of *Takara LA Taq*, (v) 1 μl of liquid bacterial culture. Thermal cycling profile was as follows: 1 cycle at 92∘C for 2 min, then 30 cycles at 50∘C for 10 s, 68∘C for 15 min, 92∘C for 10 s, and 1 cycle at 50∘C for 1 min and 68∘C for 20 min. A primer walking approach ([@B20]) was used to sequence the PCR products. The Expand High Fidelity PCR System (Roche) was used to produce PCR fragments of about 1,000 bp in size which were used as sequencing starting template to confirm sequence on the other strand. Four primer pairs were used to amplify the four fragments, ranging from 10,358 to 16,223 bp in size, containing Φ1207.3 and its chromosomal junction fragments.

DNA SEQUENCE ANALYSIS
---------------------

DNA sequence analysis was performed with the software Artemis version 11^[1](#fn01){ref-type="fn"}^. Manual gene annotation was carried out conducting BLAST homology searches of the databases available at the National Center for Biotechnology Information^[2](#fn02){ref-type="fn"}^. Protein domains were identified searching the protein family database Pfam available at the Wellcome Trust Sanger Institute^[3](#fn03){ref-type="fn"}^. The nucleotide sequence of Φ1207.3 is assigned GenBank accession no. AY657002.

RESULTS
=======

Φ1207.3 NUCLEOTIDE SEQUENCE
---------------------------

The complete nucleotide sequence of Φ1207.3 was obtained by primer walking on four long PCR fragments spanning the whole element. Inverse PCR on genomic DNA of *S. pyogenes* strain 2812A was carried out with divergent primers matching the already known 7,244-bp sequence of Φ1207.3 and its chromosomal junction sequences ([@B18]). Amplicons obtained were used as sequencing template to determine internal sequences of Φ1207.3 on which primers were designed for the amplification of the four long overlapping fragments. The nucleotide sequence of Φ1207.3 was confirmed on the other strand using short PCR fragments as sequencing templates. Φ1207.3 was found to be 52,491 bp in length and DNA sequence analysis showed the presence of 58 open reading frames (ORFs), 44 of which have the same direction of transcription (**Figure [1](#F1){ref-type="fig"}**). Regions with different GC content can be could be identified in the DNA sequence of Φ1207.3: (i) the left end, containing ORFs 1--11, with 36.4% GC; (ii) ORFs 12--16 with 31.6% GC; (iii) ORFs 17--54 with 41,4% GC; (iv) the right end, corresponding to ORFs 55--58, with 37,8% GC (**Figure [1](#F1){ref-type="fig"}**). Comparison of Φ1207.3 with sequences present in public databases showed that: (i) the first 7,244 bp of the Φ1207.3 sequence are identical to the 7,244-bp element Tn*1207.1* of *S. pneumoniae* ([@B19], [@B18]); (ii) the whole Φ1207.3 is identical to the 52,491-bp right end sequence of *S. pyogenes* Φ10394.4 ([@B2]; [@B16]); (iii) Seven sequence fragments, ranging in size from 0.2 to 9.5 kb, show 76--97% nucleotide identity to the *S. pyogenes* prophage Φm46.1 ([@B4]); (iv) homologous sequence fragments are present in the genetic elements from the genomes of *S. dysgalactiae* AC-2713 (GenBank no. HE858529), *S. suis* JS14 (GenBank no. CP002465), and *S. agalactiae* A909 (GenBank no. CP000114).

![**Schematic diagram of *Streptococcus pyogenes* Φ1207.3.** The element is 52,491 bp in size and contains 58 open reading frames (ORFs). ORFs and their direction of transcription are represented by arrows and the annotated ORFs are indicated only by their numbers. Macrolide resistance genes and resolvase genes are reported as red and yellow arrows respectively. The region homologous to Tn*1207.1* is indicated by a solid bar. The different GC content of the various regions are indicated with dotted bars. Scale is in kilobases.](fmicb-05-00687-g001){#F1}

ORFs OF Φ1207.3
---------------

For 34 out of 58 ORFs it was possible a manual homology-based annotation with functional prediction of the hypothetical gene product (**Table [1](#T1){ref-type="table"}**). Public protein databases and the Pfam protein family database were used for Blast searches of predicted gene products, taking into account significant homologies with functionally characterized proteins or good matches with Pfam domains. A putative ribosome binding sequence preceded all ORFs except *orf36* and *orf37.* The alternative start codon TTG was present in *orf34*, *orf36*, *orf43*, and *orf56*, whereas *orf37*, *orf38*, and *orf51* started with GTG. In the left arm of the element, the gene product of *orf2* was predicted to be a site-specific resolvase of the serine recombinase family, *mef*(A) (*orf4*) and *msr*(D) (*orf5*) encoded respectively the transmembrane domains and the ATP-binding domains of the ABC transporter responsible for the M-type resistance to macrolides ([@B8], Abstract C1-1188, 44th Interscience Conference Antimicrobial Agents Chemotherapy, 2004), *orf8* and *orf11* were homologous to the Tn*5253 umuC*/*umuD* operon conferring UV resistance by activation of the SOS repair system ([@B13]). SpyIM, encoded by *orf12*, is a C-5 cytosine-specific DNA methylase belonging to the SpyI type II restriction-modification cassette, and responsible for inhibition of restriction by SmaI, whereas *orf13* and *orf14* encoded the two subunits of SpyI restriction endonuclease ([@B6]). Other genes coding for putative restriction-modification proteins include *orf24* (restriction enzyme), *orf30* (endonuclease), *orf33* (cytosine-specific DNA methylase), while *orf29* gene product presented an S-adenosylmethionine synthetase domain which may act as a methyl group donor for Orf33. In the central region, *orf17, orf23,* and *orf25* encoded a DNA polymerase, a DNA primase, and a DNA helicase, possibly involved in phage DNA replication, whereas *orf38* and *orf39* encoded for the small and large subunit of the phage terminase, which, together with the portal protein encoded by *orf40*, could be involved in phage DNA packaging. *orf42*, *orf44*, *orf45,* and *orf50* code for putative structural phage proteins. A putative lysis cassette, which is typically composed by a holin and an endolysin ([@B28]) was encoded by *hol* (*orf53*) and *skl* (*orf54*). At the right end of the element, *orf56* and *orf57* gene products were homologous to resolvases of the serine recombinase family, possibly involved in excision, circularization, and site specific integration of Φ1207.3, whereas *orf58* encoded a putative ADP-ribosyltransferase toxin.

###### 

Annotated open reading frames (ORFs) of Φ1207.3.

  -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  ORF (aa)^a^             Annotation and comments (reference)                                       Pfam domains^b^\                                                                                                                                                        Homologous protein ID/origin                       Amino acid identity-similarity    *E* value^c^
                                                                                                    \[*E* value\]                                                                                                                                                                                                                                                
  ----------------------- ------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------- --------------------------------- --------------
  *orf1* (218)            CpXC protein, contains four conserved cysteines forming two CpXC motifs   CpXC (15--138) \[1.3e-31\]                                                                                                                                                                                                                                   

  *orf2* (370)            Resolvase ([@B26])                                                        Resolvase, N terminal domain (44--193) \[5.4e-25\] Recombinase (217--325) \[2.9e-20\]                                                                                                                                                                        

  *mef*(A)/*orf4* (405)   Macrolide ABC transporter, transmembrane domain                           Transmembrane secretion effector\                                                                                                                                                                                                                            
                                                                                                    (7--404) \[1.7e-24\]                                                                                                                                                                                                                                         

  *msr(D)/orf5* (487)     Macrolide ABC transporter, ATP-binding domain                             ABC transporter, ATP-binding domain\                                                                                                                                                                                                                         
                                                                                                    (20--86; 167--284; 312--437)\                                                                                                                                                                                                                                
                                                                                                    \[1.5e-6\] \[1.1e-7\] \[2e-17\]                                                                                                                                                                                                                              

  *orf7* (122)            YolD-like protein, functionally equivalent to UmuD                        YolD-like protein (28--111) \[3.2e-12\]                                                                                                                                                                                                                      

  *umuC/orf8* (471)       SOS responce UmuC protein ([@B13]; [@B8])                                                                                                                                                                                                         EU351020 orf70/Tn*5253 Streptococcus pneumoniae*   344/471 (73%) --\                 0.0
                                                                                                                                                                                                                                                                                                                               398/471 (85%)                     

  *umuD/orf11* (229)      UmuD MucA homolog ([@B13]; [@B8])                                                                                                                                                                                                                 EU351020 orf69/Tn*5253 S. pneumoniae*              110/230 (48%) --\                 2e-57
                                                                                                                                                                                                                                                                                                                               161/230 (70%)                     

  *spyIM/orf12* (408)     C-5 cytosine-specific DNA methylase ([@B6])                                                                                                                                                                                                                                                                                            

  *orf13* (555)           Restriction endonuclease subunit\                                         AAA domain (dynein-related subfamily)\                                                                                                                                                                                                                       
                                                                                                    (227--387) \[9.1e-9\]                                                                                                                                                                                                                                        

  *orf14* (415)           Restriction endonuclease subunit ([@B14])                                 LlaJI restriction endonuclease\                                                                                                                                         AAS99180/pNP40 *Lactococcus lactis*                157/406 (38%) -- 251/406 (61%)    5e-81\
                                                                                                    (13--383) \[5.7e-120\]                                                                                                                                                                                                                                       

  *orf15* (384)           DNA binding, zinc finger domain protein\                                  CGNR zinc finger (338--380) \[4.2e-5\]\                                                                                                                                                                                                                      

  *orf17* (651)\          DNA polymerase\                                                           DNA polymerase family A (247--520) \[3.7e-14\]                                                                                                                                                                                                               
                          ([@B17])                                                                                                                                                                                                                                                                                                                               

  *orf19* (190)           Conserved phage-associated protein                                        DUF2815 (11--188) \[9.7e-65\]                                                                                                                                                                                                                                

  *orf23* (761)           DNA primase ([@B27])                                                      D5 N terminal like (285--472) \[2.4e-29\]                                                                                                                                                                                                                    

  *orf24* (100)           Restriction-modification enzyme, putative ([@B9])                         VRR-NUC domain (8--88) \[3.3e-16\]                                                                                                                                                                                                                           

  *orf25* (458)           Helicase, putative                                                        SNF2 family N-terminal domain (5--293) \[8.9e-19\]                                                                                                                                                                                                           

  *orf29* (353)           *S*-adenosylmethionine synthetase ([@B23])                                *S*-adenosylmethionine synthetase: N-terminal domain Domain (9--85) \[6.9e-28\] Central domain (105--214) \[7.6e-30\] C-terminal domain Domain (216--353) \[1.2e-57\]                                                                                        

  *orf30* (121)           HNH endonuclease, putative                                                HNH endonuclease (62--110) \[1.6e-11\]                                                                                                                                                                                                                       

  *orf32* (410)           DNA methylase                                                             ParB-like nuclease domain (5--93) \[1e-10\]\                                                                                                                                                                                                                 
                                                                                                    DNA methylase (189--381) \[5.2e-29\]                                                                                                                                                                                                                         

  *orf33* (388)           C-5 cytosine-specific DNA methylase                                       C-5 cytosine-specific DNA methylase (4--382) \[1.5e-59\]                                                                                                                                                                                                     

  *orf38* (158)           Phage terminase, small subunit, putative                                  Phage terminase, small subunit (50--150) \[8.2e-31\]                                                                                                                                                                                                         

  *orf39* (530)           Phage terminase, large subunit, putative ([@B21])                         Phage Terminase (48--515) \[1.1e-103\]                                                                                                                                                                                                                       

  *orf40* (413)           Phage portal protein, putative ([@B12])                                   Phage portal protein (30--381) \[4.8e-121\]                                                                                                                                                                                                                  

  *orf41* (228)           Clp protease, putative ([@B24])                                           Clp protease (8--179) \[4.5e-40\]                                                                                                                                                                                                                            

  *orf42* (395)           Phage capsid protein, putative                                            Phage capsid family (124--393) \[6.4e-78\]                                                                                                                                                                                                                   

  *orf44* (109)           Phage head-tail adaptor, putative                                         Phage head-tail joining protein (7--105) \[8.8e-15\]                                                                                                                                                                                                         

  *orf45* (126)           Tail component protein, putative                                          Bacteriophage HK97-gp10, putative tail-component (11--92) \[1.2e-6\]\                                                                                                                                                                                        

  *orf50* (1039)          Tail tape measure protein, putative ([@B15])                              Phage-related minor tail protein (311--554) \[2.7e-14\]                                                                                                                 AAG32164.1/*Lactococcus* phage TP901-1             152/693 (22%) -- 280/693 (40%)    1e-22

  *orf52* (967)           Host specificity protein\                                                 \                                                                                                                                                                       AAK83249/*S. thermophilus* phage DT1.2             113/418 (27%) -- 186/418 (44%)    5e-40
                          ([@B5])                                                                                                                                                                                                                                                                                                                                

  *hol*/*orf53* (760)     Holin family protein ([@B10])                                             Siphovirus protein of unknown function (DUF859)\                                                                                                                        AAW27943/*S. thermophilus* phage 2972\             205/514 (40%) -- 279/514 (54%)\   8e-89
                                                                                                    (1--516) \[1.8e-214\] Holin family (637--754) \[4e-40\]                                                                                                                                                                                                      

  *skl/orf54* (260)       *N*-acetylmuramoyl-[L]{.smallcaps}-alanine amidase ([@B11])               CHAP domain (13--142) \[5.6e-10\]                                                                                                                                       CAJ13672/*S. mitis* phage SK137                    62/211 (29%) -- 96/211 (45%)      9e-8

  *orf56* (412)           Resolvase\                                                                Resolvase, N terminal domain (20--167) \[3.8e-30\] Recombinase (187--291) \[4.2e-21\]\                                                                                                                                                                       
                                                                                                    Recombinase zinc beta ribbon domain (307--368) \[1.6e-8\]                                                                                                                                                                                                    

  *orf57* (521)           Resolvase\                                                                Resolvase, N terminal domain (28--176) \[4.6e-33\] Recombinase (200--306) \[5.5e-24\]\                                                                                                                                                                       
                                                                                                    Recombinase zinc beta ribbon domain (316--377) \[2.4e-10\]                                                                                                                                                                                                   

  *orf58* (191)           ADP-ribosyltransferase toxin, putative                                    ADP-ribosyltransferase exoenzyme (66--181) \[3.7e-17\]                                                                                                                                                                                                       
  -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

a

The number of amino acids is shown in parentheses.

b

The numbers in parentheses represent the part of the protein homologous to the Pfam domain.

c

Method: compositional matrix adjust.

DISCUSSION
==========

The complete and annotated DNA sequence of the mobile genetic element previously called Tn1207.3 clearly shows that the element is a prophage which we renamed Φ1207.3. At the sequence level, Φ1207.3 (52,491 bp) shows homology to two *S. pyogenes* prophages: (i) Φ10394.4 (58,761 bp), integrated at the same chromosomal site of Φ1207.3 ([@B2]; [@B16]); (ii) Φm46.1 (55,172 bp), integrated in the *rum* gene encoding an RNA uracil methyltransferase ([@B4]). The whole Φ1207.3 is identical to the right end of Φ10394.4, whereas high homology (\>70%) to Φm46.1 is limited to 57% of the Φ1207.3 genome. Prophages similar to Φ1207.3 were also described by [@B7].

The recombination machinery of Φ1207.3 consists of three resolvases of the serine recombinase family. Serine recombinases are less common than tyrosine recombinases in prophage genomes and are usually present as a single large recombinase gene ([@B22]). Most of the *S. pyogenes* prophages present in sequenced genomes have a tyrosine recombinase (integrase) as the recombination module ([@B3]). The two resolvase genes at the right end of Φ1207.3 are probably transcribed as a single unit, as their coding sequences overlap by one nucleotide. It is likely that their gene products cooperate in mediating excision and integration of the Φ1207.3 DNA. This arrangement in tandem of two resolvase genes is also found in SSC*mec* elements (types I to IV) of *Staphylococcus aureus* ([@B25]), and in streptococcal prophage Φm46.1 ([@B7]; [@B4]).

Since Φ1207.3 can move among streptococcal species (*S. pyogenes*, *S. pneumoniae*, *S. gordonii*) by a mechanism which fits the operational definition of conjugation ([@B18]), this conjugative prophage may represent a novel class of genetic elements with a molecular mechanism of transfer that still needs to be elucidated. It is entirely possible that assembly of a complete phage particle may not be essential for the observed interspecific DNA transfer, and we hypothesize that the lysis cassette of Φ1207.3, rather than producing the bacterial cell burst after intracellular phage expansion ([@B28]), could contribute to the formation of cytoplasmic bridges between donor and recipient cell.

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This study was supported by the European Commission grant ANTIRESDEV HEALTH-F3-2009-241446.

<http://www.sanger.ac.uk/Software/Artemis/website>

<http://www.ncbi.nlm.nih.gov/sites/gquery>

<http://pfam.sanger.ac.uk>

[^1]: Edited by: *Bruna Facinelli, Università Politecnica delle Marche, Italy*

[^2]: Reviewed by: *Dmitri Debabov, NovaBay Pharmaceuticals, USA; Andrea Brenciani, Marche Polytechnic University, Italy; Pietro Emanuele Varaldo, Marche Polytechnic University, Italy*

[^3]: ^†^Present address:*Marco R. Oggioni, Department of Genetics, University of Leicester, Leicester, UK*

[^4]: ^‡^*Francesco Iannelli and Maria Santagati have contributed equally to this work.*

[^5]: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology.
