Genomic comparison between Staphylococcus aureus GN strains clinically isolated from a familial infection case: IS1272 transposition through a novel inverted repeat-replacing mechanism

A bacterial insertion sequence (IS) is a mobile DNA sequence carrying only the transposase gene (tnp) that acts as a mutator to disrupt genes, alter gene expressions, and cause genomic rearrangements. “Canonical” ISs have historically been characterized by their terminal inverted repeats (IRs), which may form a stem-loop structure, and duplications of a short (non-IR) target sequence at both ends, called target site duplications (TSDs). The IS distributions and virulence potentials of Staphylococcus aureus genomes in familial infection cases are unclear. Here, we determined the complete circular genome sequences of familial strains from a Panton-Valentine leukocidin (PVL)-positive ST50/agr4 S. aureus (GN) infection of a 4-year old boy with skin abscesses. The genomes of the patient strain (GN1) and parent strain (GN3) were rich for “canonical” IS1272 with terminal IRs, both having 13 commonly-existing copies (ce-IS1272). Moreover, GN1 had a newly-inserted IS1272 (ni-IS1272) on the PVL-converting prophage, while GN3 had two copies of ni-IS1272 within the DNA helicase gene and near rot. The GN3 genome also had a small deletion. The targets of ni-IS1272 transposition were IR structures, in contrast with previous “canonical” ISs. There were no TSDs. Based on a database search, the targets for ce-IS1272 were IRs or “non-IRs”. IS1272 included a larger structure with tandem duplications of the left (IRL) side sequence; tnp included minor cases of a long fusion form and truncated form. One ce-IS1272 was associated with the segments responsible for immune evasion and drug resistance. Regarding virulence, GN1 expressed cytolytic peptides (phenol-soluble modulin α and δ-hemolysin) and PVL more strongly than some other familial strains. These results suggest that IS1272 transposes through an IR-replacing mechanism, with an irreversible process unlike that of “canonical” transpositions, resulting in genomic variations, and that, among the familial strains, the patient strain has strong virulence potential based on community-associated virulence factors.

cut-and-paste manner [28]. Previously identified target sequences are all non-IR sequences, and no IR targets have yet been identified.
IS1272 was initially described in S. haemolyticus; it is 1,934 bp in size with 16-bp terminal IRs (sequence identity, 15 of 16 bp), lacks flanking TSDs, and contains two open reading frames (orfs): orf1 and orf2 [33]. Nosocomial isolates of S. haemolyticus are rich in IS1272 copies, suggesting the potential role of IS1272 in bacterial virulence [34]; however, whole-genomic and virulence information are currently lacking to confirm this. The initial IS1272 study [33] also showed that S. aureus contains IS1272, but these sequences are mostly incomplete fragments of IS1272, suggesting that IS1272 originally resided in S. haemolyticus and that MRSA has more IS1272-hybridizing fragments than MSSA. The MRSA type IV SCCmec (SCCmecIV) possesses the mecA-ΔmecR1-ΔIS1272 region [6]. In addition, the highly successful HA-MRSA, MRSA252 [35], carries nine copies of IS1272 on its genome (GenBank accession number NC_002952). However, IS1272 may not be common in MRSA; for example, USA300 (GenBank accession number PC000255; strain FPR3757), a successful PVL-positive CA-MRSA associated with large outbreaks, including serious invasive infections, in the United States in 2007 [7,36], has no copies of IS1272 in its genome, whereas SCCmecIVa in USA300 carries a truncated IS1272, as described above. The PVL phages, including that of USA300 [36], generally do not have any copies of IS1272 in their genomes (GenBank accession numbers PC000255).
During the course of a study on the PVL S/F genes and predicted PVL S/F amino acid sequences [37,38], we determined the sequences of not only the entire PVL gene but also of its upstream and downstream regions in clinical S. aureus isolates by PCR and sequencing. We detected an IS1272 transposition onto the PVL-converting prophage and proposed the idea of a unique "stem-loop replacing" mode of transposition, in which a target "stem-loop" structure is replaced by that of IS1272 [39] (GenBank accession numbers AB256036-39). In the present study, we elucidated the sequences of the complete circular genomes using the patient strain (GN1) and parent strain (GN3), clinically isolated from a family infection case caused by a single clone of PVL-positive ST50 community-associated S. aureus (GN), and we performed a comprehensive comparison between the GN1 and GN3 genomes. This evaluation focusd on the status of multiple IS1272 copies in the S. aureus genomes, including newly-inserted IS1272 (ni-IS1272); we unambiguously revealed that the targets of transposition for IS1272 were IR sequences (with different sequences and sizes), in contrast with the targets of previously describe ISs ("canonical" or not). For commonly-existing IS1272 (ce-IS1272) in GN1 and GN3, we searched the target site sequences using a database of previously reported genome sequences. In light of our results, we discuss, particularly from the viewpoint of basic science, potential mechanisms that could be responsible for IS1272 transposition, given that IS1272 generates a deletion of its target "stem-loop" structure.
In previous molecular epidemiology studies of clinical S. aureus infections, one clonal infection of S. aureus lineage was verified, for example, by pulsed-field gel electrophoresis (PFGE) analysis of the genomes [17,40]; however, the whole genomes of S. aureus in this type of familial infection case have not been elucidated sufficiently, particularly in terms of the IS transposition and virulence potential. Therefore, in the present study, we analyzed variations among the GN genomes as well as virulence potential differences among GN familial strains, particularly for community-associated factors, peptide cytolysins and PVL.

Ethics statement
The Ethics Review Board of Niigata University School of Medicine, Niigata, Japan (Ethics Review Board No. 748) specifically approved this study. Written informed consent was obtained from patients, where necessary.

A patient, familial infections, and bacterial strains
A 4-year-old boy was brought to a hospital (Kido Hospital, Niigata) on July 23,2005. Large skin abscesses (furuncles) were observed in his gluteal region. PVL-positive CA-MSSA was isolated from pus; this strain was designated GN1. The epidemiological definition of CA-S. aureus was based on the Centers for Disease Control and Prevention (CDC) criteria for CA-MRSA and HA-MRSA [7]. A 10% zinc oxide ointment sheet was placed on the furuncle region, and the patient was orally administered clarithromycin at 100 mg/day. The patient had frequently developed SSTIs including skin abscesses between 2003 and 2004. Nasal swabs were obtained in 2005 from eight of the patient's family members (from three families) who were living together with the boy within the same house to examine familial infections (Fig 1A). PVL- Nine family members (from three families) were living together with the 4-year-old boy (a patient who had skin abscesses) within the same house. Five members who were infected with PVL-positive S. aureus are marked with yellow. Four members, except the 4-yearold patient, did not develop any symptoms. Red square and circle indicate that the gonomes of PVL-positive S. aureus were sequenced. PVL-positive S. aureus srains: GN1, patient strain; GN2, infant strain; GN3, 27-year-old parent (female) strain; GN4, 27-year-old parent (male) strain; GN5, strain from the oldest member among infected members. (B) Familial strains (GN1 to GN5) were analyzed by pulsedfield gel electrophoresis, suggesting a clonal infection from one PVL-positive S. aureus source. Neighborhood strains were from healthy persons from neighboring families unrelated to the patient's families. (C) The genotypes, vurulence genes analyzed by PCR, and drug resistance of familial strains GN1 to GN5 are summarized, indicating the common features among strains GN1 to GN5. https://doi.org/10.1371/journal.pone.0187288.g001 Genomic, IS, and virulence analysis of S. aureus strains from a familial infection case positive MSSA was isolated from four out of the eight family members; therefore, a total of five out of the nine members (including the patient) were positive for PVL-positive MSSA, and their ages ranged from 0-60 years with a mean age of 26.9 years. Except for the 4-year-old boy (patient), the infected individuals did not show any symptoms; the PVL-positive MSSA isolated from these individuals were designated GN2 to GN5, as shown in Fig 1A. Two colonies each of GN1 to GN5, developed on initial bacterial isolation agar media, were characterized for S. aureus genotypes and drug susceptibilities.
Twenty-nine MSSA and two MRSA strains were also isolated from the nasal swabs of 78 healthy neighbors (including children) from 21 families (unrelated to the patient family); they were all PVL-negative.

Genome analysis
The PVL prophage genomes and bacterial genomes of GN1 and GN3 (patient and mother strains, respectively, Fig 1A) were analyzed. The entire genome sequences of the PVL prophages contained by GN1 and GN3, named φPVL-Sa2 GN1 and φPVL-Sa2 GN3 , respectively, were determined by PCR and sequencing; the genome sizes were 48,010 bp and 46,089 bp, respectively, and the GenBank accession numbers are LC086374 and LC086375, respectively. The bacterial genome sequences of GN1 and GN3 were analyzed by a long-read singlemolecule real-time (SMRT) sequencing platform with P5/C3 chemistry using sequencing technology and the PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA) with the assembler software SMRT Analysis v2.3.0/hierarchical genome-assembly process (HGAP) pipeline [43]. Genome coverage (sequencing depth) was 563-fold and 443-fold of each genome size for GN1 and GN3, respectively. Completion of the genome contig to construct the full circular genome sequence was performed by PCR and sequencing. For the GN1 and GN3 bacterial genomes, the sizes were 2,809,565 bp and 2,809,401 bp, respectively (Fig 2), and the GenBank accession numbers are AP018349 and AP017891, respectively.

PCR analysis of IS1272 insertions in the PVL prophage region of familial strains
Nine PCR primer sets (A to C and 1 to 6) were used to investigate the presence of an IS1272 insertion at a region located downstream of the PVL S/F genes (luk PV SF) on the PVL prophage DNA; primer sequences are summarized in S1 Fig [37,44]. PCR primers, PVL-1 and NPVL-2, were based on reference [44]. Other PCR primers were initially designed based on the DNA sequence of a PVL prophage lacking an IS1272 insertion that was carried by the ST30 CA-MRSA strain NN1 [37]; later primers were designed based on the sequences of φPVL-Sa2 GN1 and φPVL-Sa2 GN3 . The primers for IS1272 were designed based on the φPVL-Sa2 GN1 sequence, which has an IS1272 insertion. Of the three primer sets (A to C), primer set B detects the terminal IRs of the target sequence of IS1272; thus, in the present study, GN1, GN2, and GN5 (in which the target was replaced by IS1272) each produced negative results in PCR assays using primer set B. Of the othe six PCR primer sets (1 to 6), primer set 1 detects the PVL S gene and also its upstream region (thus, in the present study, GN1 to GN5 each gave PCR products of the same size, indicating no IS1272 insertion); primer sets 2-5 each detect an IS1272 inserted downstream of the PVL S/F genes (thus, in the present study, GN1, GN2, and GN5 each produced positive results); and primer set 6 detects the 3'-end region of the PVL F gene and a region located downstream of the PVL F gene (thus, in the present study, GN1 to GN5 all yielded PCR products, but GN1, GN2, and GN5 each produced ca. 2-kb bigger PCR products, due to the presence of an IS1272 insertion). mRNA expression assay S. aureus strains were cultured on 5% sheep blood agar (Becton Dickinson, Tokyo, Japan) for 8 h at 37˚C. The mRNA expression levels of the psmα, δ-hemolysin (hld), PVL (luk PV SF), and 16S rRNA genes were then examined using an RT-PCR assay [20][21][22]. The psmα hld, and luk PV SF expression levels were then normalized to 16S rRNA expression levels. ST5/SCCmecII HA-MRSA strains (Mu50 and N315) were used as low psmα hld expression control strains, and the ST8/SCCmecIVa PVL + CA-MRSA type strain USA300-0114, ST30/SCCmecIVc PVL + CA-MRSA strain RS08, and ST121/agr4 CA-MSSA strain KT1 were used as stronger psmα/hld expression control strains [20][21][22]. Experiments were repeated six times for each strain.

Phylogenetic analysis
Multiple alignments were performed up to 1,000 times using default settings with ClustalW software (version 2.1), and a phylogenetic tree analysis was performed using TreeViewX software (version 0.5.0) (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Analysis of the target sequences of ce-IS1272 transposition
A database composed of previously reported S. aureus genomes was searched for target sequences for ce-IS1272 transposition. The analysis of homology between the S. aureus genome sequences containing the target sequences of ce-IS1272 transposition and the GN1/ GN3 genome sequences carrying ce-IS1272 was performed using the software BLAST (http:// blast.ddbj.nig.ac.jp/top-e.html).

Statistical analysis
Data were evaluated by a Student's t-test, a Fisher's exact test, or an analysis of variance with repeated measurements for the mRNA expression assay. The level of significance was defined as a P value of <0.05.

Familial infection from PVL-positive MSSA
Among the nine members from three families who were living together within the same house, five were positive for PVL-positive CA-MSSA, and the strains isolated from these individuals were named strains GN1 to GN5 (Fig 1A). A PFGE analysis revealed that the five PVLpositive CA-MSSA strains GN1 to GN5 were the same (Fig 1B), indicating the intrafamilial spread proton/sodium-glutamate symport protein, sbi, second binding protein of immunoglobulin, hlg, γ-hemolysin (Hlg); fnb, fibronectin-binding protein; ica, intercellular adhesion protein A (biofilm formation); cna, collagen adhesin. The DNA helicase gene and eight other helicase genes, dnaB, recQ1, comFA, recG, recQ, ruvB, rubA, and dnaB2, were also mapped on the genomes; they are shown in purple. GN1 and GN3 had PVL-converting φSa2 (φPVL-Sa2 GN1 and φPVL-Sa2 GN3 , respectively), carrying the PVL genes (luk PV SF). Unique genetic structures were carried by φSa3 remnant, the immune evasion cluster (IEC), composed of three immune evasion genes, sak (staphylokinase, SAK), chp (chemotaxis inhibitory protein of S. aureus, CHIPS), and scn (staphylococcal complement inhibitor, SCIN), being present on the GN genome as IEC/PR. Penicillin resistance was encoded by a chromosomal bla-tnp structure, carrying an array of blaI-blaR1-blaZ and tnpC-tnpB-tnpA. The GN1 and GN3 genomes carried 14 and 15 copies of IS1272, respectively. Of those, 13 copies were commonly-existing IS1272 (ce-IS1272), and named 1 to 13; moreover, GN1 had one copy of newly-inserted IS1272 (ni-IS1272), which was named P1, while GN2 had two copies of ni-IS1272, which were named C1 and C2, as shown in A and B, respectively. ce-IS1272 copy 6 and ni-IS1272 copy C1 formed a larger structure (designated as IS1272L). ce-IS1272 copy 2, labeled in green, had the transposase gene (tnp) encoding for a long fusion form of transposase; ce-IS1272 copy 7, marked with an asterisk, had tnp encoding for a truncated form of transposase. The direction of the IS1272 insertion is shown by + or -. The targets of IS1272 transposition are indicated by color: red, IRs; blue, "non-IRs"; yellow and green, unknown (target sequences are too big in size). The 3-kb region, located close to IS1272 copy 3, in GN1 (A) was deleted in GN3 (B). of the single PVL-positive CA-MSSA clone (GN). The PVL-positive CA-MSSA GN (strains GN1 to GN5) belonged to ST50 (CC50), exhibited spa108 (t185), agr4, and coagulase VI, carried toxin genes such as luk PV SF, hld, psmα and egc (an entrotoxin gene cluster carrying sei, selm, seln, selo, and selu, but lacking seg), carried 12 adhesin genes including cna (encoding for collagen adhesin), and was only resistant to ampicillin/penicillin G ( Fig 1C). The above characteristics of GN strains (GN1 to GN5) were confirmed for two initially-isolated colonies of each strain.
Among the five family members infected with GN, only a 4-year-old boy, infected with strain GN1, developed furuncles, whereas the four other members, a <1-year-old infant, 27-year-old female and male parents, and the oldest (57-year-old) infected member (infected with strains GN2, GN3, GN4, and GN5, respectively), did not develop any symptoms.
PVL-positive MSSA was not isolated from the healthy individuals of neighboring families; MSSA (or MRSA) strains isolated from neighboring family members were divergent, as shown in Fig 1B. The complete circular genome structures of patient strain GN1 and parent strain GN3 The GN1 and GN3 genomes were estimated to be 2,809,565 bp and 2,809,401 bp, respectively. Based on the GN1 and GN3 complete circular genome sequences, the GN1 and GN3 circular genome maps were constructed, as shown in Fig 2A and 2B, respectively, with a focus on IS1272, some virulence genes, some regulatory genes or regulons, genes used for genotyping (spa, agr, and coa), resistance genes, and phages.
Regarding ISs, 14 and 15 copies of IS1272 were distributed along the GN1 and GN3 genomes, respectively. In addition to IS1272, the GN1 and GN3 genomes carried several other ISs: eight copies of ISSep3, designated as ISSep3 (GN1) or (GN3), and two copies of ISSep2, designated as ISSep2 (GN1) or (GN3), suggesting that IS1272 was the most prevalent IS in the GN1 and GN3 genomes. GN1 and GN3 did not have any copies of IS256, which exhists as a multi-copy system in epidemic MRSA [29].
Regarding virulence genes, the immune evasion cluster (IEC), which is generally present on the left-end side of φSa3 [21,27,29,45], was found in the GN1 and GN3 genomes. IEC (GN1) and IEC (GN3) each carried three immune evasion genes: sak for staphylokinase (SAK), chp for the chemotaxis inhibitory protein of S. aureus (CHIPS), and scn for staphylococcal complement inhibitor (SCIN), as well as the φSa3 remnant. Therefore, the IEC in the GN1 and GN3 genomes was designated as IEC/PR. The location of IEC/PR was not hlb, which provides the insertion site (att) for φSa3; IEC/PR was located distant from hlb. Thus, the hlb in the GN1 and GN3 genomes was intact (Figs 1C, 2A and 2B).
Regarding resistance, three penicillin resistance-related genes, blaZ, blaR1, and blaI, which are carried by a penicillinase plasmid in S. aureus [46], were present in the GN1 and GN3 genomes.

Status of multiple IS1272 copies on the GN1 and GN3 genomes
The IS1272 copies of GN1 and GN3, IS1272 (GN1) and IS1272 (GN3), only carried one transposase gene (tnp), unlike IS1272 (S. haemolyticus), which has two orfs [33], as shown in Fig 3A  [ 33,47]. The orf1 sequence of S. haemolyticus IS1272 contains a premature stop codon due to a frame shift caused by a one-base deletion, relative to the transposase gene sequences of IS1272 (major form) of GN1 and GN3. Regarding the 16-bp terminal IR sequences, IS1272 (S. haemolyticus) and IS1272-major form (GN1, GN3) were divergent by two nucleotides. The IR L and  [33]. In contrast, a major form of IS1272 in GN1 and GN3 had only one tnp and homogeneous IRs (sequence identity, 16 of 16 bp). (B) The structures of IS1272, major form and minor forms (2, 3, and 4), were compared. Homologous regions are shaded in each comparison. IS1272/minor form-2 (copy 7) had a premature stop codon in tnp, thus its product was predicted to be truncated Tnp. IS1272/minor form-4 (copy 2) had tnp of a larger size, which started at an ATG codon located upstream of tnp/major form; the ribosome binding sequence (AAGGA), which can potentially pair with the complementary sequence at the 3'-end of 16S rRNA [47], is shown in red. (C) The genetic statuses of 16 IS1272 copies on the GN1/GN3 genomes are summarized. IS1272/major form had 1,647-bp (548-aa) tnp and 16-bp homogeneous IRs (sequence identity, 16 of 16 bp). IS1272/minor form-1 had a nonsynonymous substitution in tnp. IS1272/minor form-2 had a premature stop codon in tnp. IS1272/minor form-3 had 16-bp heterogeneous IRs (sequence identity, 14 of 16 bp) and tnp which was divergent in comparison with tnp/major form. IS1272/minor form-4 had 16-bp more-divergent IRs (sequence identity, 10 of 16 bp) and tnp with a 450-bp (150-aa) longer N-terminal side. nt, nucleotide.
The 14 copies of IS1272 (GN1) and 15 copies of IS1272 (GN3) were not uniform. Based on the sequence divergence in the transposase gene and terminal IRs, the IS1272 forms were classified into a major form and four minor forms (Fig 3B and 3C). The major form contained copies 1, 3-6, 8, 11, C1, C2, and P1 (Fig 2A and 2B); these copies encoded for 548-amino acid (aa) transposase, had terminal IRs of the same sequences (sequence identity of IR L and IR R , 16/16), and showed an over all sequence identity of 100% (Fig 3B and 3C). Minor form-1 (copy 12) (Fig 2A and 2B) had a point mutation (nonsynonymous substitution, which caused an amino acid change) in the transposase gene ( Fig 3C); minor form-2 (copy 7) (Fig 2A and  2B) had a premature stop codon in the transposase gene, resulting in a truncated (328-aa) transposase (Fig 3B and 3C); minor form-3 (copies 9, 10, and 13) (Fig 2A and 2B) had two or three nonsynonymous substitutions in the transposase gene and also heterogeneous terminal IRs (sequence identity of IR L and IR R , 14/16) (Fig 3B and 3C); and minor form-4 (copy 2) (Fig  2A and 2B) used a new start codon for the transposase gene, resulting in a 150-aa larger (698-aa) transposase (Fig 3B), and also had heterogeneous terminal IRs (sequence identity of IR L and IR R , 14/16) (Fig 3C).
IS1272 copy C1, on the GN3 genome (Fig 2A), and IS1272 copy 6, on both the GN1 and GN3 genomes (Fig 2A and 2B), formed a larger structure (IS1272L) with tandem duplications of the 25-bp left side sequence including IR L ; IS1272L was 1,970 bp in size and had terminal IR sequence (IR L2 ) in addition to an ordinary major form of 1,945-bp IS1272 with IR L and IR R (Fig 4). Unlike IS256 [29,48], IS1272 did not form an extrachromosomal circular DNA molecule.

Targets and the mode of transposition for ni-IS1272
A comparison of the GN1 and GN3 genome sequences revealed three cases of ni-IS1272; one case (copy P1) occurred in the GN1 genome and two cases (copies C1 and C2) in the GN3 genome (Fig 5). This new IS1272 insertion on GN1 (copy P1 transposition) occurred at a 24-bp IR structure (with 9-bp terminal IRs and a 6-bp intervening region), which was located 66 bp downstream of the PVL F gene (luk pv F) in the prophage φPVL-Sa2 (GN3) region ( Fig   Fig 4. Structure of IS1272 with tandem duplications of its left side sequence. ce-IS1272 copy 6 in GN1 and GN3 and ni-IS1272 copy C1 in GN3 formed a larger IS1272 structure (designated as IS1272L) with the same sequence. IS1272L had tandem duplications of the 25-bp left side sequence including IR L ; the size of IS1272L with IR L2 and IR R is 1,970 bp. https://doi.org/10.1371/journal.pone.0187288.g004 Genomic, IS, and virulence analysis of S. aureus strains from a familial infection case One new IS1272 insertion in GN3 (copy C2 transposition) occurred at a 30-bp IR structure (with 12-bp terminal IRs and a 6-bp intervening region), which was located 15 bp downstream of rot and also down stream of the rRNA methyltransferase gene (Fig 5B); the other new IS1272 insertion in GN3 (copy C1 transposition) occurred at a 22-bp IR structure (with 9-bp terminal IRs and a 4-bp intervening region), which was located within the DNA helicase gene (at the 3'-end region) (Fig 5C). Thus, the targets of all three ni-IS1272 transpositions were IR sequences, albeit with different sizes and different sequences, suggesting that the target is an IR structure (a potential stem-loop structure).
For all three cases, the insertion process may have included the complete separation of the IS1272 IR structure from the flanking donor sequence, the insertion of IS1272 into the target IR site, and the complete separation of the target IR structure from the flanking recipient sequence ("cut-paste-and-cut"), thereby resulting in an IR-replacing model of transposition (or potential stem-loop replacing model of transposition) (Fig 5). There were no TSDs for ni-IS1272 in GN1 and GN3.

Search for possible targets of ce-IS1272
The GN1 and GN3 genomes each had 13 copies of ce-IS1272 (Fig 2). To elucidate the targets and modes of the previous transpositions that produced the 13 current copies of ce-IS1272, a database of previously reported S. aureus genome sequences was searched for possible target sequences. Seven sets of possible target-containing "database" sequences and IS1272-containing GN1/GN3 sequences suggested the presence of a target IR structure and an IR-replacing mode of transposition, as shown in Fig 6A (for IS1272 copies 1, 3, 4, 7-10).
Based on data from a total of 10 IS1272 copies, ni-IS1272 copies P1, C1, and C2 and ce-IS1272 copies 1, 3, 4, and 7-10, the size of the target IR structure ranged between 21 and 38 bp, while that of the IRs ranged between 7 and 13 bp (Fig 7). In contrast, three ce-IS1272 cases (6, 11, and 12) suggested that there were no IR structures as a target, as shown in Fig 6B; the mode of transposition is not clear for these cases. In the remaining three ce-IS1272 cases (copies 2, 5, and 13), the target sequences were not specific or were too big in size in the present database searches.

Database searches for the IS1272 targets and transposition modes that support the IR-replacing mode of transposition
Database searches yielded 19 sets of IS1272 target and transposition sequences, that support the IR-replacing mode of transposition, as shown in S3 Fig. Based on these results, the estimated target IR structure (target terminal IRs) and IS1272 IRs are summarized in Fig 8. The length of IS1272 IRs may be 16 bp (model A), yielding a target IR structure size that ranges between 21 and 85 bp and a target terminal IR size ranging between 5 and 17 bp. These results strongly indicate that the IS1272 transposase recognizes targets as an IR structure (or a stemloop structure), rather than as a specific sequence. Regarding the sequence of IS1272 IRs, there was an IS1272 copy P1 type (the major form shown in Fig 3C), however, further divergence was noted (Fig 8, model A).
in a region downstream of rot (and also downstream of the rRNA methyltransferase gene), targeting a 30-bp sequence with 12-bp IRs and yielding IS1272-C1 (on GN3). In C, IS1272 transposition occurred within the DNA helicase gene, targeting a 22-bp sequence with 9-bp IRs and yielding the larger IS1272 structure (IS1272L) of IS1272-C1 (on GN3); this IS1272L may have occurred by the tandem duplication of the 25-bp left side sequence upon transposition or by transposition of IS1272L. In all the three cases, the target IRs are replaced with IS1272. https://doi.org/10.1371/journal.pone.0187288.g005 Genomic, IS, and virulence analysis of S. aureus strains from a familial infection case Analysis of previous transposition modes for current IS1272 copies. For 13 copies of commonlyexisting IS1272 (ce-IS1272) on the GN1 and GN3 genomes, possible targets of transposition were searched in the database. Color: red, target inverted repeats (IRs); yellow, IS1272 with terminal IRs; blue, the same DNA sequence region between the GN1/GN3 genome and the genome searched in the database. In A, the possible targets were IRs for seven copies of ce-IS1272 (copies 1, 3, 4, 7-10). In B, the possible targets were "non-IRs" for three copies of ce-IS1272 (copies 6,11,12). For the remaining three copies of ce-IS1272 (copies 2, 5, 13), the possible targets were unknown (target sequences not specific or too big in size).

Sizes of the IS1272 tnp genes
The sizes of the IS1272 tnp genes analyzed in the present study are summarized in Fig 9A [33]. The majority of the tnp genes encoded for a 548-aa transposase. However, there were two cases of a premature stop codon, resulting in truncated transposases (S. haemolyticus IS1272 and IS1272-copy 7) and one case of a larger sequence (IS1272-copy 2), as also shown in Fig 3A  and 3B.
The transposase of IS1272-copy 2 was a fusion transposase, which was constructed by isochorismatase (putative) and IS1272 transposase (Fig 9B). There was a link peptide region (PI) between the isochorismatase (putative) domain and IS1272 transposase domain that was also shared by isochorismatase (putative) and IS1272 transposase. The nucleotide sequence corresponding to the link peptide region (PI) was 5'-CCAATA in any case, providing a possible hot spot sequence for the genetic fusion event. The GN1 and GN3 genomes both lacked the isochorismatase (putative) gene, suggesting that the genetic fusion event occurred in other bacterial cells or that the isochorismatase (putative) gene was deleted in GN1 and GN3.

Characteristic distribution of IS1272 on the GN genomes
The location of IS1272 may affect other genes. In the GN genomes, IS1272 was located as follows: copy P1, at a position 66 bp downstream of the PVL F gene, luk pv F; copy 7, at a position 3 bp downstream of scn in IEC/PR; copy C2, at a position 15 bp downstream of rot; and copy 11, at a position 66 bp downstream of blaZ in blaZ-blaR1-blaI (bla-tnp). Additionally, the   , T-ORC_001, SA957, and DAR4145 (they were from the database) and S. haemolyticus was from [33]. The majority of tnp genes encoded for a 548-aa product. However, for example, GN1/3 copy 7 tnp had a premature stop codon, resulting in a truncated product (328 aa); and S. haemolyticus IS1272 tnp had one base deletion, resulting in a frame shift mutation and two smaller ORFs (229 aa and 273 aa), due to one more deletion [31]. Moreover, GN1/3 copy 2 tnp encoded for a larger product (698 aa). (B) This figure shows that the tnp gene of GN1/3 IS1272 copy 2 encodes for a fusion protein, constructed by isochorismatase (putative), shown in red, and GN1 IS1272 copy P1 transposase, shown in blue. There was a link peptide region (PI), shown in black, between the isochorismatase (putative) domain and IS1272 P1 transposase domain; the same link peptide region was IS1272 (copy C1) insertion into the DNA helicase gene resulted in a truncated DNA helicase, due to the introduction of an internal stop codon (TAA), which was present in the IR L2 of IS1272 copy C1 (IS1272L) (Fig 10A). Furthermore, a small (3-kb) deletion was present close to IS1272 (copy 3) on the GN3 genome, but not on the GN1 genome (Fig 10B).
IS1272 (copy 7) was associated with novel genetic structure ICE/PR in the GN1 and GN3 genomes (Fig 10C). The size of IEC/PR was 21,063 bp. IEC/PR was identified as a φSa3 remnant; it corresponded to the end region of φSa3 that carries IEC, it carried φSa3 att (although 5 out of 13 nucleotides were divergent), and it lacked the integrase gene on the other side. Its location on each of the GN1 and GN3 genomes was far from the φSa3 insertion site (att) in hlb. IS1272 may have played a role in the translocation of this remnant.

Detection of the PVL prophage with an IS1272 insertion by PCR
The GN1 genome, but not the GN3 genome, had an IS1272 insertion on a region downstream of the PVL F gene on the PVL prophage, as described above. When the other three GN genomes were analyzed by PCR and sequencing for the presence of this IS1272 insertion, GN4 was negative for the IS1272 insertion, while GN2 and GN5 were positive for it (S1 Fig).

Expression of virulence factors
The PVL production levels of strains GN1 and GN2 were comparative to that of CA-MRSA USA300-0114, used as a control strain, while those of GN3, GN4, and GN5 were two-fold lower (Fig 11). Addition of serum to bacterial culture medium resulted in two fold-higher levels of PVL production in any case (Fig 11).
Regarding mRNA expression (Fig 12), all GN strains except for GN5 expressed the PVL gene at high levels that were similar to (or more than) that of CA-MRSA USA300-0114. Among GN strains, the PVL gene expression levels of GN1 and GN2 were significantly higher than those of GN3 to GN5 (P< 0.05). GN1 to GN5 each expressed the PSMα gene at higher levels than did HA-MRSA Mu50 (P < 0.05), similar to USA300-0114; notably, GN1 showed higher levels of PSMα gene expression than did the remaining GN strains (GN2 to GN5) (P < 0.05). Regarding the Hld gene expression, GN1 to GN5 also expressed higher levels compared with HA-MRSA Mu50 (P < 0.05), similar to USA300-0114, and the Hld gene expression level by GN1 was higher than that by the remaining GN strains (GN2 to GN5) (P < 0.05).

Discussion
In the present study, we elucidated the complete circular genome sequences of GN, a community-associated PVL-positive S. aureus of genotype ST50/spa108(t185)/agr4, because it was associated with strong colonization and skin abscesses in a case that was part of a familial infection. ST50 S. aureus may not be a globally disseminated lineage; however, the GN agr type was the same as that of globally disseminated, hyper-virulent PVL-positive ST121/agr4 MSSA [49,50]. The genome of ST50/spat185 S. aureus was reported previously; this strain (6850) was isolated from a skin abscess case in Belgium, which progressed to further invasive infections also present in both isochorismatase (putative) and IS1272 P1 transposase; the nucleotide sequence corresponding to the link peptide region was 5'-CCAATA. https://doi.org/10.1371/journal.pone.0187288.g009 Genomic, IS, and virulence analysis of S. aureus strains from a familial infection case The GN3 genome, but not GN1 genome, had IS1272 insertion within the DNA helicase gene, yielding newly-inserted IS1272 (ni-IS1272) copy C1. The copy C1 formed a larger IS1272 structure (IS1272L) with a tandem duplication of the 25-bp left side sequence including IR L . Due to the IS1272 C1 insertion, particularly the internal stop codon TAA which was present in IR L2 of IS1272L, the DNA helicase gene product was estimated to be truncated (973-aa) DNA such as bacteremia and osteomyelitis [51] (GenBank accession number, CP006706). Notably, the clinically important and, thus, well-characterized strain 6850 was PVL-negative and lacked phage 2, but it also carried multiple copies of IS1272 (11 copies/genome). Having multiple IS1272 copies may be linked with this lineage. Characteristic virulence factors for CA-MSSA GN include PVL [9,[11][12][13][14][15]17], the stronger expressions of psmα and hld compared with HA-MRSA [12,19], collagen adhesin [52,53], and the immune evasion factors SAK, CHIPS, and SCIN [9,27,45,54].
In 2007 [39], based on the findings obtained by PCR and sequence analyses of PVL gene regions with and without IS1272 (GenBank accession numbers AB256036-39, 2006), we proposed the concept of stem-loop-replacing transposition. Here, to further investigate the IRreplacing transposition of IS1272, we attempted to elucidate the underlying mechanisms in helicase. (B) The GN3 genome had a small (3-kb) deletion close to IS1272 (copy 3); the 3-kb region was present on the GN1 genome and also on the genome of related ST50 MSSA strain 6850. (C) The immune evasion cluster (IEC), carrying sak for staphylokinase (SAK), chp for chemotaxis inhibitory protein of S. aureus (CHIPS), and scn for staphylococcal complement inhibitor (SCIN), was carried by the φSa3 remnant. This structure, IEC/PR, had the att of φSa3; 5 out of 13 nucleotides were divergent (divergent nucleotides are underlined). Its location was not hlb (insertion site of φSa3). GN1 and GN3 lacked φSa3, and hlb was intact. The IEC/PR structure carried one IS1272 copy (copy 7). (D) The blaZ,R1,I-tnpA,B,C structure encodes for penicillin resistance. This structure is flanked by short (6-bp) direct repeats. This structure also carried one IS1272 copy (copy 11).
https://doi.org/10.1371/journal.pone.0187288.g010 Fig 11. Panton-Valentine leukocidin (PVL) production levels of familial strains GN1 to GN5. Bacteria were grown in a liquid medium for 18 h with or without 5% fetal bovine serum (FBS), serial doubling dilutions of the culture supernatants were made, and the amounts of PVL in the supernatants were serologically measured. Bars (color): black, PVL production in the absence of FBS; red, PVL production in the presence of FBS. The PVL production levels of GN1 and GN2 were two-fold higher than those of GN3, GN4, and GN5. Addition of serum to the bacterial culture medium resulted in two fold-higher PVL production levels in any case. USA300-0114 was used as a PVL-positive control strain. https://doi.org/10.1371/journal.pone.0187288.g011 Genomic, IS, and virulence analysis of S. aureus strains from a familial infection case more detail by performing a comprehensive comparison between GN genomes focusing on the status of multiple IS1272 copies, including ni-IS1272 and ce-IS1272.
Based on the results of the present study, together with previous findings reported by others [23,28,55], we proposed a potential transposition mechanism (shown in Fig 13). Basically, in the transposition of transposable elements (TEs) catalyzed by DDE transposase, the first step is the hydrolysis of the phosphodiester backbone at each end of the TE to generate free 3' OH ends [55]. The exposed 3' OH ends are then joined to the target DNA in a trans-esterification reaction. Thus, neither of the 5' ends of the TE are joined with the 3' ends of target DNA. In cases in which the attacked site of the top strand is upstream of the site of the bottom strand, the single-stranded sequences at both sides of the TE need to be repaired. Therefore, if the 3' ends of TE are joined to the target DNA at the 5' foot of the stem-loop, it may result in the duplication of the whole stem-loop (Fig 13, pathway b). If the 3' ends of the TE are joined to the target DNA at the 5' end of the loop, it may result in the duplication of the loop only ( Fig  13, pathway c). However, if the 3' ends of the TE (IS1272 in this case) are joined to the target DNA at the 3' foot of the stem-loop (Fig 13 3A and 13 4A), the stem-loop cannot be replicated unless the opposite strands are cleaved. It is more likely that the two stem-loop sequences become removed by 3' exonucleases, which results in the deletion of stem-loop sequences. We propose that this is a potential transposition mechanism for IS1272, and we have designated this potential mechanism as "replacement by structure-dependent transposition (RST)", "stem-loop replacing transposition", or "cut-paste-and-cut". Additional evidence for this potential mechanism is currently being investigated.
In general, the transposition of ISs or any DDE-type transposons generates TSDs upon transposition [23,28]. The feature of stem-loop replacement in the IS1272 transposition is an irreversible process, in contrast with the "canonical" transposition that can be reversed by recombinational deletions between two target site duplications [23,28,56]. The basis of the differences between "canonical" IS transposition and IS1272 transposition is likely in their transposition mechanisms. Therefore, we may be able to use such differences to control IS1272 without affecting other IS elements or to control other ISs without affecting IS1272. Saturation mutagenesis using IS1272 is one possible direction for assessing the effect of promoting IS1272 transposition.
It is possible that inhibitors capable of blocking the transposition of IS1272 could help overcoming the spread of MRSA. Alternately, the promotion rather than inhibition of IS1272 transposition may help overcome the spread of MRSA. Given that the supercoiling of genomic DNA enhances the cruciform form, intercalates that can enhance supercoiling could be candidate promoters of IS1272 transposition, and antagonizing intercalation may be a way to inhibit the transposition of IS1272. Although the clinical application of these modifications is not  (psmα and hld) and Panton-Valentine leukocidin (PVL) genes (luk PV SF) in familial strains GN1 to GN5. Bacteria were grown on sheep blood agar for 8 h, and the mRNA expression levels were examined by an RT-PCR assay. PVL-positive CA-MRSA USA300-0114 was used as a control strain which shows high expression levels for psmα and hld, and PVL-negative HA-MRSA Mu50 was used as a control strain which shows low expression levels for psmα and hld. In (A), the products in an RT-PCR assay were visualized on 2% agarose gels after electrophoresis. In (B), the expression data of each strain were normalized to those of USA300-0114.  GN2, GN3, GN4, and GN5), P<0.05. For PVL genes: L1 (group of GN1 and GN2) vs. L2 (group of GN3, GN4, and GN5), P<0.05. The LVL mRNA expression level of GN5 was unexpectedly low, compared with that of other familial strains (P<0.05). PVL-positive CA-MRSA strain RS08 and CA-MSSA strain KT1 were also used as strong psmα/hld expression control strains, the data being comparable to that of CA-MRSA USA300-0114 (for psmα, 1.00, 1.09, and 0.92 for USA300-0114, RS08, and KT1, respectively; and for hld, 1.00, 0.85, and 0.79 for USA300-0114, RS08, and KT1, respectively); and HA-MRSA strain N315 was also used as a low psmα/hld expression control strain, the data being comparable to that of HA-MRSA Mu50 (for psmα, 0.45 and 0.38 for Mu50 and N315, respectively; and for hld, 0.41 and 0.30 for Mu50 and N315, respectively). https://doi.org/10.1371/journal.pone.0187288.g012 Genomic, IS, and virulence analysis of S. aureus strains from a familial infection case immediate, the advance in understanding the diversity in transposition mechanisms of ISs and transposons contributes to the future control of bacterial infections.
During the preparation of this manuscript, Furi et al. [57] published a study on the transposition of two composite Tns (TnSha1 and TnSha2), which had IS1272 as a component, and they demonstrated that these two composite Tns removed the stem-loop sequences upon transposition. However, the tnp of composite Tn (TnSha1) has no stop codon, resulting in a larger predicted product, and has three IS1272 IRs in its complex structure, thereby making it difficult to elucidate the precise mechanism underlying IS1272 transposition. Siguier et al. [28] described an IS1182 family that included heterogeneous members (including IS1272), in which some members targeted palindromic sequences, but they did not provide detailed data. Thus, the present study is the first to demonstrate the precise structure, transposition, and model of IS1272.
The cluster analysis of IS1272 from S. aureus, S. epidermidis, and S. haemolyticus (Fig 14) indicated that our IS1272 (GN1) showed high homology to IS1272 from S. aureus MRSA252, but, it exhibited more divergence from the IS1272 sequences of S. epidermidis ATCC12228 and S. haemolyticus Y176 (the original strain in which IS1272 was initially characterized) [33]. IS1272 from S. aureus QBR-102278-1619 is closely related to S. haemolyticus IS1272; however, since IS1272 from S. aureus QBR-102278-1619 is a constituent of a composite Tn carrying S. haemolyticus gene, fabI [57], there is a possibility that it originated in S. haemolyticus.
Although IS1272 was first found and characterized in S. haemolyticus, and is considered to have originally resided in S. haemolyticus [33], IS1272 can also be an important constituent in S. aureus, for example as a multi-IS1272 copy system. We speculate that IS1272 has been evolving through adaptation to S. aureus.
Regarding unique genetic traits, the GN1 and GN3 genome each contained the immune evasion cluster carried by a phage 3 remnant (IEC/PR) for virulence and blaZ,R1,I-tnpA,B,C for drug resistance. IS1272 was a constituent of these genetic structures. GN3, but not GN1, had a small deletion within the genome, relatively close to IS1272; however, the relation of IS1272 to this deletion is not clear. IS1272 may play a role in chromosomal rearrangements in S. aureus.
Regarding the location of IS1272, there were four cases of insertion that occurred very close to genes; in all four cases, IS1272 was inserted immediately downstream of the genes.
IS1272 is a flexible IS, but it also behaves in a strictly regulated manner. Its strict features include 16-bp IRs [33] [this study] and targets of IRs [28,39,57]  Among the five GN familial strains, the patient strain (GN1) manifested the highest levels of PVL production and of psmα and hld expression. The importance of PSMα in SSTIs has been increasingly reported [58,59]. Although GN5 had a PVL prophage with an IS1272 insertion, the GN5 PVL production level was comparable to those of GN3 or GN4, whose PVL prophages did not have an IS1272 insertion. Interestingly, the GN5 PVL mRNA expression level was low compared with that of other GN strains. The background mechanism for the high virulence potential of GN1 needs to be elucidated.
In conclusion, this study is the first to present evidence for a novel IR-replacing mode of transposition and sequence data that suggests a potential stem-loop-replacing transposition Genomic, IS, and virulence analysis of S. aureus strains from a familial infection case mechanism for IS1272. We performed a comprehensive comparison of the whole-genomic sequences of familial strains of one S. aureus clone (GN) that was isolated from a five-person familial infection case. Notably, the IS1272 transposition appeared to have occurred via an irreversible process, unlike that of the reversible "canonical" transposition of IR and Tn. IS1272 sequences existed as a multi-IS1272 system in the S. aureus genome, accompanying strictly regulated but also flexible structures. Basic investigation of the IS1272 mechanism may lead to a new method of MRSA control in the future. IS1272 was linked to IEC and drug resistance segments and was located both near a deletion and close to several genes, suggesting its possible role in chromosomal rearrangements. Although IS1272 was originally isolated and characterized in S. haemolyticus, it plays a role in clinically important S. aureus. The present study also demonstrates that the patient strain in this familial infection case had an increased virulence potential based on community-associated virulence factors.
Supporting information S1 Fig. PCR detection of IS1272 insertions on the Panton-Valentine leukocidin (PVL)-prophage using GN familial strains. The applied familial strains were GN1 (patient strain) and GN2 to GN5. In the course of the previous study on the PVL S/F genes (luk PV SF), we determined the sequences of not only the entire PVL gene but also its upstream and downstream regions of clinical S. aureus isolates by PCR and sequencing, and detected an IS1272 transposition in a region distal of the PVL F gene. PCR primers, PVL-1 and NPVL-2, were from reference [44]. Other PCR primers were designed based on the DNA sequence of a PVL prophage carried by ST30 CA-MRSA strain NN1 [37] and φPVL-Sa2 GN1 and φPVL-Sa2 GN3 . The primers for IS1272 were designed based on the φPVL-Sa2 GN1 sequence, which had an IS1272 insertion. Of three primer sets (A to C), B detects the terminal IRs of the target of IS1272; thus, GN1, GN2, and GN5 (in which the target was replaced by IS1272) produced negative results in PCR using primer set B. Six PCR primer sets (1 to 6) were used to investigate the presence of an insertion at the region located downstream of the PVL genes. The insertion of IS1272 (size, ca. 2 kb) was checked by PCR product sizes and PCR product sequences. GN1, GN2, and GN5 had a ca. There was no specific funding during this study and any authors did not receive a salary for this specific study.