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J Bacteriol. 2007 March; 189(5): 2086–2100.
Published online 2006 December 15. doi:  10.1128/JB.01637-06
PMCID: PMC1855768

First Complete Genome Sequence of Two Staphylococcus epidermidis Bacteriophages[down-pointing small open triangle]


Staphylococcus epidermidis is an important opportunistic pathogen causing nosocomial infections and is often associated with infections in patients with implanted prosthetic devices. A number of virulence determinants have been identified in S. epidermidis, which are typically acquired through horizontal gene transfer. Due to the high recombination potential, bacteriophages play an important role in these transfer events. Knowledge of phage genome sequences provides insights into phage-host biology and evolution. We present the complete genome sequence and a molecular characterization of two S. epidermidis phages, [var phi]PH15 (PH15) and [var phi]CNPH82 (CNPH82). Both phages belonged to the Siphoviridae family and produced stable lysogens. The PH15 and CNPH82 genomes displayed high sequence homology; however, our analyses also revealed important functional differences. The PH15 genome contained two introns, and in vivo splicing of phage mRNAs was demonstrated for both introns. Secondary structures for both introns were also predicted and showed high similarity to those of Streptococcus thermophilus phage 2972 introns. An additional finding was differential superinfection inhibition between the two phages that corresponded with differences in nucleotide sequence and overall gene content within the lysogeny module. We conducted phylogenetic analyses on all known Siphoviridae, which showed PH15 and CNPH82 clustering with Staphylococcus aureus, creating a novel clade within the S. aureus group and providing a higher overall resolution of the siphophage branch of the phage proteomic tree than previous studies. Until now, no S. epidermidis phage genome sequences have been reported in the literature, and thus this study represents the first complete genomic and molecular description of two S. epidermidis phages.

Staphylococcus epidermidis, a member of the novobiocin-susceptible coagulase-negative staphylococci, is an important opportunistic pathogen and a major cause of nosocomial infections (42, 55). S. epidermidis predominantly colonizes the mucous membranes, groin, and axillar areas, as well as the cutaneous system of human body, with bacterial counts of up to 10 to 103 CFU/cm2 (26). S. epidermidis is usually considered a harmless commensal microorganism; however, infections can occur in immunocompromised individuals and in patients with indwelling or implanted medical devices such as prosthetic heart valves and joint prostheses, where the staphylococci penetrate cutaneous and mucosal barriers (79). With the increasing use of such devices in medical practice, several million people are affected by complications arising from S. epidermidis infections (49). The widespread use of various antimicrobial agents, including penicillins, macrolides, aminoglycosides, and semisynthetic penicillins such as methicillin, has now led to the emergence of multiple-drug-resistant S. epidermidis strains (21, 72).

Two S. epidermidis genomes have been sequenced: those of the non-biofilm-forming, non-infection-associated strain ATCC 12228 and the infectious biofilm-forming strain RP62A (ATCC 35984) (21, 80). The difference between commensalism and pathogenicity was attributed to various factors: (i) single-nucleotide polymorphisms in pathogenicity-related genes (75), (ii) differential expression of genes contributing to S. epidermidis virulence (78), and (iii) acquisition of novel virulence factors and a high recombination and lateral gene transfer potential. These factors contributed toward the evolution of S. epidermidis from a commensal pathogen to a more aggressive opportunistic pathogen (21). Comparative genomic studies between staphylococcal species indicated that the majority of novel and unique genes could be accounted for by the presence or absence of prophages and genomic islands (21).

Bacteriophages are among the most abundant inhabitants of the biosphere, considering that an environmental sample contains nearly 10-fold more phage particles than prokaryotes (9). The bacteriophage population is estimated to be on the order of ≥1031, comprising approximately 108 distinct species (65, 77). The phage population is very dynamic, with rapid population turnover occurring within a relatively short period of time (7). They represent a rich and unique source of genetic and protein diversity, since less than 0.0002% of the global phage metagenome has been sampled (65) and a majority of genes have no assigned functions or matches in GenBank (6, 62). Bacteriophages also confer novel biological and physiological traits allowing host strains to adapt to new environments or obtain virulence determinants, thereby driving bacterial speciation and adaptation. The role of bacteriophages in bacterial biology, evolution, and diversity is now being truly appreciated due to concerted efforts toward characterizing bacteriophage genomes and analyzing their overall impact on global genetic and proteomic diversity, ecology, and recycling of organic matter (76).

Complete genomic sequences and molecular characterization of several bacteriophages infecting important pathogenic bacteria, such as Bacillus anthracis (69), Pseudomonas aeruginosa (34), Mycobacterium tuberculosis (62), and Staphylococcus aureus (35), have been reported in recent years. This wealth of information has provided researchers a better understanding of the dynamics of phage-host interactions, mechanisms of virus evolution, acquisition of bacterial virulence determinants, and ecological and evolutionary changes of their hosts. Bacteriophages infecting Staphylococcus epidermidis have also been isolated (63) and are typically used for typing S. epidermidis strains (24, 73). However, to our knowledge, no S. epidermidis phage sequences have been reported in the literature.

In this study, we determined the ultrastructures and the complete genome sequences of two S. epidermidis phages, [var phi]PH15 (PH15) and [var phi]CNPH82 (CNPH82). The two genomes are highly similar and display typical modular organization seen in phages. Differential superinfection inhibition corresponded with nucleotide sequence differences and overall gene content within the lysogeny module. Detailed sequence analysis suggested the presence of two introns in PH15 which are spliced in vivo. We also conducted comparative phylogenetic analyses of the two phages with other sequenced phages of the family Siphoviridae reported in GenBank.


Bacterial strains and phages.

Staphylococcus epidermidis 414 (Felix d'Herelle Reference Center for Bacterial Viruses), referred as HER 1292 in this study, as well as phages PH15 and CNPH82 were donated by the Health Protection Agency (United Kingdom) and obtained from the Centers for Disease Control and Prevention, Atlanta, GA. HER 1292 was grown in Trypticase soy broth (TSB) (Difco) at 37°C in a shaking incubator at 250 rpm. Phages were propagated on strain HER 1292 in TSB supplemented with 4 mM CaCl2 and 3 mM MgCl2.

Preparation of phage lysates.

PH15 and CNPH82 phage lysates were prepared by incubating phage stock with early-log-phase HER 1292 cells until complete lysis occurred (approximately 50 min). The lysates were cleared by centrifugation at 10,000 × g for 10 min, sterile filtered, and stored at 4°C. Phage titers were determined as described previously, using soft-agar overlay assays (68). The typical phage titers obtained by this method were in the range of 108 to 109 PFU/ml.

Electron microscopy of phages.

Phages were pelleted by centrifugation at 100,000 × g for 1 h, washed twice with 100 mM ammonium acetate, and resuspended in 4 mM CaCl2. The sample was adsorbed onto a carbon grid, which was floated on a small drop of 2% Na-phosphotungstate, pH 7.0, for 1 min and then blotted off. The sample was air dried and examined in a JEOL 100CX2 transmission electron microscope.

Production of HER 1292/PH15 and HER 1292/CNPH82 lysogens.

Serial dilutions of PH15 and CNPH82 were spotted on HER 1292 lawns and incubated overnight at 37°C. Bacterial colonies growing within the plaques were harvested and passaged for several generations on TSB agar plates. After each passage, 25 PH15 lysogens and 20 CNPH82 lysogens were tested by colony PCR using the PH15-specific helicase primer set and the CNPH82-specific portal protein primer set for the presence of phage (primer sequences are available on request). Additionally, induction of PH15 and CNPH82 was determined by mitomycin C (5 μg/ml) treatment of the lysogenized HER 1292 cells followed by soft-agar overlays.

Preparation of phage DNA.

Phage DNA was prepared from the lysates after treatment with RNase and DNase (10 μg each) at 37°C for 30 min. Phage were precipitated by adding NaCl (0.5 M final concentration) and polyethylene glycol 8000 (10%, wt/vol) and incubating on ice for 30 min. Phage were pelleted by ultracentrifugation at 100,000 × g for 1 h, resuspended in 10 mM Tris-Cl (pH 7.5) plus 50 mM MgCl2, and lysed by adding sodium dodecyl sulfate (0.5% final concentration) and 20 mM EDTA. DNA was purified by phenol-chloroform extraction as previously described (68).

Sequencing of phage DNA.

Complete genome sequencing of both phages was done by Macrogen Inc., Seoul, Korea. Briefly, phage genomic DNA was sheared using a nebulizer (Invitrogen, CA), and blunt-end repaired and dephosphorylated. DNA fragments of desired size (1 to 6 kb) were blunt-end ligated into pCR4Blunt-TOPO vector (Invitrogen, CA) and electroporated into Escherichia coli DH10B cells according to the manufacturer's instructions. Clones were sequenced until >10-fold redundancy was obtained. The complete genomes were assembled using the SeqMan II (DNASTAR, Inc.) sequence analysis software.

DNA sequence and bioinformatics analyses.

Open reading frames (ORFs) in the final genome sequences were predicted using GeneMark.hmm for prokaryotes ( (47) and ORF Finder ( software. In our analyses, putative ORFs contained either AUG (methionine), UUG (leucine), or GUG (valine) as the starting codon, preceded by a Shine-Dalgarno sequence optimally placed (3 to 12 bp) upstream of the start codon and containing sequence encoding at least 50 amino acids before the termination codon. ORF numbers are preceded by either ph or cn designation for phage PH15 or CNPH82, respectively (Tables (Tables11 and and2).2). The predicted proteins were searched against the protein database by using BLAST (1) and PSI-BLAST (2) algorithms. Structural predictions and motif searches of proteins were performed with the proteomic tools at ExPASy ( and Pfam ( as well as the software COILS (48), and PSORT-B (48). Transmembrane domains were predicted with the TMHMM version 2.0 program ( (71).

Genome organization of PH15
Genome organization of CNPH82

Reverse transcription-PCR (RT-PCR).

HER 1292 cells were infected at a multiplicity of infection of 5, and the corresponding phage and RNA were isolated at 15 and 25 min postinfection (p.i.). RNA was isolated using the RNeasy kit (QIAGEN Sciences, MD) according to the manufacturer's instructions. Contaminating DNA was removed with the TURBO DNA-free kit (Ambion Inc., TX). First-strand cDNA synthesis was performed using the Superscript III first-strand Synthesis kit (Invitrogen, CA) with random hexamers according to the manufacturer's instructions. PCR was conducted with 2 μl each of cDNA and PH15 and CNPH82 large terminase subunit- and lysin-specific primers (sequences are available on request). The PCR conditions were as follows: 94°C for 3 min, 94°C for 30 s, 55 to 57°C for 30 s, and 72°C for 1 min 30 s, for 35 cycles. PCR products were separated on 1% agarose gels.

Phylogenetic analysis.

The DOTTER program (70) was used to compare the nucleotide sequences of PH15 and CNPH82 with 35 S. aureus phage sequences present in the GenBank database, with a sliding window of 25 bp.

Proteomic distances were calculated using an approach similar to that described by Rohwer and Edwards (66). Predicted protein sequences for PH15 and CNPH82 were compared to all known siphophage protein sequences in GenBank (see Table S1A in the supplemental material for accession numbers). Protein distance scores were estimated using the same methods and parameters as described by Rohwer and Edwards (66). Like in that study, we considered protein distance scores (SPD) significant if −1 < SPD < 5, and we applied a penalty of 5 for pairwise protein comparisons that did not have a significant score. Proteomic distance was calculated using the formula {[Σ(significant SPD) × 100] + (penalty × number of nonhits)}/[(number of hits × 100) + number of nonhits], where a “hit” is a pairwise comparison that had a significant SPD and a “nonhit” is a pairwise comparison that did not have a significant SPD. We did not use a length correction, as this caused a small number of phages to cluster in inappropriate places. For example, when using length, two members of Mycoplasma were drawn into the outer edge of the Staphylococcus branch. These phages clustered completely separately from Staphylococcus when length was not included in the proteomic distance formula. Because most SPD ranged from 0.1 to 5, we applied a 100× amplification in the calculation of the proteomic distance score. This amplification of scores facilitated tree generation and resulted in a tree that was less dense. It did not affect relationships between genomes.

An unrooted phylogenetic tree was generated using FITCH (22) with the default parameters plus the additional options of randomizing the input order of sequences and global rearrangement optimization. The representative tree was produced using the MEGA 3.1 software package (33).

Nucleotide sequence accession numbers.

The genome sequences of PH15 and CNPH82 were submitted to GenBank and were assigned accession numbers DQ834250 and DQ831957, respectively.


We report the complete annotated genome sequence of two temperate S. epidermidis phages, PH15 and CNPH82. These phages, along with several other phages such as [var phi]PH48, [var phi]PH456, and [var phi]PH459, were obtained as a gift from the CDC (17) and have been previously used for S. epidermidis phage typing. To our knowledge, our study is the first report of genomic sequences of S. epidermidis phages with characterization at the molecular level.

Phage ultrastructure and host range studies.

PH15 and CNPH82 were selected for further analyses because EcoRI and BamHI restriction profiles showed substantive sequence variation between these two phages (data not shown). Purified PH15 and CNPH82 particles observed by electron microscopy revealed that the two phages had similarly sized small icosahedral heads (diameter of approximately 55 nm) and long flexible noncontractile tails (length of approximately 160 nm) with dual disc baseplates (Fig. 1B and E), suggesting that they both belong in the Siphoviridae family within the order Caudovirales. A small collar-like structure between the head and tail was visible in phage particles, particularly where the tail had separated from the head (Fig. 1B and E). Similar collar-like structures have been observed in Lactococcus lactis phage TP901-1 (27). The phage particles appeared to aggregate and form a “bouquet-like” arrangement, possibly due to tail fiber adherence to wall fragments (Fig. 1A and D), similar to those observed in Bacillus anthracis γ phage (69).

FIG. 1.
Ultrastructures of phages PH15 (A to C) and CNPH82 (D to F). (A and D) Transmission electron micrographs of phages PH15 and CNPH82, showing their isometric heads and noncontractile tails. (B and E) Tails of PH15 and CNPH82 detached from the virion head, ...

Host range analysis of PH15 and CNPH82 revealed that both phages formed plaques within infected bacterial lawns of HER 1292, while no plaques were observed on lawns of S. epidermidis strains ATCC 12228 or RP62A. Additionally, no plaques were observed on 10 S. aureus strains tested, including NCTC 8325 (58) and RN4220 (31).

Both PH15 and CNPH82 were able to form stable lysogens in HER 1292, suggesting the presence of a functional lysogeny module in both phages. Phages could be induced from the respective lysogens by mitomycin C induction, and the induced phages formed plaques on HER 1292 lawns. The lysogens did not show any differences in colony morphology or growth curve characteristics in liquid media compared to the host (data not shown). The lysogens were tested for superinfection by infecting PH15-lysogenized HER 1292 and CNPH82-lysogenized HER 1292 with PH15 and CNPH82. PH15 formed plaques on CNPH82-lysogenized HER 1292 lawns. The efficiency was similar to that on HER 1292 lawns. CNPH82 was unable to form plaques on CNPH82-lysogenized HER 1292 (data not shown). Neither PH15 nor CNPH82 was able to form plaques on a PH15-lysogenized HER 1292 lawn.

The results of superinfection studies suggest that the PH15-lysogenized host exhibits superinfection immunity whereby the integrated PH15 prophage prevents infection by a homoimmune phage (PH15 itself and CNPH82). However, the ability of PH15 to infect CNPH82-lysogenized strain could be attributed to differences in the genomic organization within the lysogeny control module (see below). Such differences have been shown to be important in Streptococcus thermophilus phage Sfi19 for eliminating Sfi21 prophage control of superinfection immunity (46). We conclude that the genomic differences observed in the lysogeny module of PH15 may eliminate superinfection immunity by CNPH82 prophage.

Genome features of PH15 and CNPH82.

The complete genome sequences of PH15 and CNPH82 were determined. The PH15 genome comprised 44,047 bp containing 68 putative ORFs, while the CNPH82 genome comprised 43,420 bp containing 65 putative ORFs (Fig. (Fig.2).2). The G+C contents of PH15 and CNPH82 were 34.91% and 34.67%, respectively, which are comparable to that of the S. epidermidis genome (80).

FIG. 2.
Schematic representation of CNPH82 and PH15 genomes, showing the genome organization, predicted ORFs and some putative functions. The ORFs are depicted by arrows or arrowheads pointing in the direction of transcription and are numbered consecutively (see ...

Restriction analysis with endonucleases that have one cut site within the phage genomes (XhoI and SacI for PH15 and ApaLI and MscI for CNPH82) produced a single band on agarose gels, while double digestion produced two fragments (data not shown). These results suggested that both the PH15 and CNPH82 genomes were circularly permuted, since circularly permuted (and terminally redundant) linear phage chromosomes behave as do circular chromosomes during restriction analysis (5). We also observed submolar fragments of both PH15 and CNPH82 DNAs after heating the restriction endonuclease digestion mixture (data not shown), suggesting that both phages belonged to the pac-type group. In this group of phages, which typically have circularly permuted linear chromosomes, packaging of concatemeric phage DNA is initiated processively in a headful manner after the terminase cuts the DNA at a pac site (5).

Bioinformatic analyses revealed that the gene coding potentials for PH15 and CNPH82 were 91% and 94%, respectively, indicating tight packing and few intergenic regions, with approximately 1.5 genes/kbp of nucleotide sequence. The majority of ORFs initiated translation from an AUG start codon in both genomes, except for five ORFs in PH15 (ph15, ph49, ph55, ph56, and ph57) and eight ORFs in CNPH82 (cn3, cn6, cn10, cn19, cn46, cn52, cn54, and cn55) that initiated with UUG start codon and three ORFs (ph34, ph37, and ph42) in PH15 and five ORFs (cn26, cn32, cn37, cn59, and cn61) in CNPH82 that initiated with GUG start codon (Tables (Tables11 and and2).2). Both genomes were annotated using comparisons to current databases. However, biological function could be assigned to only 43% of the PH15 proteome and only 41% of the CNPH82 proteome. Both genomes displayed organizations similar to those of the phages of the Siphoviridae family (8). The genomes were modularly organized and consisted of DNA packaging, head-and-tail morphogenesis, host cell lysis, lysogeny, and DNA replication and modification modules (Fig. (Fig.22).

PH15 and CNPH82 genome comparison.

Database searches with the putative ORFs of PH15 and CNPH82 revealed that the gene orders were similar for the two genomes, and the proteins encoded by the two genomes showed one-to-one correspondence (Fig. (Fig.2).2). Pairwise nucleotide comparison revealed that the two genomes displayed similarity at the nucleotide level, with ~60% identity genome-wide and ~85% identity over the regions corresponding to the head-and-tail morphogenesis module and between the tail morphogenesis and lysis modules (Fig. (Fig.3).3). The interruption of the straight line seen within the head morphogenesis module corresponded to the swapping of ORF ph7 (Table (Table2)2) with ORF cn8. A straight line was also observed in the DNA packaging region, except for a lateral shift marked by the presence of an intron (terL-I) (see below) within the putative large terminase subunit (Fig. (Fig.3).3). Another lateral shift of the straight line in the otherwise highly similar lysis module corresponded to the lys-I intron (see below). A gap corresponding to ORFs ph25/26 and ORFs cn26/27 was also observed. The lysogeny module did not show nucleotide similarity, which corresponded with the presence of numerous unique ORFs in the PH15 genome (Tables (Tables11 and and2;2; Fig. Fig.3).3). Likewise, there was a lack of similarity in the DNA replication module due to the presence of numerous unique ORFs in the two genomes (Fig. (Fig.33).

FIG. 3.
Dot plot comparison of PH15 and CNPH82 genomes. Dot plot analysis was conducted for the genomic DNA sequences of PH15 (x axis) and CNPH82 (y axis) by using the DOTTER program (70) with a sliding window of 25 bp. The color-coded schematic genome maps of ...

DNA-packaging and morphogenesis modules.

BLAST and Pfam searches suggested that ORFs ph1 and cn1 encoded putative small terminase subunits. Sequence analysis indicated that the putative large terminase subunit of PH15 (and not that of CNPH82) was interrupted by an intron. An ORF (ph3) was also predicted within this intron. The DNA segment encoding the remainder of a large terminase subunit was downstream of the intron, which was spliced in vivo (see below). Thus, ORF ph2 was subdivided into ph2a and ph2b, with ph3 between them (Table (Table2;2; Fig. Fig.2).2). ORF cn2 also encoded a putative large terminase subunit but was not interrupted by an intron. Terminases are enzymes involved in phage DNA packaging into an empty capsid (13). These enzymes function as heteromultimers, with the small subunit involved in DNA binding and the large subunit, with its associated ATPase activity, involved in DNA packaging (19, 67). The highly conserved GKT/S Walker box (P loop) in the N termini of phage terminases (54) was predicted in both Ph2 and Cn2.

The overall organization of genes encoding the structural module in PH15 displayed similarity to that of S. aureus phage 37, while the CNPH82 genome showed similarity to that of S. aureus phage 52A (35). ORFs ph4 and cn3 were 99% identical and showed homology to Bacillus subtilis phage SPP1 Gp6-like putative portal protein (18). Portal proteins enable DNA passage into phage heads during packaging by forming a 12-fold-symmetrical ring (40, 74). ORFs ph5 and cn4 showed homology to the phage Mu protein F-like putative minor head protein.

The organization of genes in the CNPH82 genome downstream of cn4 differed from that in the PH15 genome in that two CNPH82 ORFs, cn5 and cn6, were unique to the CNPH82 genome (Fig. (Fig.2).2). These ORFs encoded proteins of unknown function. Similarity searches indicated that whereas Cn6 displayed 38% to 54% identity (over 27 to 37 amino acids) with ORF products of S. aureus phages of clade IIC, such as phages 96, 55, 29, and 52A, Cn5 displayed 72% identity (24/33) only with ORF147 of S. aureus phage EW (35). ORFs ph6 and cn7 encoded a putative phage minor capsid protein. ORF ph7 was predicted to encode a putative major capsid protein (MCP). No MCP was predicted from the CNPH82 genome. The PH15 MCP, which shares high sequence identity with the S. aureus phage phiETA MCP, falls within the group of HK97-like MCPs due to the presence of nine strictly conserved amino acid residues (25). Neither a prohead protease nor a scaffolding protein was predicted in the genome, suggesting that the assembly of capsid differs from that in lambda-like phages. In lambda-like phages, a viral protease is involved in the processing of the prohead protein (25).

ORFs ph16 and cn17 were identified as putative tape measure proteins (TMPs). The TMP is one of the largest proteins in the phage genome and is found in almost all Siphoviridae phages (62). Phylogenetic analyses have revealed that phage TMPs frequently contain a soluble lytic transglycosylase domain (66), and lytic transglycosylases have been proposed to be involved in phage DNA entry during early stages of infection (30, 39). However, the soluble lytic transglycosylase domain was not predicted in Ph16 and Cn17, suggesting that these phages utilize a different mechanism to deliver phage DNA during the early infection stages.

In phage λ, the length of the phage tail is directly proportional to the size of the TMP, where one amino acid of the TMP equals 0.15 nm of tail length (28, 61). Using this equation, the length of the tails in PH15 and CNPH82 corresponds to 155 nm (TMP, 1,034 amino acids), which correlates well with our estimation from the electron micrographs. Similar calculations of tail length based on TMP size have also been made for phages that are unrelated to λ (11, 44, 62, 81, 82).

Lysis module.

The lysis module was located after the structural module and included ORFs ph24a and ph24b, ph27, and ph28 in PH15 and cn25, cn29, and cn30 in CNPH82 (Fig. (Fig.2).2). The holin-endolysin dual-lysis system responsible for cell lysis and phage progeny release in double-stranded DNA phages was present in both phages. The putative holins of both phages (Ph27 and Cn29) were composed of 90 amino acids, displayed 100% sequence identity with each other, and contained two transmembrane regions. The stop codons of ORFs ph27 and cn29 overlapped the corresponding downstream ORFs by 1 bp in a different reading frame. A similar organization is seen in the S. aureus phage K (60).

The endolysins encoded by ORFs ph28 and cn30 shared 99% sequence identity with each other and contained a CHAP domain (residues 14 through 144) (3) as well as an N-acetylmuramoyl-l-alanine amidase domain (residues 170 through 350). Both PH15 and CNPH82 encoded an additional peptidoglycan hydrolase (amidase) (Ph24a and -24b and Cn25, respectively) which contained an N-terminal CHAP domain as well as a C-terminal endo-β-N-acetyl-glucosaminidase domain. This amidase exhibited 73% identity with the amidase from S. aureus phage EW (35) and 65% identity with Ply187 from S. aureus phage 187 (43). ORF ph24 (product of ORFs ph24a and ph24b) contained an intron which is spliced in vivo (see below). The arrangement of genes in the lysis module of CNPH82 resembled that of the clade IIC of S. aureus phages, with a putative tail fiber protein (Cn26) immediately downstream of amidase Cn25 (35). RT-PCR analysis revealed that while both endolysins were expressed during the PH15 infection cycle, only the endolysin encoded by ORF cn30 was expressed during the CNPH82 infection cycle (Fig. (Fig.44).

FIG. 4.
In vivo splicing of intron DNA from the PH15 lys gene. (A) RT-PCR was conducted on RNA isolated from PH15- or CNPH82-infected host strain HER 1292 with primer pairs Lys-F/Ph24b-Lys-R (lanes 1 to 5) and Lys-F/Cn25-Lys-R (lanes 6 to 10) (Table ...

Lysogeny module.

The overall genetic organization of the lysogeny modules of both phages was similar to that of other Siphoviridae infecting low-GC-content gram-positive bacteria. In these phages, the lysogeny-related genes are compactly organized, unlike the case for Siphoviridae infecting high-GC-content gram-positive bacteria, where the lysogeny-related genes are more loosely organized and interspersed by many ORFs (45). The PH15 lysogeny module had more unique ORFs (orf32, orf33, orf37, and orf38) than CNPH82, while CNPH82 had only two unique ORFs (orf33 and orf37). These differences could be accounted for by gene transfer among S. aureus phages (35) or DNA rearrangements/recombinational events similar to those observed in S. thermophilus phages, where the lysogeny module is shown to be a recombination hot-spot (45).

The nucleotide sequences of ORFs ph31 and cn32 were nearly identical and were predicted to encode site-specific phage recombinases (int). Preliminary experiments suggested that unlike those in other phages (23, 41, 82), the attPP′ site might not be located close to the int gene (unpublished data). An excisionase-encoding gene (xis) was not identified in either the PH15 or CNPH82 genome, similar to the case for other phages where the xis gene is absent but an int gene is predicted (23, 32, 82).

ORFs ph35 and cn35 did not share sequence similarity; however, both ORFs showed homology to putative cI-like repressor protein involved in suppression of the phage lytic cycle. Likewise, ORFs ph36 and cn36 did not share sequence similarity, but were homologous to the putative Cro-like repressor protein of the HTH-XRE family, which is required for the lytic cycle. However, ORFs ph39 and cn38 shared sequence similarity and were predicted to encode putative antirepressors.

Two adjacent and outward-facing putative promoters for the putative cI- and cro-like repressor genes were identified in both phages. Additionally, a 19-bp overlapping direct repeat between the two putative promoters of cn35 and cn36 was also identified. Sequence repeats within the promoter region have been reported to be present in L. lactis phage r1t (56) as well as Listeria monocytogenes phage A118 (44). These repeats have been shown to regulate lysogeny module gene expression in phage r1t (56). The order and orientation of the cI- and cro-like repressor genes with corresponding outward facing promoters are seen in other phages, including Lactobacillus phage [var phi]g1e (29), S. thermophilus phages TPJ34 and Sfi21 (10, 57), L. monocytogenes phage A118 (44), and few S. aureus phages (35). The Cro-like repressor could play a role during the lytic cycle in lysogenized hosts, as suggested by Lucchini et al. (45).

An interesting biochemical property of the PH15 cI-like repressor is that the predicted isoelectric point (pI) is 8.3 (basic protein), unlike the CNPH82 cI-like repressor (pI 5.5) and other λ-like cI repressors that are acidic. Therefore, assignment of a possible function on the basis of non-sequence-alignment-based homology parameters as suggested by Chandry et al. (15) cannot be applied for the PH15 cI-like repressor.

DNA replication and metabolism module.

Both PH15 and CNPH82 contained ORFs encoding proteins involved in DNA replication and metabolism (Tables (Tables11 and and2).2). ORF ph54 displayed homology to a Holliday junction resolvase (RusA) homolog, while ORF cn57 displayed homology to a staphylococcal nuclease homolog. A 13-bp direct repeat was present in cn45. ORFs ph51 and cn47 showed homology to the DnaB_C family of helicases and also contained the ATP/GTP-binding P loop. A putative dUTPase gene (ORFs ph62 and cn60) was predicted in both genomes. The ORFs had 99% sequence identity and displayed high homology with dUTPase genes of several staphylococcal and lactococcal phages. Several regulatory proteins were also identified in both genomes. ORFs ph65 and cn62 displayed similarity to the RinB family of transcriptional regulators, while ph68 and cn65 displayed similarity to the RinA family of transcriptional regulators.

PH15 has two group I introns.

Two introns interrupting genes with crucial enzymatic functions were identified in the PH15 genome. The first intron, referred to as terL-I, was present within the sequence for the putative large terminase subunit (TerL)-encoding ORFs ph2a and ph2b. The terL-I intron harbored a putative endonuclease-encoding ORF ph3, which belonged to the GIY-YIG family of class I homing endonucleases typically found in introns (4). The second intron, referred to as lys-I, was present between the putative endolysin (Lys)-encoding ORFs ph24a and ph24b. A similar example of introns interrupting terL and lys genes within the same genome has been reported for Streptococcus thermophilus phage 2972 (41). Additionally, interrupted lys genes have been reported to occur in S. thermophilus and the S. aureus phage K genomes (20, 59). Also, an intron interrupting the terL gene has been characterized for Lactobacillus delbrueckii phage LL-H (52), L. delbrueckii phage JCL1032 (64), and Lactobacillus plantarum phage LP65 (16).

The presence of introns in crucial genes of PH15 confirms a common theme seen in many phage genomes, i.e., that phage introns target essential function genes, unlike eubacterial introns that are present in tRNA genes. The introns target conserved regions within these genes (lys-I targets the CHAP domain, and terL-I targets the nuclease motif) (59). This is particularly true for lysin genes, which contain a defined homing sequence (20). However, unlike the streptococcal intronless lysin genes that have a conserved 5′ATTT3′ sequence immediately upstream of the intron insertion site (20), both the intronless CNPH82 lysin gene (cn25) and the intron-containing PH15 lysin gene (ph24) have the sequence 5′GTGT3′, suggesting that intron homing in staphylococcal lysin genes may depend on a different homing sequence(s).

In vivo splicing in the lys RNA transcripts was tested by RT-PCR using cDNA prepared from HER 1292 RNA. Primers were designed based on identical sequences in lys-containing ORFs ph24a/ph24b and cn25 (primer sequences are available on request). Primers specific for the PH15 helicase gene (ph51) and the CNPH82 portal protein gene (cn3) were designed as positive controls. PH15 and CNPH82 genomic DNAs were also included in the analysis. A 2.1-kb PCR product was amplified from PH15 genomic DNA, while a ~1.9-kb RT-PCR product was amplified from PH15-infected HER 1292 cDNA (Fig. (Fig.4A),4A), indicating that a 246-bp intron was excised from the RNA precursor. In contrast, both CNPH82 genomic DNA and CNPH82-infected HER 1292 cDNA produced a 1.9-kb product. The PCR and RT-PCR products of the PH15 helicase and CNPH82 portal protein, included as positive controls, showed identical 1.2-kb bands (Fig. 4B and C).

RT-PCR analysis was also conducted to test in vivo splicing in the terL RNA transcripts by using cDNA prepared as described above. Primers recognizing identical sequences in both the PH15 and CNPH82 genomes that were located in terL-carrying ORFs ph2a/ph2b and cn2 were designed. As a control, PCR was conducted using the same primer set with PH15 and CNPH82 genomic DNAs. A 750-bp RT-PCR product was amplified for PH15-infected HER 1292 cDNA, in contrast to the 1,720-bp PCR product amplified from the PH15 genomic DNA (Fig. (Fig.5),5), suggesting that a 971-bp intron was excised from the RNA precursor. The spliced terL mRNA was 1,263 bp long. In contrast, a 750-bp product was obtained when PCR was conducted with CNPH82 genomic DNA and CNPH82-infected HER 1292 cDNA (Fig. (Fig.55).

FIG. 5.
In vivo splicing of intron DNA from the PH15 terL gene. RT-PCR was conducted on RNA isolated from PH15- or CNPH82-infected host strain HER 1292 with a terL-specific primer pair. The template used for the PCR was as follows: lane 1, PH15 genomic DNA; lane ...

Secondary structure of the introns.

The RT-PCR product was sequenced to determine the exact exon-intron boundary. As is the case with other group I introns, splicing of lys-I occurred after uridine (coordinate 21315, within ph24a) and guanosine (coordinate 21561, upstream of ph24b) residues. No new codon was generated, while the reading frame was maintained with a tryptophan residue following the conserved cysteine residue of the CHAP domain (Fig. (Fig.6,6, left panel). Similar to the case for lys-I, splicing of terL-I occurred after uridine (coordinate 1518, within ph2a) and guanosine (coordinate 2488, 2 bp upstream of ph2b) residues. A new codon, UAC rather than UAU, with the conserved coding potential for tyrosine was created at the splice junction (Fig. (Fig.6,6, right panel). The absence of an intron in CNPH82 could be due to sequence variation between PH15 and CNPH82 near the integration site, similar to that seen in S. thermophilus phages (20, 41).

FIG. 6.
Secondary structure predictions for lys-I (left) and terL-I (right) introns in the PH15 genome. The secondary structures are represented according to the structural convention of Burke et al. (12). Lower- and uppercase letters denote exon and intron sequences, ...

The putative secondary structures of lys-I and terL-I were determined by using comparative sequence analysis based on secondary structure predictions (51) as well as the mFOLD software ( (83). Both introns could be folded into a secondary structure containing the entire canonical group I structural features essential for self-splicing and formation of the catalytic core (Fig. (Fig.6).6). The overall structure of corresponding introns were similar to that of the S. thermophilus phage 2972 introns, although closer structural comparisons revealed that some elements were swapped between the two PH15 introns (41). For example, in PH15 terL-I, P3.1 and P3.2 stems were predicted, similar to those in the phage 2972 lys-I intron, while P9 and P9.1 stems predicted in PH15 lys-I were predicted in the phage 2972 ter-I intron (Fig. (Fig.6).6). Also, ORF ph3 was present in the terL-I intron, similar to the phage 2972 lys-I intron, which contained a putative endonuclease-encoding ORF (orf28) (41). A poly(U) loop was predicted in P6a of terL-I (Fig. (Fig.6,6, right panel). A putative tertiary interaction (P12) and the internal guide sequence, which facilitates the splicing process by bringing P1 and P10 together, were identified in the lys-I intron (Fig. (Fig.6,6, left panel) (14).

A BLASTN search with the lys-I intron sequence revealed significant matches to phage Twort orf142 introns I2 and I3 (E value = 2.4e−04; identity, 39/43 bases). Since Twort orf142 introns I2 and I3 are closely related to the phage T4 nrdB intron (37), pairwise comparisons of the orf142 I2 and I3 introns and the T4 nrdB intron to lys-I intron revealed 72 to 75% and 60% overall identity, respectively (Table (Table33).

Sequence identity between the Ph15 lys-I intron, Twort orf142 introns, and T4 nrdB intron

The structural differences between lys-I and terL-I indicated that the two introns were from different sources. The predicted secondary structures of both terL-I and lys-I contain all group I canonical structures. However, terL-I belongs to the IA1 subgroup while lys-I belongs to the IA2 subgroup, because terL-I contains the P7.1-P7.1a stems while lys-I has the decorative P7.1-P7.2 stems separated by a G-U-A sequence. The introns of subgroup IA2 occur more commonly in phage genomes than those of subgroup IA1 (51), although introns belonging to subgroup IA1 recently have been reported to occur in Bacillus (36, 38), Lactobacillus (64), Streptococcus (41), and Synechococcus (53) phages. PH15 terL-I is similar to most subgroupIA1 introns because they contain an endonuclease-encoding ORF (ph3 in PH15), unlike the Streptococcus thermophilus phage 2972 (41) and Lactobacillus delbruekii phage JCL1032 (64) introns, which do not contain an ORF.

Comparative genomics and phylogenetic analysis.

Pairwise nucleotide sequence comparison of all S. aureus phage genomes reported in the GenBank database was conducted using dot plot analysis (Fig. (Fig.7).7). PH15 and CNPH82, along with S. aureus phage phiETA, show high sequence similarity with class II clade C phages and therefore belong in this clade. We also identified a novel clade within class II, clade D, consisting of S. aureus phages phi13, PV83, PVL, and N315. This fourth clade generated by our analyses also included phage 77, which previously could not be classified due to its similarity to members of multiple clades (35).

FIG. 7.
Dot plot matrix of PH15 and CNPH82 with S. aureus phages. The nucleotide sequences of PH15 and CNPH82 along with those of all S. aureus phages reported in the database to date were compared using the DOTTER program (70). The sliding window was set at ...

A phylogenetic tree based on proteomic distances between all known Siphoviridae phages in GenBank was generated using the approach detailed by Rohwer and Edwards (66). All staphylococcal phages, including PH15 and CNPH82, were present on one major branch of the tree (Fig. (Fig.8).8). The secondary grouping of phages within the staphylococcal branch correlated well with the results of our pairwise nucleotide analysis. Every phage of the corresponding clades clustered within a branch in the proteomic tree (Fig. (Fig.8).8). PH15 and CNPH82 clustered with other phages of class II clade C, and the members of our newly described clade D clustered together, further confirming the close relationship of these phages.

FIG. 8.
The Siphoviridae section within the phage proteomic tree. The relationship between PH15, CNPH82, and all phages belonging to Siphoviridae available in the database is presented. The tree was constructed using 153 phage genomes within Siphoviridae collated ...

The number of complete genome sequences of Siphoviridae phages in the GenBank database has increased exponentially since the first phage proteomic tree was published (66). Rohwer and Edwards (66) observed that the siphophage branch within the proteomic tree was most problematic due to rampant lateral gene transfer among siphophages. However, due to the large increase in the number of siphophage genomes available (153 genomes), our tree shows much higher resolution, which confirms the general findings and further refines the siphophage branch of the phage proteomic tree. Rohwer and Edwards (66) identified five separate groups of siphophages, in which lambda-like, D29-like, SK-1-like, and TP901-like phages formed monophyletic taxa and Sfi21-like phages formed a polyphyletic group. We confirm this structure and observe that the mycobacterial and gram-negative groups are the most diffuse and therefore the most diverged of the five groups. Lactococci and streptococci group together in the broader context of the tree but show distinct clusters within the group. Bacilli and staphylococci show distinct and more intragroup clustering, indicating younger lineages. In fact, we were able to define a novel clade within the Staphylococcus grouping which was also confirmed by our pairwise sequence comparison. One intriguing finding in our phylogenetic analyses was the presence of Bacillus subtilis phage SPP1 clustering at the edge of the Staphylococcus branch of the tree. This could be due to high similarity between the head and tail morphogenesis proteins. This result may stem from a common ancestry for these proteins or a more recent gene transfer event.


In this study, we have presented the first detailed genomic and molecular characterization of two S. epidermidis phages. We have demonstrated that the two phages are highly similar in gene content, although differences exist within the lysogeny and DNA replication modules. We have identified two introns within essential genes in the PH15 genome, one of which belongs to subgroup 1A1. Phylogenetic analysis revealed that PH15 and CNPH82 are very similar to the S. aureus phages, and based on comparative sequence analysis, we propose a new clade D within class II to classify staphylococcal phages. Sequence information for more S. epidermidis phages will provide better insight into the role of phages in S. epidermidis pathogenesis and evolution.

Supplementary Material

[Supplemental material]


We thank John Curtin, CDC, Atlanta, GA, for his gifts of S. epidermidis strain HER 1292 and phages PH15 and CNPH82; Barry Kreiswirth for S. epidermidis strain ATCC 12228; Xuning Wang at the Department of Information Technology at The Rockefeller University for bioinformatic analyses; Elena Sphicas at the Bio-Imaging Resource Center at The Rockefeller University for electron microscopy; and Chad Euler, Daniel Nelson, and Ray Schuch for helpful discussions.

This research was supported by USPHS grants AI-057472 and AI-056510 to V.A.F.


[down-pointing small open triangle]Published ahead of print on 15 December 2006.

Supplemental material for this article may be found at


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