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. 2007 Mar;189(5):2086-100.
doi: 10.1128/JB.01637-06. Epub 2006 Dec 15.

First complete genome sequence of two Staphylococcus epidermidis bacteriophages

Anu Daniel  1 , Penelope E Bonnen, Vincent A Fischetti
Affiliations

First complete genome sequence of two Staphylococcus epidermidis bacteriophages

Anu Daniel et al. J Bacteriol. 2007 Mar.

Abstract

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, phiPH15 (PH15) and phiCNPH82 (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.

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Figures

FIG. 1.
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, showing the attached collar (arrowheads) and dual disc plates (arrows). (C and F) Single intact phage virions of PH15 and CNPH82. The collar is marked by arrowheads. Bars, 100 nm.
FIG. 2.
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 Tables 1 and 2, respectively). ORFs identical in both the PH15 and CNPH82 genomes are shown in red, while ORFs unique to either genome are shown as hatched arrows. The terL and lys ORFs of both phages are shown as yellow arrows. The phage modules determined by database matches and genome organization are labeled. The ruler marks the relative positions of the ORFs within the 44,047-bp PH15 genome and the 43,420-bp CNPH82 genome.
FIG. 3.
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 PH15 and CNPH82 along with the numbers of the corresponding modules (see Fig. 2) are presented at the respective axes for easy orientation. Specific regions of differences between the two genomes are marked and annotated. The int gene within the lysogeny module is identical in both genomes and is marked by broken arrow.
FIG. 4.
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 1). RT-PCR was also done using primer pairs Ph28-Lys-F/Ph28-Lys-R (lanes 11 to 15) and Cn30-Lys-F/Cn30-Lys-R (lanes 16 to 20) (Table 1). The template used for the PCR was as follows: lanes 1 and 11, PH15 genomic DNA; lanes 2 and 12, RNA isolated at 15 min p.i. from the PH15-infected host (cDNA); lanes 3 and 13, Similar to lanes 2 and 12 but with no RT; lanes 4 and 14, RNA isolated at 25 min p.i. from the PH15-infected host (cDNA); lanes 5 and 15, similar to lanes 4 and 14 but with no RT; lanes 6 and 16, CNPH82 genomic DNA; lanes 7 and 17, RNA isolated at 15 min p.i. from the CNPH82-infected host (cDNA); lanes 8 and 18, similar to lanes 7 and 17 but with no RT; lanes 9 and 19, RNA isolated at 25 min p.i. from the CNPH82-infected host; lanes 10 and 20, similar to lanes 9 and 19 but with no RT. (B and C) RT-PCR on RNA isolated from the PH15-infected host with helicase-specific primers (B) and from the CNPH82-infected host with portal protein-specific primers (C). Lanes: 1, genomic DNA; 2, RNA isolated at 15 min p.i. (cDNA); 3, similar to lane 2 but with no RT; 4, RNA isolated at 25 min p.i. (cDNA); 5, similar to lane 4 but with no RT; L, 1-kb plus DNA ladder (Invitrogen).
FIG. 5.
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 2, RNA isolated at 15 min p.i. from the PH15-infected host (cDNA); lane 3, similar to lane 2 but with no RT; lane 4, RNA isolated at 25 min p.i. from the PH15-infected host (cDNA); lane 5, similar to lane 4 but with no RT; lane 6, CNPH82 genomic DNA; lane 7, RNA isolated at 15 min p.i. from the CNPH82-infected host (cDNA); lane 8, similar to lane 7 but with no RT; lane 9, RNA isolated at 25 min p.i. from the CNPH82-infected host; lane 10, similar to lane 9 but with no RT.
FIG. 6.
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, respectively. The 5′ and 3′ splice sites (ss) are indicated by arrows. The conserved structural elements P1 through P10 and sequences P, Q, R, and S are indicated, and a putative tertiary interaction, P12, within the lys-I intron is shown (50). The putative guanosine binding site in P7 is shaded. The structural elements are connected with bold lines with pointers indicating the 5′-to-3′ direction. The stop codon of ORF ph3 in terL-I is boxed. The predicted internal guide sequence (IGS) within the lys-I intron is marked. The nucleotide position in PH15 genome is shown in parentheses.
FIG. 7.
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 25 bp.
FIG. 8.
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 from the GenBank database as well as the website http://phage.sdsu.edu/∼rob/phage. The staphylococcal subbranch was expanded to show the phages clearly. PH15 and CNPH82 are highlighted by filled circles. Representative phages of different groups are highlighted in larger font. A high-resolution version of the tree is presented in Fig. S1A in the supplemental material. The phage abbreviations are listed in Table S1A in the supplemental material.
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