Identification of an Inducible Bacteriophage in a Virulent Strain of Streptococcus suis Serotype 2

SSH.

The virulent S. suis serotype 2 strain 89-999 (EF⁻ MRP⁻ hemolysin⁻), isolated from a diseased pig in Canada and causing septicemia in experimentally inoculated pigs (20), was selected as the DNA tester source. The driver DNA was extracted from the avirulent S. suis serotype 2 strain 90-1330 (20). Standard methods were used for growth of and genomic DNA extraction from S. suis (5). Adaptor ligation, SSH, and PCR amplification were performed essentially as recommended by the supplier of the Clontech PCR-select bacterial genome subtraction kit (BD Biosciences Clontech, Mississauga, Canada). Nested-PCR products were inserted into pBluescript II KS (Stratagene, La Jolla, Calif.) or into pCR2.1 vector by using a T/A cloning kit (Invitrogen, Burlington, Canada). Plasmid DNAs were prepared by a using QIAprep Spin Miniprep kit (Qiagen Inc., Mississauga, Canada) according to the manufacturer's protocol. The cloned SSH products were amplified by PCR in 25 μl of reaction mixture containing 1 μl of diluted recombinant plasmid, 10 pM concentrations of the M13F and M13R primers, 200 μM concentrations of deoxynucleoside triphosphates (Amersham Biosciences Corp., Baie d'Urfé, Canada), 2.5 μl of 10× PCR buffer, and 1 U of Taq polymerase (Amersham Biosciences Corp.). The PCR was performed in GeneAmp PCR system 9700 (Perkin-Elmer Applied Biosystems, Foster City, Calif.) using the following parameters: 94°C for 3 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 7 min. Biotin labeling of the inserts was conducted as described above but with the addition of biotin-21-dUTP (BD Biosciences Clontech). For filter hybridization purposes, total genomic DNAs of S. suis strains were randomly labeled with ³²P by using Ready-To-Go DNA labeling beads (Amersham Biosciences Corp.). To assess the specificity of the tester-subtractive clones, we carried out the hybridization in both directions. First, PCR products were placed on a nylon membrane and probed with ³²P-labeled DNA of tester (S. suis serotype 2 virulent strain 89-999) and driver (S. suis serotype 2 avirulent strain 90-1330) strains (Fig. 1). Second, the clones hybridizing only with the tester DNA were biotin labeled and used to probe dot blot and Southern membranes containing immobilized DNA of tester and driver strains. Thus, after eliminating the clones that hybridized with 90-1390 DNA, the clones harboring sequence specific and unique to the virulent strain 89-999 were recovered.

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FIG. 1.

Representative dot blot hybridization of SSH products. Cloned SSH products were PCR amplified and probed with ³²P randomly labeled genomic DNAs of S. suis serotype 2 strains 89-999 (virulent) and 90-1330 (avirulent). A, E, and R, clones from AluI-, EcoRV-, and RsaI-subtracted libraries, respectively; pKS1, -2, -4, and -6, RsaI-subtracted fragments cloned in pBluescript II KS; 999, DNA from virulent Canadian strain 89-999; 1330, DNA from avirulent Canadian strain 90-1330; S-735, DNA from virulent European strain S-735. Circles indicate the PCR products belonging to phage sequences.

DNA sequences and analysis of the selected clones.

Only clones hybridizing to the genome of the virulent strain 89-999 were selected, giving a total of 35 clones. These clones were also confirmed by hybridization with the genome of S. suis strain 89-1591, another virulent strain from Canada (20). Fifteen of the 35 clones gave a stronger hybridization signal with both virulent strains, 89-999 and 89-1591, and hence were selected for further analysis. DNA sequencing of the clones was performed at the Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono. Nucleotide homology searches were performed using the BLASTX program of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/ ). The average size of the subtracted fragments was approximately 600 to 700 bp. Eleven of 15 clones studied were unique.

Bioinformatic analysis.

Nine of the 11 clones showed significant database matches with accessory genetic elements such as transposons, plasmids, and bacteriophages of gram-positive bacteria (Table 1). For instance, two clones (A#3 and A#15) shared homology with deduced proteins found in plasmids of the pMV158 family, including the small cryptic plasmid pSSU1 previously isolated from a S. suis serotype 2 strain (29, 30). This finding suggests that the plasmid carried by strain 89-999 was closely related to pSSU1.

Only two clones (R#23 and A#4) shared homology with the sequenced genome of S. suis strain P1/7 (http://www.sanger.ac.uk/Projects/S_suis/blast_server.shtml ), which is a European strain with the phenotype MRP⁺ EF⁺ suilysin⁺. These results suggest that most clones obtained in this study may be specific to pathogenic Canadian strains 89-999 and 89-1591 that do not carry the above-mentioned phenotype. Clone R#23 showed homology with the open reading frame of a haloacid dehalogenase-like hydrolase found in the genome of strain P1/7. Interestingly, the gene encoding this protein is located upstream of the suilysin gene (sly) in S. suis strain P1/7. Recently, King et al. (15) showed that in strains lacking the sly gene, the genome arrangement of the region was conserved despite the presence of another gene of unknown function in the sly equivalent position.

The sequences of clones A#16 (AY198215) and E#67 (AY198225) did not share significant homology with the sequences of any proteins in the databases. It can be speculated that these fragments could potentially encode virulence factors or a new class of proteins involved in pathogenicity or virulence processes.

Bacteriophages in S. suis.

Sequence homologies to conserved hypothetical phage (clone A#38) or phage-associated proteins (clones R#32 and R#13) from Streptococcus pyogenes (6, 25) as well as phage resistance systems (clone R#P4) (18, 19) were found (Table 1). In S. pyogenes, phage-like elements account for the great majority of the variation in gene content relative to the genomes of S. pyogenes between the sequenced serotypes M3, M1, and M18 (6, 25). Recombination may have produced chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. S. pyogenes strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen, and a previously uncharacterized phospholipase A (2) (designated Sla) (6). Phages in other pathogenic Streptococcus species have also been described (21, 27). Recently, Artiushin et al. (4) found that the genes encoding the pyrogenic mitogens in Streptococcus equi were acquired via a phage-mediated horizontal transfer. Interestingly, the genomic region that codes for a phage in S. equi showed extensive similarity to a defective phage sequence in S. pyogenes.

Since phage sequences were found in the selected clones, the presence of a prophage in the virulent strain 89-999 as well as in the avirulent strain 90-1330 was investigated by induction with mitomycin C. Both S. suis strains were grown in 500 ml of Todd-Hewitt broth supplemented with 0.2% maltose and 10 mM MgSO₄ until an optical density at 600 nm of 0.2 was reached. The phage induction was attempted via overnight incubation at 37°C in the presence of mitomycin C (1 μg/ml). Complete cell lysis was observed with strain 89-999 but not with strain 90-1330.

Bacterial lysates of strain 89-999 were then treated as described previously (17) and were observed with a JEOL 1200 electron microscope. Electron microscopy analysis of the lysate from S. suis strain 89-999 revealed the presence of phage particles (Fig. 2). The phage, named Ss1, has a long noncontractile tail of approximately 150 nm and is therefore a member of the Siphoviridae family (9). Phage Ss1 has a small isometric nucleocapsid approximately 50 nm in diameter and thus belongs to the morphotype B1. Phage genome was extracted using a Lambda Midi kit (Qiagen Inc.). Restriction fragment analysis of EcoRI- and HindIII-digested phage DNA indicated that the genome size of Ss1 is approximately 32 kb (data not shown).

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FIG. 2.

Electron micrograph of mitomycin-induced temperate phage Ss1 from S. suis strain 89-999. Phosphotungstic acid staining was used. Magnification, ×100,000.

Phage sequences in the genomes of other S. suis strains.

Bacterial and phage DNAs were then digested with EcoRI and HindIII, run on an agarose gel, and transferred onto a nylon membrane. The membrane was hybridized with radioactively labeled DNA from phage Ss1 or from S. suis strain 89-999. The hybridization results confirmed that S. suis 89-999 is a lysogenic strain (Fig. 3). Preliminary data indicated that the labeled phage Ss1 DNA also hybridized with most (9 of 12 strains were positive) of the Canadian S. suis strains isolated from diseased pigs or with proven virulence in mouse or pig models (data not shown). However, the Ss1 sequence was not detected in the genomes of five strains from Europe, including the reference strain of serotype 2 (S-735) and strain P1/7 (data not shown), and one strain from Mexico (data not shown).

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FIG. 3.

Southern blots of digested phage and chromosomal DNAs from Ss1 and S. suis strain 89-999, respectively, hybridized with radioactively labeled Ss1 phage genomic DNA (A) or chromosomal DNA of S. suis strain 89-999 (B). Lanes: 1 and 4, uncut Ss1 phage DNA; 2 and 5, EcoRI-treated Ss1 phage DNA; 3 and 6, HindIII-treated Ss1 phage DNA; 7, HindIII-treated chromosomal DNA.

The presence of Ss1 suggests that phage could be involved in the acquisition of virulence genes by strain 89-999 and might have contributed to the genetic diversity observed for S. suis (7). The dissemination of genes encoding virulence factors by such horizontal transfer of DNA is critical in the emergence of new pathogenic organisms. Recent findings suggest that genetic exchange is a significant process involved in the evolution of the population of S. suis strains, which contains various combinations of virulence markers such as suilysin (22, 28). Furthermore, the capsular genes may be moving horizontally through the S. suis population, as has been suggested by a multilocus sequence typing study (16).

Conclusion.

PCR-based SSH was adapted to isolate unique DNA sequences which were specific to virulent strains of S. suis isolated in Canada. In addition, we have demonstrated for the first time the presence of an inducible phage in a virulent S. suis serotype 2 strain from Canada.

Nucleotide sequence accession numbers.

The GenBank accession numbers for the clones from S. suis strain 89-999 are as follows: AY198216 (A#3), AY198222 (A#4), AY198217 (A#15), AY198215 (A#16), AY198218 (A#38), AY198225 (E#67), AY198220 (R#13), AY198223 (R#14), AY198224 (R#23), AY198219 (R#32), and AY198221 (R#P4).