Streptococcus pyogenes Φ1207.3 Is a Temperate Bacteriophage Carrying the Macrolide Resistance Gene Pair mef(A)-msr(D)...

Streptococcus pyogenes prophage Φ1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if Φ1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the Φ1207.3-carrying lysogen were treated with mitomycin C to assess if Φ1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if Φ1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of Φ1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 × 10⁴ and 6.06 × 10⁴ excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 × 10³ and 7.68 × 10³ phage particles per milliliter of supernatant. The phage preparations of Φ1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 × 10^-6 lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 10³ phage particles. IMPORTANCE Bacteriophages play an important role in bacterial physiology and genome evolution. The widespread use of genome sequencing revealed that bacterial genomes can contain several different integrated temperate bacteriophages, which can constitute up to 20% of the genome. Most of these bacteriophages are only predicted in silico and are never shown to be functional. In fact, it is often difficult to induce the lytic cycle of temperate bacteriophages. In this work, we show that Φ1207.3, a peculiar bacteriophage originally from Streptococcus pyogenes, which can lysogenize different streptococci and carries the macrolide resistance mef(A)-msr(D) gene pair, is capable of producing mature virions, but only at a low level, while not being able to produce plaques. This temperate phage is probably a partially functional phage, which seems to have lost lytic characteristics to specialize in lysogenization. While we are not used to conceiving phages separately from lysis, this behavior could actually be more frequent than expected.