Streptococcus pyogenes Φ1207.3 is a temperate bacteriophage carrying the macrolide efflux gene pair mef(A)-msr(D) and capable to lysogenise different Streptococci

Abstract

Streptococcus pyogenes prophage {phi}1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) efflux resistance genes, responsible for type M macrolide resistance. To investigate if {phi}1207.3 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 {phi}1207.3-carrying lysogen were treated with mitomycin C to assess if {phi}1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment resulting in early entrance in the stationary phase. To determine if {phi}1207.3 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, non-contractile tail, typical of a siphovirus. Quantification of {phi}1207.3 was performed by qPCR and semi-quantitatively by TEM. PCR quantified 3.34 x 104 and 6.06 x 104 excised forms of phage genome per ml of supernatant obtained from the untreated and mitomycin C treated cultures, respectively. By TEM, we estimated 3.02 x 103 and 7.68 x 103 phage particles per ml of supernatant. The phage preparations of {phi}1207.3 infected and lysogenised pneumococcal recipient strains at a frequency of 7.5 x 10-6 lysogens/recipient, but did not show sufficient lytic activity to form plaques. Phage lysogenisation efficiently occurred after 30 minutes of contact of the phages with the recipient cells and required a minimum of 103 phage particles. ImportanceBacteriophages 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 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 {phi}1207.3, a peculiar bacteriophage originally from Streptococcus pyogenes, which can lysogenise 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 into lysogenisation. While we are not used to conceive phages separately from lysis, this behavior could actually be more frequent than expected.