Streptococcus pyogenesϕ1207.3 is a temperate bacteriophage carrying the macrolide efflux gene pairmef(A)-msr(D) and capable to lysogenise different Streptococci

Abstract

AbstractStreptococcus pyogenesprophage ϕ1207.3 (formerly Tn1207.3) carries themef(A)-msr(D) efflux resistance genes, responsible for type M macrolide resistance. To investigate if ϕ1207.3 is a functional bacteriophage, we transferred the element from the originalS. pyogeneshost in a prophage-free and competence-deficientS. pneumoniaestrain. 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 in 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, non-contractile tail, typical of a siphovirus. Quantification of ϕ1207.3 was performed by qPCR and semi-quantitatively by TEM. PCR quantified 3.34 × 104and 6.06 × 104excised forms of phage genome per ml of supernatant obtained from the untreated and mitomycin C treated cultures, respectively. By TEM, we estimated 3.02 × 103and 7.68 × 103phage particles per ml of supernatant. The phage preparations of ϕ1207.3 infected and lysogenised pneumococcal recipient strains at a frequency of 7.5 × 10−6lysogens/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 103phage 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 predictedin silicoand 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 fromStreptococcus pyogenes, which can lysogenise different Streptococci and carries the macrolide resistancemef(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.