The phage {Phi}13-encoded transcriptional regulator Ltr controls phage assembly in Staphylococcus aureus

Temperate phages play a central role in evolution and pathogenicity of Staphylococcus aureus. Sa3int phages, in particular, contribute highly human-specific virulence factors that promote immune evasion and survival within the host. The reversible excision of these phages which occurs without phage production and bacterial lysis allows the simultaneous expression of phage virulence genes and the hlb gene where they usually integrate. However, the regulatory mechanisms controlling phage assembly and the cross-talk with host factors remain poorly understood. In this study, we analyzed the regulatory mechanism controlling late gene transcription in Sa3int phage {Phi}13. We identified a functional promoter, P23, located upstream of the late phage genes that control DNA processing and packaging, capsid assembly, bacterial lysis and immune evasion. SAOUHSC_02200, the gene located upstream of P23, encodes for a late transcriptional regulator (Ltr). Mutating the P23 TATA-box or the ltr gene abolished P23 activity and formation of mature intact phage particles, thus confirming the role of Ltr in regulating P23 activity. Four direct repeats upstream of the P23 transcriptional start site were identified as potential Ltr binding sites. RT-qPCR analysis confirmed that Ltr-dependent P23 activation is essential for expression of late genes and the subsequent propagation of {Phi}13. Furthermore, comparative analysis of P23 activity and ltr expression in different host strain backgrounds revealed strain-specific differences that appear to depend on the alternative sigma factor SigB and its downstream effector SpoVG. These findings establish Ltr as the major regulator of late gene expression in {Phi}13 and reveal bacterial host factors that control successful phage assembly, and bacterial lysis. ImportanceThe dynamic integration and excision of highly prevalent Sa3int phages in Staphylococcus aureus is considered a regulatory switch that enables bacterial adaptation to specific niches. These phages carry several human-specific virulence genes and integrate into the hlb virulence gene. It was assumed that they undergo a mechanism termed active lysogeny, which allows the phages to be excised reversibly without phage production or bacterial lysis. Here, we have identified a new phage-encoded late transcriptional regulator (Ltr) that controls the expression of all late phage genes and thus phage assembly and lysis. We found that Ltr activity is regulated by the alternative sigma factor B and its downstream effector SpoVG. Restriction of SpoVG, and consequently phage assembly, likely contributes to the maintenance of Sa3int phages, even under phage-inducing conditions. This may be relevant in certain infectious conditions where both the phage-encoded virulence genes and the gene that is usually interrupted by the phage are required for infectivity.