A novel temperate phage from Alicyclobacillus: first evidence in this genus of genomic identity to a sigK-integrated prophage
Carvalho Leonard, Inês et al.
Bacteriophages are increasingly explored as clean-label, environmentally friendly tools for sustainable food processing. Several phage-based products have been approved for agri-food applications, all of which rely on strictly lytic phages to ensure safety, regulatory compliance, and rapid bactericidal action. Despite its industrial relevance, only two phages targeting the genus Alicyclobacillus, thermoacidophilic spore-formers responsible for fruit product spoilage, have been reported to date, leaving a very limited arsenal for their control. This study reports the Alicyclobacillus phage MMB025, infecting Alicyclobacillus acidoterrestris, a species associated with spoilage. Transmission electron microscopy revealed a contractile-tailed morphotype, and host range assays demonstrated specificity for a narrow subset of Alicyclobacillus isolates. MMB025 displayed stability across a wide pH range (3-12) and moderate heat (≤60°C). Hybrid sequencing identified a 105 kb dsDNA genome encoding structural, replication, and lysogeny modules, the latter indicating a temperate lifestyle. Consistently, the phage genome showed 99.99% identity to a chromosomal region of A. acidoterrestris DSM 3922T, most likely corresponding to MMB025 prophage state. Genome mapping showed prophage integration within the sporulation sigma factor gene sigK, reminiscent of the Bacillus subtilis skin element, suggesting a novel phage-sporulation interaction in Alicyclobacillus. Despite the presence of lysogeny-related features, bioinformatic lifestyle predictions were inconclusive, emphasizing current limitations of in silico classification. Although temperate phages like MMB025 are not ideal for direct biocontrol, the genome encodes putative lysins and enzymes with potential for spoilage mitigation. This study contributes to expanding the still-limited diversity of Alicyclobacillus phages and provides new insights into phage-host interactions in thermoacidophilic spore-forming bacteria. Importance: The genus Alicyclobacillus remains a persistent problem for the fruit juice industry, as its thermoacidophilic nature leads to spoilage events that cause product recalls, loss of consumer confidence, and significant economic losses. Yet remarkably little is known about the phages infecting these bacteria, with only two described to date. This study reports the first infectious phage with genomic identity to a prophage in the genus, the Alicyclobacillus phage MMB025. This finding broadens the extremely limited record of Alicyclobacillus phages and reveals integration within the sporulation sigma factor sigK, suggesting a novel link between prophage activity and sporulation regulation in Alicyclobacillus. By combining stability assays, host range testing, and genome analysis, MMB025 represents both a valuable model for phage-spore-former biology and a genetic resource for candidate lysins with potential applications in food preservation.
