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bioRxiv · 10.1101/2025.06.28.662109

Characterisation of a temperate phage induced from Alicyclobacillus acidoterrestris DSM 3922T: the Alicyclobacillus phage MMB025

Leonardo, I. C.·Ferreira, H.·Quendera, A. P.·Crespo, M. T. B.·Sao-Jose, C.·GASPAR, F. B.
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Abstract

The fruit juice industry has increasingly faced spoilage incidents attributed to Alicyclobacillus (ACB) bacteria. These spore-forming bacteria produce off-flavours and odours that compromise product quality and lead to significant food waste. Their resistance to acidic pH and the pasteurisation processes commonly used in the food industry demands the investigation of new preservation strategies. Bacteriophage-related treatments have emerged as a promising alternative, being considered safe, green, and sustainable. In this study, the type strain A. acidoterrestris DSM 3922T and a food isolate, A. acidoterrestris MMB007, were used as hosts to isolate new ACB-targeting phages. A phage was isolated from a soil sample in contact with the type strain, purified, and characterised for its genetic and phenotypic features. The isolated phage, named Alicyclobacillus phage MMB025, belongs to the Caudoviricetes class and exhibits a narrow host range. Also, phage MMB025 was revealed to be stable under different environmental conditions, maintaining its lytic activity across a wide range of pH values (3 to 12) and high temperatures (up to 1 h at 60 {degrees}C). The hybrid assembly of short and long reads revealed a phage genome of 105,243 bp with a GC content of 43.25 %. Sequence mapping to the A. acidoterrestris DSM 3922 genome indicated phage DNA integration within the bacterial chromosome, disrupting the sigK open reading frame. This suggests that MMB025 may have originated from a prophage induction event potentially linked to sporulation regulation. Despite the limitations derived from its likely temperate nature, phage MMB025 remains a valuable source of lysins capable of specifically and effectively eliminating ACB bacteria. More importantly, this study highlights the potential of phages as an alternative preservation strategy against ACB bacteria. The phage MMB025 characterisation opens venues to develop strategies for mitigating ACB-related spoilage, thereby enhancing the sustainability of food production.

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Leonardo, I. C., Ferreira, H., Quendera, A. P., Crespo, M. T. B., Sao-Jose, C., GASPAR, F. B.. 2025-06-29. Characterisation of a temperate phage induced from Alicyclobacillus acidoterrestris DSM 3922T: the Alicyclobacillus phage MMB025. https://doi.org/10.1101/2025.06.28.662109

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