Genomic and functional analysis of stress-responsive prophages in Lactobacillus helveticus

Introduction: Bacteriophages are pervasive components of natural whey starters (NWS) used in traditional hard cheese production, yet their functional role under cheesemaking-related stresses remains poorly understood. Methods: In this study, six temperate bacteriophages associated with Lactobacillus helveticus were isolated from Trentingrana NWS. Classified within the Caudoviricetes class, they harbored gene sets associated with the typical phage cycle together with a large number of open reading frames (ORFs) with hypothetical or unknown function. By combining quantitative PCR targeting hallmark phage genes with detection of extracellular phage particles, we investigated prophage induction dynamics under abiotic conditions relevant to dairy processing, including heat shock, nutrient limitation, lactic acid, and oxidative stress. Results: Across conditions, stress exposure elicited phage-specific and dosedependent responses, frequently revealing a decoupling between early increases in phage genomic copy number and subsequent virion release. Heat shock emerged as the most consistent inducer of prophage activation, whereas lactic acid and oxidative stress produced threshold-dependent or inhibitory effects, particularly at higher intensities. Nutrient limitation alone had limited impact, suggesting a permissive rather than triggering role. Discussion: Overall, these findings support a non-binary model of prophage induction, spanning intermediate states from partial genome replication to productive lytic cycles, strongly constrained by host physiological status. Our results indicate that L. helveticus prophages respond heterogeneously to cheesemakingrelated stresses and may act as context-dependent modulators of NWS stability rather than direct disruptors. Such stress-responsive prophage dynamics are likely to influence microbial population turnover and resilience during back-slopping, ultimately contributing to the long-term stability and functional robustness of Trentingrana natural whey starters