Fate, Inducibility, and Behavior of Latilactobacillus curvatus Temperate Phage TMW 1.591 P1 during Sausage Fermentation.

AIMS: Temperate phages insert their genome into the host's chromosome. As prophages, they remain latent in the genome until an induction event leads to lytic phage production. When this occurs in a starter culture that has been added to food fermentation, this can impair the fermentation success. This study aimed to analyze prophage inducibility in the Latilactobacillus curvatus TMW 1.591 strain during meat fermentation and investigate whether an induction signal before cryopreservation is maintained during storage and can lead to phage-induced lysis after culture activation. METHODS AND RESULTS: A prophage-free isogenic derivative of the model starter organism, L. curvatus TMW 1.591, was developed as a negative control (L. curvatus TMW 1.2406). Raw meat fermentation was performed with the wild-type (WT) and phage-cured strains. The WT strain produced high numbers of phages (5.2 ± 1.8 × 107 plaque-forming units g-1) in the meat batter. However, the prophage did not significantly affect the meat fermentation process. Induction experiments suggested an acidic environment as a potential trigger for prophage induction. Phage induction by ultraviolet light before strain cryopreservation remains functional for at least 10 weeks of storage. CONCLUSIONS: Intact prophages are active during meat fermentation. However, in this study, this has no measurable consequences for fermentation, suggesting a high resiliency of meat fermentation against phages. Inadequate handling of lysogenic starter strains, even before preservation, can lead to phage introduction into food fermentation and unintended host lysis.

Distribution, inducibility, and characteristics of Latilactobacillus curvatus temperate phages.

Aim: Temperate phages are known to heavily impact the growth of their host, be it in a positive way, e.g., when beneficial genes are provided by the phage, or negatively when lysis occurs after prophage induction. This study provides an in-depth look into the distribution and variety of prophages in Latilactobacillus curvatus (L. curvatus). This species is found in a wide variety of ecological niches and is routinely used as a meat starter culture. Methods: Fourty five L. curvatus genomes were screened for prophages. The intact predicted prophages and their chromosomal integration loci were described. Six L. curvatus lysogens were analysed for phage-mediated lysis post induction via UV light and/or mitomycin C. Their lysates were analysed for phage particles via viral DNA sequencing and transmission electron microscopy. Results: Two hundred and six prophage sequences of any completeness were detected within L. curvatus genomes. The 50 as intact predicted prophages show high levels of genetic diversity on an intraspecies level with conserved regions mostly in the replication and head/tail gene clusters. Twelve chromosomal loci, mostly tRNA genes, were identified, where intact L. curvatus phages were integrated. The six analysed L. curvatus lysogens showed strain-dependent lysis in various degrees after induction, yet only four of their lysates appeared to contain fully assembled virions with the siphovirus morphotype. Conclusion: Our data demonstrate that L. curvatus is a (pro)phage-susceptible species, harbouring multiple intact prophages and remnant sequences thereof. This knowledge provides a basis to study phage-host interaction influencing microbial communities in food fermentations.

Distribution, inducibility, and characterisation of prophages in Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria (LAB) are used as starters in a wide variety of food fermentations. While the number of reports of phages infecting other LAB steadily increased over the years, information about phage associated with Latilactobacillus sakei, a frequently used meat starter, remains scarce. RESULTS: In this study, a predictive genomic analysis of 43 Latilactobacillus sakei genomes revealed the presence of 26 intact, eleven questionable and 52 incomplete prophage sequences across all analysed genomes with a range of one to five predicted prophage sequences per strain. Screening 24 sakei strains for inducible prophages by utilising UV light or mitomycin C, we identified seven lysogenic strains showing lysis after induction during subsequent growth monitoring. Electron microscopic analysis revealed fully assembled virions in the purified lysates of four samples, thus confirming successful prophage induction. All virions featured icosahedral, isomeric heads and long, most likely non-contractile tails indicating siphoviruses. By performing phylogenetic analyses with various marker genes as well as full prophage sequences, we displayed a remarkably high diversity of prophages, that share a similar gene module organisation and six different chromosomal integration sites were identified. By sequencing viral DNA purified from lysates of Latilactobacillus sakei TMW 1.46, we demonstrate that simultaneous induction of multiple prophages is possible. CONCLUSIONS: With this work, we not only provide data about the incidence of prophages harboured by the meat starter Latilactobacillus sakei, we also demonstrated their potential to impact growth of their host after induction, as well as forming seemingly fully assembled virions.