Whole-Genome Sequencing Uncovers Metabolic and Immune System Variations in Propionibacterium freudenreichii Isolates

Propionibacterium freudenreichii plays a crucial role in the production of Swiss-type cheeses; however, genomic variability among strains, which affects their technological traits, remains insufficiently explored. In this study, whole-genome sequencing and comparative analysis were performed on five industrial P. freudenreichii strains. Despite their overall high genomic similarity, the strains proved different in gas production and substrate metabolism. Phylogenetic analysis revealed a close relationship between strain FNCPS 828 and P. freudenreichii subsp. shermanii (z-score = 0.99948), with the latter being unable to reduce nitrates but being able to metabolize lactose. The narG gene encoding the nitrate reductase alpha subunit was detected in only one of the five analyzed strains - FNCPS 828 - and in 39% of previously described P. freudenreichii genomes, suggesting its potential as a marker of nitrate-reducing capability. Analysis of 112 genomes showed that the I-G CRISPR-Cas system was present in more than 90% of the strains, whereas the type I-E system was found in approximately 25%. All the five study strains harbored the type I-G system; strain FNCPS 3 additionally contained a complete type I-E system with the highest number of CRISPR spacers, some of which matched previously published bacteriophage sequences. The most prevalent anti-phage defense systems included RM I, RM IV, AbiE, PD-T4-6, HEC-06, and ietAS. These findings highlight the genetic diversity of P. freudenreichii strains, which is of great importance in their industrial applications. The identification of narG as a potential marker of nitrate-reducing activity, along with detailed mapping of CRISPR- Cas systems, boosts opportunities for the rational selection and engineering of starter cultures with tailored metabolic properties and increased resistance to bacteriophages.