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

## TL;DR

This study uses whole-genome sequencing to reveal genetic differences in Propionibacterium freudenreichii strains, focusing on metabolism and immune systems.

## Contribution

The study identifies narG as a potential marker for nitrate reduction and maps CRISPR-Cas systems in P. freudenreichii strains.

## Key findings

- Strains differ in gas production and substrate metabolism despite high genomic similarity.
- The narG gene is present in only 39% of P. freudenreichii genomes and one of the five studied strains.
- Most strains have the I-G CRISPR-Cas system, with some also containing the I-E system and phage-matching spacers.

## Abstract

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.