Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

Abstract

Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

Fig. 1. Assembly, annotation, and functional properties of the two metagenome-assembled genomes (MAGs) from the Swiss hard cheese starter culture RMK202.

A The Metagenome-assembled-genomes of S. thermophilus and L. delbrueckii with different genetic features highlighted (see legend). B Functional properties potentially involved in the metabolic interaction of the two species. The coloring of the circles indicate the ratio of nonsynonymous vs synonymous substitution rates (pN/pS) of the genes, while striped circles indicate the absence.

Fig. 2. Metagenomic sampling design and species abundance.

A The starter culture propagation scheme as applied in the cheese starter culture production. The samples subjected to metagenomic sequencing are indicated by darker colors and labelled with numbers. Every propagation cycle includes a freeze drying (lyophilization), reactivation, and working stock step. From the working stock, commercial starter cultures for weekly shipments to cheesemakers are produced. The propagation experiment was carried out in the same way as in the production plant and in five replicates corresponding to samples 7–11. The numbers between the working stock (x) indicate the number of cycles in between. Between 1996 and 2012 there are between 1 and 3 propagtion cycles. B The relative abundance of the two bacterial species in the 11 starter cultures samples (as illustrated in Fig. 2A). C Bacterial counts throughout the propagation experiment for both species and the five replicates (lines are colored according to species and points according to samples within Fig. 2A). D Acidification potential throughout the propagation experiment, as measured by pH reached after 18 h incubation at 37 °C in milk.

Fig. 3. Strain-level diversity of S. thermophilus in cheese starter cultures.

A Alternative allele frequencies of all S. thermophilus SNVs over the metagenomic samples. Recurring SNVs from different samples are connected with a line. Clustering of lines indicates a large amount of SNVs with similar frequencies suggesting genomic coupling. Sample labels on the x-axis correspond to samples highlighted in Fig. 2A. B The phylogeny of the isolated S. thermophilus strains based on maximum likelihood analysis on 1788 core genes. The isolates split into four lineages indicated by different color shadings. Strains sequenced with Nanopore are labelled with an asterisk. Values on branches indicate bootstrap values (100 replicates). C The relative abundance of each of the four sub-lineages of S. thermophilus across the 11 metagenomes as based on the average frequency of lineage-specific SNVs identified on the basis of the isolates in Fig. 3B.

Fig. 4. Phenotypic properties of individual strains, pairwise combination of strains, and original starter culture.

A Colony forming units (CFUs) of S. thermophilus and L. delbrueckii over 18 h of growth when cultured alone, in pairwise combinations, or in the original starter cultures (RMK). The ribbons illustrate the interquartile range and the lines the modeled growth curves. B Acidification curves of the same samples. The ribbons illustrate the min and max pH of the different samples. C Principal component analysis of the metabolic profiles after 24 h of growth at 37 °C. Different treatments are highlighted in colors and with the surrounding eclipse.

Fig. 5. CRISPR spacer diversity of L. delbrueckii and S. thermophilus.

A The correlation of fraction of shared CRISPR spacers and ANI of all L. delbrueckii and S. thermophilus with the corresponding densities and heatmaps on the x-and y-axis. B The heatmap of the genomic and CRISPR spacer diversities of S. thermophilus illustrated with ANI (top heatmap; from white to red) and percent shared CRISPR spacers (bottom heatmap; from white to blue). C The amount of metagenomic (metaG) and genomic (isolates) CRISPR spacers according to the five arrays.

Fig. 6. Characteristics of the phages identified in the cheese starter cultures.

A Gene annotation of the two Streptococcus starter culture phages, RMK202_1 and RMK202_2, and the two closest relatives (illustrated in lighter colors). Protein similarity between genes are indicated in gray (80–95% identity) and black (95–100%). B Relative abundance of bacteria and phages over all metagenomic samples based on genome read coverage. C Fraction of Streptococcus genomes with an integrated phage as based on the read coverage of phage-bacteria spanning regions relative to the coverage of the S. thermophilus genome. D Fraction of Streptococcus phages which show signs of integration as based on the read coverage of phage-bacteria spanning regions relative to the coverage of the Streptococcus phage genomes. E The number of spacers mapping against the different phage types. F The Streptococcus phage network with the protospacer containing phages colored or labeled according to phage type. G The spacer abundance vs the protospacer abundance from all phage spacers. The database specific linear regression and distributions are indicated in the figure and the axis figures accordingly.