Specialized adaptation of a lactic acid bacterium to the milk environment: the comparative genomics of Streptococcus thermophilus LMD-9

Abstract

Background: Streptococcus thermophilus represents the only species among the streptococci that has "Generally Regarded As Safe" status and that plays an economically important role in the fermentation of yogurt and cheeses. We conducted comparative genome analysis of S. thermophilus LMD-9 to identify unique gene features as well as features that contribute to its adaptation to the dairy environment. In addition, we investigated the transcriptome response of LMD-9 during growth in milk in the presence of Lactobacillus delbrueckii ssp. bulgaricus, a companion culture in yogurt fermentation, and during lytic bacteriophage infection.

Results: The S. thermophilus LMD-9 genome is comprised of a 1.8 Mbp circular chromosome (39.1% GC; 1,834 predicted open reading frames) and two small cryptic plasmids. Genome comparison with the previously sequenced LMG 18311 and CNRZ1066 strains revealed 114 kb of LMD-9 specific chromosomal region, including genes that encode for histidine biosynthetic pathway, a cell surface proteinase, various host defense mechanisms and a phage remnant. Interestingly, also unique to LMD-9 are genes encoding for a putative mucus-binding protein, a peptide transporter, and exopolysaccharide biosynthetic proteins that have close orthologs in human intestinal microorganisms. LMD-9 harbors a large number of pseudogenes (13% of ORFeome), indicating that like LMG 18311 and CNRZ1066, LMD-9 has also undergone major reductive evolution, with the loss of carbohydrate metabolic genes and virulence genes found in their streptococcal counterparts. Functional genome distribution analysis of ORFeomes among streptococci showed that all three S. thermophilus strains formed a distinct functional cluster, further establishing their specialized adaptation to the nutrient-rich milk niche. An upregulation of CRISPR1 expression in LMD-9 during lytic bacteriophage DT1 infection suggests its protective role against phage invasion. When co-cultured with L. bulgaricus, LMD-9 overexpressed genes involved in amino acid transport and metabolism as well as DNA replication.

Conclusions: The genome of S. thermophilus LMD-9 is shaped by its domestication in the dairy environment, with gene features that conferred rapid growth in milk, stress response mechanisms and host defense systems that are relevant to its industrial applications. The presence of a unique exopolysaccharide gene cluster and cell surface protein orthologs commonly associated with probiotic functionality revealed potential probiotic applications of LMD-9.

Figure 1

Genome maps of the S. thermophilus LMD-9 chromosome and two cryptic plasmids, pSTER_A and pSTER_B. From the innermost towards the outermost circles of the chromosome map: Circle 1, COG functional classification of ORFs on the forward strand; Circle 2 (blue), ORFs on the forward strand; Circle 3 (red), ORFs on the reverse strand; Circle 4, COG functional classification of ORFs on the reverse strand; Circle 5 (salmon), GC skew; Circle 6 (green), GC composition (%); Circle 7, rRNAs and tRNAs, shown in brown and orange, respectively; Circle 8 (dark grey), pseudogenes; Circle 9, intact and truncated transposases, shown in red and green, respectively; Circle 10, key features encoded in the chromosome – urease gene cluster (blue), CRISPR regions (dark red), prophage remnant (yellow), eps and rgp clusters (fuchsia), gal-lac cluster (cyan) and blp cluster (green); Circle 11 (green), S. thermophilus-specific genes not present in S. salivarius SK126 draft genome; Circle 12 (red), LMD-9 specific genes or chromosomal regions not present in both CNRZ1066 and LMG 18311. Selected features within the unique regions are indicated. Genes encoding the Pep1357C cyclic peptide was previously identified by Ibrahim et al. [15]. Color designation for COG classification of ORFs in Circle 1 and 4 is shown at the bottom right legend.

Figure 2

Alignment of S. thermophilus genomes. Genome alignment of S. thermophilus LMD-9 with CNRZ1066 and LMG 18311 strains was performed using the GenomeComp software tool [32]. Alignment lengths are color-coded as follows: red, >10,000 bp; blue, 5,000 to 10,000 bp; cyan, 1,000 to 5,000 bp; yellow, 500 to 1,000 bp, and pink, <500 bp. Unique regions are in green. LMD-9 specific regions are indicated with black arrowheads above the LMD-9 linear chromosome, with region designation shown at the top of the upper canvas. ORFs present within these regions are described in Additional file 3.

Figure 3

Functional genome distribution (FGD) tree of 39 Streptococcus genomes. FGD analysis of 39 Streptococcus strains for which genome sequences are available, depicting the functional relationship among the species and strains based on the amino acid sequence similarities of the predicted ORFeomes. S. thermophilus strains formed a functional cluster with S. salivarius as the closest relative. The FGD analysis also predicted functional sub-clusterings among the S. pneumoniae and S. pyogenes strains. Approximated branch lengths, representing the level of functional similarity between genomes are depicted by distance units (du).

Figure 4

The exopolysaccharide (EPS) biosynthetic gene clusters in S. thermophilus and S. salivarius SK126. ORFs were color-coded according to their prediction functions (see legend at lower left panel). Regions exhibiting sequence similarities among the eps clusters are shown in light blue-shaded boxes. The size of each eps cluster is indicated in bracket.