In silico prediction of horizontal gene transfer events in Lactobacillus bulgaricus and Streptococcus thermophilus reveals protocooperation in yogurt manufacturing

Abstract

Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.

FIG. 1.

Phylogenetic tree of the cbs-cblB(cglB)-cysE gene cluster in LAB genomes. The tree is constructed on the basis of the alignment of concatenated sequences of the cbs and cblB or cglB genes. Since the gene cysE is a pseudogene in a few genomes, it is not taken into account in the phylogenetic analysis. Bootstrap values are reported for a total of 1,000 replicates. Truncated cbs and cblB or cglB genes from Lactobacillus acidophilus NCFM are also included in the tree. Genes from Bacillus subtilis 168 are used as the outgroup. The HGT event is indicated by an arrow. Lb., Lactobacillus; St., Streptococcus; Lc., Lactococcus.

FIG. 2.

Gene context of cbs-cblB(cglB)-cysE in L. bulgaricus, L. helveticus, and S. thermophilus. The clusters have the same gene context, while cysE in L. bulgaricus strains is truncated due to the presence of an insertion sequence (see Fig. S4 in the supplemental material). PSG, pseudogene. Dashed arrows also represent the pseudogenes. Predicted terminators are indicated by mushroom symbols. Transposase genes are shadowed by gray diagonal stripes. Modified from an image created with the Microbial Genome Viewer application (26).

FIG. 3.

Methionine and cysteine interconversion pathway in S. thermophilus and L. bulgaricus. Filled red boxes represent the presence of the genes in the three S. thermophilus strains. Filled blue boxes represent the presence of the genes in both L. bulgaricus strains. The half-filled blue box represents a gene present only in L. bulgaricus ATCC BAA365. Open boxes indicate that the genes either are absent or are pseudogenes. Arrows between boxes represent the HGT events and the directions of the transfers. The cysE gene in L. bulgaricus is truncated after the HGT; thus, it is also shown as an open box. The enzymes shown are as follows: CysE, serine acetyltransferase; CysK, O-acetylserine sulfhydrylase; CBL, cystathionine beta-lyase; CGL, cystathionine gamma-lyase; MetH, homocysteine S-methyltransferase; MetE, homocysteine methyltransferase; HSK, homoserine kinase; HSST, homoserine O-succinyltransferase; CGS, cystathionine gamma-synthase; CBS, cystathionine beta-synthase. The distribution of the above-mentioned genes in the S. thermophilus and L. bulgaricus genomes is derived from our previous study (31). Suc-CoA, succinyl coenzyme A; Pi, phosphate.