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Appl Environ Microbiol. Jun 2009; 75(12): 4120–4129.
Published online Apr 24, 2009. doi:  10.1128/AEM.02898-08
PMCID: PMC2698337

In Silico Prediction of Horizontal Gene Transfer Events in Lactobacillus bulgaricus and Streptococcus thermophilus Reveals Protocooperation in Yogurt Manufacturing[down-pointing small open triangle]


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.

Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) and Streptococcus thermophilus have been used in starter cultures for yogurt manufacturing for thousands of years. Both species are known to stably coexist in a milk environment and interact beneficially. This so-called protocooperation, previously defined as biochemical mutualism, involves the exchange of metabolites and/or stimulatory factors (38). Examples of biochemical protocooperation between L. bulgaricus and S. thermophilus include the action of cell wall-bound proteases, produced by L. bulgaricus strains, and formate, required for growth of L. bulgaricus and supplied by S. thermophilus (6, 7). An overview of the interactions between the two yogurt bacteria, including the exchange of CO2, pyruvate, folate, etc., can be found in a recently published review by Sieuwerts et al. (43). Putative genetic mechanisms underlying protocooperation, however, so far have not been studied in detail.

The genomes of two L. bulgaricus strains and three S. thermophilus strains, all used in yogurt manufacturing, have been fully sequenced (3, 32, 33, 34, 39, 44, 46). The available genomic information could provide new insights into the genetic aspects of protocooperation between L. bulgaricus and S. thermophilus through the identification of putative horizontal gene transfer (HGT) events at the genome scale. HGT can be defined as the exchange of genetic material between phylogenetically unrelated organisms (23). It is considered to be a major factor in the process of environmental adaptation, for both individual species and entire microbial populations. Especially HGT events between two species existing in the same niche can reflect their interrelated activities and dependencies (13, 17). Nicolas et al. (36) predicted HGT events between Lactobacillus acidophilus and Lactobacillus johnsonii by analyzing 401 phylogenetic trees, also including the genes of L. bulgaricus. Several HGT events have been predicted in the S. thermophilus strains CNRZ1066 and LMG 18311 (3, 10, 18) as well as in L. bulgaricus ATCC 11842 (46). Moreover, a core genome of S. thermophilus and possibly acquired genes were identified by a comparative genome hybridization study of 47 strains (40).

In this study, we describe an in-depth bioinformatics analysis in which we combined gene composition (GC content and dinucleotide composition) and gene transfer mechanism-associated features. Thus, we predicted horizontally transferred genes and gene clusters in the five sequenced L. bulgaricus and S. thermophilus genomes, with a focus on niche-specific genes and genes required for bacterial growth. Identification of HGT events led to a list of putative transferred genes, some of which could be important for bacterial protocooperation and the adaptation to their environment. The evolution and function of the transferred gene cluster cbs-cblB(cglB)-cysE (originally called cysM2-metB2-cysE2 in S. thermophilus), involved in the metabolism of sulfur-containing amino acids, were analyzed in detail. On the basis of our analysis, it can be concluded that both species probably agonistically coevolved at the genetic level to optimize their combined growth in a milk environment and that protocooperation thus includes both biochemical and genetic aspects.


Genome sequences.

The complete genome sequences of L. bulgaricus ATCC 11842 (46), L. bulgaricus ATCC BAA365, S. thermophilus LMD9 (33), S. thermophilus CNRZ1066, S. thermophilus LMG 18311 (3), and Lactobacillus helveticus DPC 4571 (4) were obtained from the NCBI GenBank Entrez Genome database (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) under GenBank accession numbers CR954253, CP000412, CP000419, CP000024, CP000023, and CP000517, respectively (Table (Table1).1). The L. bulgaricus and S. thermophilus strains are isolated from either yogurt or industrial yogurt starter cultures.

General features of the published L. bulgaricus and S. thermophilus genomesa

Whole-genome comparison.

Genome sequences of the two L. bulgaricus strains and three S. thermophilus strains were aligned using the software package Mauve 2.0 (http://asap.ahabs.wisc.edu/mauve/) (8). Mauve 2.0 can efficiently construct multiple genome sequence alignments with modest computational resource requirements. The tool is used for identifying genomic recombination events (such as gene loss, duplication, rearrangement, and horizontal transfer) and characterizing the rates and patterns of genome evolution. Mauve 2.0 uses an anchored alignment technique to rapidly align genomes and allows the order of those anchors to be rearranged to detect genome rearrangements. The anchors, local collinear blocks (LCBs), represent homologous regions of sequence shared by multiple genomes. Mauve 2.0 requires that each collinear region of the alignment meet “minimum weight” criteria in order to identify and discard random matches. The weight of an LCB is defined as the sum of the lengths of matches in the LCB, and the minimum weight is a user-definable parameter. The minimum weight of the LCB used in this analysis was 41 and 46 for the S. thermophilus and L. bulgaricus genomes, respectively. After removing the low-weight LCBs from the set of alignment anchors, Mauve 2.0 could complete a gapped global alignment for each LCB.

HGT analysis.

Putative HGT events between L. bulgaricus and S. thermophilus strains were first detected by whole-genome comparison using Mauve 2.0. The whole-genome alignments were manually inspected to identify putative horizontally transferred genes. Sequence composition analysis was carried out, including the calculation of GC composition (of 600-bp fragments) and dinucleotide dissimilarity value δ (of 1,000-bp fragments) along the whole genome, using the δρ-Web tool (48) (see Fig. S2 and S3 in the supplemental material). Identification of HGT events by using composition differences is based on previous observations by Karlin et al. (24, 25) that each genome has a typical dinucleotide frequency and that related species have similar genome signatures. A high genomic dissimilarity between an input sequence and a representative genome sequence of the species from which the sequence was isolated suggests a heterologous origin of the input sequence. In other words, horizontally acquired genes can have a very different sequence dinucleotide composition compared to that of the genome in which they presently reside, and the difference can be expressed by the δ value. DNA fragments with significantly different GC composition and/or dinucleotide composition (average ± two standard deviations) compared to those of the whole genomes were predicted to be HGT regions.

The predicted horizontally transferred genes and gene clusters were checked for HGT mechanism-associated features such as neighboring mobile elements or tRNA genes using Artemis (41) and Mauve 2.0. Homologs of the genes that were predicted to be transferred between S. thermophilus and L. bulgaricus were collected by performing BLASTP searches (1) against all the available genomes of lactic acid bacteria (LAB) or the nonredundant NCBI protein database. Homologous sequences were aligned with MUSCLE (12), and phylogenetic trees were constructed using the neighborhood-joining method implemented in ClustalW (30). The phylogenetic trees were visualized in LOFT (47). The positions of orthologs from L. bulgaricus and S. thermophilus in the phylogenetic trees were checked to confirm whether the predicted genes are genes that are horizontally transferred between the two genomes.


Through HGT, a genome can be rearranged by the integration and/or deletion of genetic elements, one of the driving forces in the evolution of organisms (50). HGT events can be detected using phylogenetic and compositional approaches. Information on gene transfer mechanisms, for instance, transposases or bacteriophage-related genes found in the neighborhood of the target genes, can improve the prediction of HGT events (50). In order to reveal protocooperation between the two coexisting yogurt species on the genetic level and to understand the rationale of their coevolution, putative HGT events were predicted and analyzed. HGT events, detected by combining composition analysis or phylogenetic analysis and gene transfer mechanism-associated features, are described for both S. thermophilus and L. bulgaricus strains.

Putative HGT from foreign origin to S. thermophilus.

Previously, Hols et al. (18) identified putative HGT events in the genomes of S. thermophilus strains CNRZ1066 and LMG 18311. In this study, we predicted the horizontally transferred genes in the S. thermophilus LMD9 genome and also thoroughly reanalyzed the genomes of S. thermophilus strains CNRZ1066 and LMG 18311 based on both gene composition and gene transfer mechanism-associated features. In order to identify strain-specific regions and indications for gene transfer or locus rearrangement, genome alignments of the strains from each species were performed (see Fig. S1 in the supplemental material). The alignment of the three S. thermophilus genomes showed that strain LMD9 has undergone more genome rearrangements than both other strains. Strain CNRZ1066 and strain LMG 18311 share a more conserved genome context (see Fig. S1A in the supplemental material).

Gene composition analysis, including GC content (see Fig. S2 in the supplemental material) and dinucleotide composition (see Fig. S3 in the supplemental material) analyses, revealed a list of putative horizontally transferred genes in the three S. thermophilus strains (Table (Table2;2; see also Table S1 in the supplemental material). In total, 197 genes were predicted as potentially acquired in the three S. thermophilus genomes, of which 118 genes are located in 28 gene clusters (Table (Table2).2). Over 60% of those genes were found to be associated with gene transfer mechanism-associated features, such as transposase, bacteriophage, and tRNA genes (see Table S1 in the supplemental material). Compared with the core gene set of 47 S. thermophilus strains, identified by a recent comparative genomic hybridization study (40), 30 of the core genes overlapped with our HGT prediction. Since most of these core genes are not found to be associated with any mobile element or located in a predicted HGT gene cluster, they may be incorrectly predicted to be horizontally transferred by the gene composition analysis (see Table S1 in the supplemental material).

Proposed horizontally transferred genes and gene clusters in S. thermophilus genomesa

The three genomes have several putative horizontally transferred gene clusters in common, including clusters S1, S5, S23, S24, S25, and S26. Cluster S1 encodes several lantibiotic/bacteriocin biosynthesis proteins and an exporter, i.e., labB, labC, and labT. These genes in S. thermophilus strains CNRZ1066 and LMG 18311 have been described by Hols et al. (18) as horizontally transferred genes. An ABC-type transporter in cluster S5 (stu0324, str0324, STER_1694) was found to have the best homolog (40% identity) in L. helveticus DPC 4571. Cluster S26 encodes enzymes involved in the metabolism of sulfur-containing amino acids, which will be described in detail below.

We also found examples of strain-specific HGT events such as exopolysaccharide (EPS) biosynthesis genes. According to the distinct groups of polysaccharides defined by Delcour et al. (9), EPSs in this study refer to extracellular polysaccharides which are released into the medium or attached to the cell surface (capsular polysaccharides). EPSs are important for the rheology, texture, and “mouthfeel” of yogurt. In addition, they are believed to contribute to probiotic effects (35). Previous studies revealed that EPS biosynthesis genes can vary enormously in different LAB strains (49). The EPS biosynthesis genes either are obtained by HGT or may have evolved much faster than other genes. The clusters S10, S12, and S13 of S. thermophilus strains LMD9, CNRZ1066 and LMG 18311, respectively, have different gene compositions and contexts and share limited sequence similarities (Table (Table3),3), suggesting that their origins differ.

Predicted horizontally transferred EPS biosynthesis clustersa

Cluster S21 of S. thermophilus LMD9 contains STER_1698, encoding a phage resistance protein with a Pfam Abi_2 domain. Homologs of this protein were found to be involved in bacteriophage resistance, mediated through abortive infection in Lactococcus species (2). They belong to the AbiD, AbiD1, or AbiF family of proteins encoded by lactococcal plasmids and are active mainly against bacteriophage groups 936 and C2. The unique presence of this phage resistance gene in strain LMD9 might indicate a strain-dependent antiphage characteristic. Moreover, the genes present in cluster S19 of the same strain encode a novel phage resistance system, CRISPR3 (for clustered regularly interspaced short palindromic repeats), with a low GC content (31%) indicative of a foreign origin. It agrees with the observation of Horvath et al. that CRISPR loci were possibly horizontally transferred and that they may play an important role in adaptation to a specific environment (19, 20).

Putative HGT from foreign origin to L. bulgaricus.

A similar analysis of the genomes of L. bulgaricus strains ATCC 11842 and ATCC BAA365 also revealed several putative HGT events (Table (Table4;4; see also Table S1 and Fig. S2 and S3 in the supplemental material). The chromosomal regions with significantly lower GC content could be the result of HGT from either S. thermophilus or other low-GC organisms. The high δ values, showing higher dissimilarity with dinucleotide composition, and the features of the flanking regions related to mobile elements are indicative for the HGT event (Table (Table4;4; see also Table S1 in the supplemental material). In total, 149 genes (including pseudogenes) were identified as being putatively horizontally transferred, 120 of which were found to be associated with gene transfer elements. The large majority (130 of the 149 genes) were distributed in 24 gene clusters.

Proposed horizontally transferred genes and gene clusters in L. bulgaricus genomesa

Among the predicted HGT events in L. bulgaricus, genes encoding transporter systems, restriction-modification systems, and EPS biosynthesis clusters are highly represented (Table (Table4).4). Genes present in the EPS biosynthesis clusters L18, L19, and L20 (Table (Table3)3) have low-GC content values (from 34% to 37%) and relatively high δ values (between 0.08 and 0.11), which are higher than those of about 90% of the genome fragments. This indicates that most EPS biosynthesis genes in L. bulgaricus are possibly derived from phylogenetically unrelated low-GC organisms.

Of the predicted horizontally transferred gene clusters, 15 out of 24 are found in both L. bulgaricus genomes (Table (Table4).4). For example, cluster L6, harboring genes encoding an ABC-type transporter, has the best blast hit with Streptococcus pneumoniae TIGR4. Another example is the folate biosynthesis operon folB-folKE-folC-folP (cluster L3) which has been described previously by van de Guchte et al. (46). L. bulgaricus strains are not able to produce folate, one of the essential components in human diets (45). This could be due to the absence of the genes involved in the biosynthesis of p-aminobenzoic acid (PABA), a precursor of folate. The PABA biosynthesis genes are present in the genomes of S. thermophilus. During the cogrowth of the two organisms, the S. thermophilus cells may provide PABA to the L. bulgaricus cells, thereby enabling them to produce folate (43, 46). A glutamine transporter system (LBUL_0214-0217 or ldb0251-0254) present in the same cluster, L3, could also be acquired by HGT. All the putative horizontally transferred genes indicate that the region harboring gene cluster L3 could be an active region, with respect to genomic recombination. The ISL5-like (29) transposase gene (LBUL_0221) in this region provides additional suggestions for the predicted HGT events.

Another interesting finding is the identification of three eukaryotic-type serine/threonine protein kinase genes (ldb0301, ldb0334, and ldb2095 or LBUL_0255, LBUL_0290, and LBUL_1939) in gene clusters L5, L6, and L21, respectively. Their protein sequences share about 30% identity with S. thermophilus homologs. Notably, BLAST analysis found the homologs of these genes present only in the genomes of S. thermophilus, L. bulgaricus, and one Lactococcus strain but absent in the other sequenced LAB genomes. The nucleotide sequence compositions of these protein kinase genes were not very similar to those of the S. thermophilus or L. bulgaricus genomes, suggesting a putative horizontal transfer event between the kinase genes from a foreign origin to S. thermophilus and L. bulgaricus genomes. Serine/threonine protein kinases are commonly found in eukaryotes, and they have been reported to play a role in the bacterial growth and development in many bacterial species (28, 51). The function of the serine/threonine protein kinases in L. bulgaricus and S. thermophilus remains to be discovered.

HGT between Lactobacillus bulgaricus and Streptococcus thermophilus: cbs-cblB(cglB)-cysE as an example.

In addition to the HGT events in L. bulgaricus and S. thermophilus genomes from foreign origins, we also identified putative HGT events between the L. bulgaricus and S. thermophilus genomes. The EPS biosynthesis genes epsIM and epsIL (ldb1998-2000) in cluster L20 of L. bulgaricus were possibly acquired from S. thermophilus, since they share high sequence homology with the eps15 gene (stu1092) of S. thermophilus LMG 18311 and eps3I of another S. thermophilus strain, with 46% and 49% identity at the amino acid level, respectively (Table (Table3).3). epsIL (ldb1999-2000) is truncated due to an in-frame stop codon. A phylogenetic analysis using the homologs of epsIM and epsIL supported the hypothesis that the genes were transferred from S. thermophilus to L. bulgaricus (data not shown). The transfer of EPS biosynthesis genes between S. thermophilus and L. bulgaricus could play a role in (the optimization of) the physical interaction between the species in mixed yogurt cultures and, thus, in the exchange of metabolites and/or stimulatory factors. This hypothesis is supported by the observation that EPS production is also influenced by the cocultivation of the two species (S. Sieuwerts, C. Ingham, and J. van Hylckama Vlieg, personal communication). For kefir, similar findings have been obtained for Lactobacillus kefiranofaciens and Saccharomyces cerevisiae (5). The physical contact between the two species enhanced the capsular kefiran production of L. kefiranofaciens.

Another example of an HGT event between S. thermophilus and L. bulgaricus is the cbs-cblB(cglB)-cysE gene cluster encoding enzymes involved in sulfur-containing amino acid metabolism. The gene cluster was originally named cysM2-metB2-cysE2 (3) but was renamed in our previous study of the basis of a consistent functional annotation in LAB (31). Since this HGT event could give insight into the coevolution in milk and protocooperation between the two bacterial species during yogurt fermentation, we performed an in-depth comparative analysis on the evolution of this gene cluster, in light of phylogeny, gene context, and sequence features.

The cbs-cblB(cglB)-cysE gene cluster is located in a 3.6-kb region (including a 600-bp upstream noncoding region) in S. thermophilus cluster S26. The corresponding genes in L. bulgaricus and S. thermophilus share more than 94% nucleotide sequence identity. Using the δρ-Web tool, we analyzed the sequence similarity between this DNA fragment and the whole genomes of S. thermophilus, respectively: the cbs-cblB(cglB)-cysE fragment has a very high δ value compared to the value for rest of the genome, indicating lower sequence composition similarity between the DNA fragment and the S. thermophilus genomes. The δ values of the cbs-cblB(cglB)-cysE cluster against the S. thermophilus genomes are much higher than their δ values against the L. bulgaricus genomes. Moreover, phylogenetic analysis also supported the prediction of this HGT event and provided more insight into the evolutionary events leading to the transfer of this cluster (Fig. (Fig.1).1). The cbs-cblB(cglB) gene clusters of S. thermophilus are closely linked to those of L. bulgaricus and L. helveticus (lactobacillus branch) instead of L. lactis in the classic phylogenetic tree, suggesting an HGT event from L. bulgaricus or L. helveticus to S. thermophilus. Although the sequence composition of this gene cluster is more similar to that of the L. bulgaricus genomes than to that of the L. helveticus genome (lower δ values and more-similar GC content), the difference is not significant enough to conclude that the cluster originates from L. bulgaricus.

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 ...

Gene context analysis indicates that L. bulgaricus, L. helveticus, and S. thermophilus have the same gene organization in this region (Fig. (Fig.2).2). Moreover, the locus is flanked by transposase genes, supporting recombination events to occur in this region. Multiple DNA sequence alignment of the cbs-cblB (cglB)-cysE cluster revealed a 179-bp DNA fragment with low GC content (34%) inserted into the open reading frame of the cysE gene in the L. bulgaricus genomes (see Fig. S4 in the supplemental material). This sequence is responsible for the truncation of the cysE gene in L. bulgaricus. An inverted repeat and a direct repeat are found in close proximity to the boundaries of the insertion sequence (see Fig. S4 in the supplemental material). The 179-bp DNA sequence shares 98% identity with a transposase gene located in the vicinity of an EPS biosynthesis cluster in L. bulgaricus ATCC BAA365 (cluster L18). We found that this insertion element frequently occurs in nonfunctional genes (pseudogenes) of L. bulgaricus genomes (see Table S2 in the supplemental material). For instance, in L. bulgaricus ATCC BAA365, it was found to be responsible for the truncation of the sucrose-6-phosphate hydrolase gene, a gene important for sucrose metabolism (22).

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 ...

The cbs-cblB(cglB)-cysE cluster in L. helveticus shares over 90% nucleotide sequence identity to the counterparts in L. bulgaricus. Interestingly, the cysE gene in L. helveticus is intact. This suggests that S. thermophilus could have acquired this gene cluster from L. helveticus. Both species are used in conjunction in the manufacture of Bulgarian-type yogurt (11).

Role of the horizontally transferred cbs-cblB(cglB)-cysE cluster in protocooperation.

The enzymes encoded by the cbs-cblB(cglB)-cysE gene cluster are involved in the metabolism of sulfur-containing amino acids. The metabolic pathway of the interconversion between cysteine and methionine containing these enzymes is shown in Fig. Fig.3.3. Serine acetyltransferase, encoded by cysE, initiates cysteine biosynthesis by converting l-serine to O-acetylserine. The enzymes encoded by cblB or cglB and cbs are involved in the conversion of homocysteine to cysteine. As indicated, CysE, CblB/CglB, and CBS in S. thermophilus are probably obtained by HGT from L. bulgaricus, while the cysE gene is truncated in L. bulgaricus. It should be noted that another copy of the cysE and cbl or cgl genes [in addition to those in the cbs-cblB(cglB)-cysE operon] is present in the S. thermophilus genomes.

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 ...

The sulfur-containing amino acids cysteine and methionine are present in low concentrations in milk proteins, 0.89% and 2.4%, respectively (42). In milk, only traces of methionine and cysteine can be detected as free amino acids (14). As the amount of sulfur-containing amino acids in milk either as free amino acids or derived from proteolysis may not meet the requirements for bacterial growth, bacteria need to synthesize these amino acids de novo. S. thermophilus strains are already equipped with most genes required for methionine and cysteine biosynthesis. However, under the evolutionary pressure, S. thermophilus might need to produce more cysteine/methionine, and a “foreign” gene cluster could probably have added value, for instance, when under the control of an alternative regulatory mechanism. In Streptococcus strains, it was found that the promoter region of cysK, the cbs paralog, has a conserved motif for binding to the transcriptional regulator CmbR (27). This activator regulates the metC-cysK operon encoding a cystathionine lyase and cysteine synthase in Lactococcus lactis (15). This regulatory motif is not found upstream of the cbs-cblB(cglB)-cysE gene cluster. However, we did find a GC-rich motif to be conserved in the upstream region of the cbs gene in the S. thermophilus and L. bulgaricus genomes, as well as in the Lactobacillus plantarum, Oenococcus, and Leuconostoc genomes, which may be a binding site for an alternative transcriptional regulator (see Fig. S5 in the supplemental material). After transferring the intact gene cluster to S. thermophilus, the L. bulgaricus strains may have inactivated their cysE genes, thereby losing the ability to synthesize cysteine. In contrast to S. thermophilus, L. bulgaricus lacks an active serine biosynthesis pathway; it is unnecessary to maintain a functional gene involved in this pathway for synthesizing cysteine from serine.

A recent proteomics study of S. thermophilus LMG 18311 revealed the upregulation of sulfur-containing amino acid biosynthesis genes, including cbs (cysM2) and cblB or cglB (metB2), when grown in coculture with L. bulgaricus ATCC 11842 (16). The stimulatory effect of L. bulgaricus on this biosynthetic pathway in S. thermophilus suggests that the enzymes are indeed of importance for protocooperation in yogurt manufacturing.


The protocooperation between L. bulgaricus and S. thermophilus in yogurt manufacturing has been previously described but with respect mainly to their dependency on growth factors and metabolic interactions. In this study, we identified HGT events between the yogurt bacteria and revealed protocooperation on the basis of exchanged and/or acquired genetic elements during evolution. The genome-wide analysis generated a list of genes and gene clusters, most probably obtained by HGT. The EPS biosynthesis proteins EPSIM and EPSIL in L. bulgaricus genomes were likely acquired from S. thermophilus. A gene cluster encoding the enzymes involved in sulfur-containing amino acid metabolism [cbs-cblB(cglB)-cysE] in S. thermophilus was probably transferred from L. bulgaricus strains. The predicted HGT events in bacteria used in yogurt manufacturing, S. thermophilus and L. bulgaricus, provide important information on their coevolution and their protocooperation strategies for adaptation to the milk environment. The new insights could be used advantageously to improve the control of cogrowth of both species in yogurt manufacturing and, accordingly, to improve product characteristics.

Supplementary Material

[Supplemental material]


This work was supported by grant CSI4017 from the Casimir program of the Ministry of Economic Affairs, The Netherlands.

We thank Ellen Looijesteijn for critically reading the manuscript and Celine Prakash for the analysis of the conserved regulatory motifs.


[down-pointing small open triangle]Published ahead of print on 24 April 2009.

Supplemental material for this article may be found at http://aem.asm.org/.


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