BackgroundLactic acid bacteria (LAB) are a group of gram-positive, lactic acid producing Firmicutes. They have been extensively used in food fermentations, including the production of various dairy products. The proteolytic system of LAB converts proteins to peptides and then to amino acids, which is essential for bacterial growth and also contributes significantly to flavor compounds as end-products. Recent developments in high-throughput genome sequencing and comparative genomics hybridization arrays provide us with opportunities to explore the diversity of the proteolytic system in various LAB strains.ResultsWe performed a genome-wide comparative genomics analysis of proteolytic system components, including cell-wall bound proteinase, peptide transporters and peptidases, in 22 sequenced LAB strains. The peptidase families PepP/PepQ/PepM, PepD and PepI/PepR/PepL are described as examples of our in silico approach to refine the distinction of subfamilies with different enzymatic activities. Comparison of protein 3D structures of proline peptidases PepI/PepR/PepL and esterase A allowed identification of a conserved core structure, which was then used to improve phylogenetic analysis and functional annotation within this protein superfamily.The diversity of proteolytic system components in 39 Lactococcus lactis strains was explored using pangenome comparative genome hybridization analysis. Variations were observed in the proteinase PrtP and its maturation protein PrtM, in one of the Opp transport systems and in several peptidases between strains from different Lactococcus subspecies or from different origin.ConclusionsThe improved functional annotation of the proteolytic system components provides an excellent framework for future experimental validations of predicted enzymatic activities. The genome sequence data can be coupled to other "omics" data e.g. transcriptomics and metabolomics for prediction of proteolytic and flavor-forming potential of LAB strains. Such an integrated approach can be used to tune the strain selection process in food fermentations.

4Lactic acid bacteria (LAB) have been widely used as starter or nonstarter cultures in the dairy industry for over a thousand years. They play an essential role in flavor formation during the fermentation of dairy products. Several metabolic routes can lead to the formation of flavor compounds when LAB are growing in milk. One of the main precursors for flavor compounds in milk is casein, although they can also be derived from fatty acids and sugars. The proteolytic system of LAB degrades casein into its constituent amino acids, which can be converted to flavor compounds. Although amino acid catabolism by LAB has been well researched (5, 31, 65, 78), many flavor-forming routes are yet to be discovered.Over 20 LAB genomes have been fully sequenced (1,11,12,16,19,41,44,45,53, 68,71). The available genomic information provides us with new opportunities to study the flavorforming potential of LAB. However, one of the main problems that one encounters while reconstructing flavor-forming routes based on the genomic information stored in the public databases is the inconsistency in the functional annotation for many of the relevant genes. These genes are mostly members of larger protein families. Moreover, even when the functional annotation in databases is appropriate, it sometimes reflects only part of the protein's full functional potential, since broad substrate specificities are often not taken into consideration in the annotation.We have now improved the functional annotation of the key enzymes in the formation of flavor compounds from amino acids by applying comparative genomics approaches that have been developed within our group to specify the annotation of homologous proteins, by combining phylogeny, gene context, and experimental evidence (32). We focused especially on the enzymes involved in the metabolism of sulfur-containing amino acids since these are known to be precursors of many flavor compounds in dairy fermentations. Comparative analysis of the various sequenced LAB species and strains resulted in an overall view of differences in their flavor-forming capacities.

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 CO 2 , 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.

The incompleteness of genome-scale metabolic models is a major bottleneck for systems biology approaches, which are based on large numbers of metabolites as identified and quantified by metabolomics. Many of the revealed secondary metabolites and/or their derivatives, such as flavor compounds, are non-essential in metabolism, and many of their synthesis pathways are unknown. In this study, we describe a novel approach, Reverse Pathway Engineering (RPE), which combines chemoinformatics and bioinformatics analyses, to predict the “missing links” between compounds of interest and their possible metabolic precursors by providing plausible chemical and/or enzymatic reactions. We demonstrate the added-value of the approach by using flavor-forming pathways in lactic acid bacteria (LAB) as an example. Established metabolic routes leading to the formation of flavor compounds from leucine were successfully replicated. Novel reactions involved in flavor formation, i.e. the conversion of alpha-hydroxy-isocaproate to 3-methylbutanoic acid and the synthesis of dimethyl sulfide, as well as the involved enzymes were successfully predicted. These new insights into the flavor-formation mechanisms in LAB can have a significant impact on improving the control of aroma formation in fermented food products. Since the input reaction databases and compounds are highly flexible, the RPE approach can be easily extended to a broad spectrum of applications, amongst others health/disease biomarker discovery as well as synthetic biology.

f Sulfuric volatile compounds derived from cysteine and methionine provide many dairy products with a characteristic odor and taste. To better understand and control the environmental dependencies of sulfuric volatile compound formation by the dairy starter bacteria, we have used the available genome sequence and experimental information to systematically evaluate the presence of the key enzymes and to reconstruct the general modes of transcription regulation for the corresponding genes. The genomic organization of the key genes is suggestive of a subdivision of the reaction network into five modules, where we observed distinct differences in the modular composition between the families Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, on the one hand, and the family Streptococcaceae, on the other. These differences are mirrored by the way in which transcription regulation of the genes is structured in these families. In the Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, the main shared mode of transcription regulation is methionine (Met) T-box-mediated regulation. In addition, the gene metK, Many of the characteristic flavors in fermented dairy products such as cheese and yoghurt are the result of metabolic reactions involving sulfur-containing amino acids. The microorganisms applied in these products degrade cysteine and methionine, resulting in the production of flavor components such as methanethiol, dimethyl sulfide (DMS), dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS). Insight into the regulatory signals and pathways that control the corresponding metabolic fluxes involved in the formation of these flavor compounds and their precursors is essential to rationally control and steer the flavor profiles of said dairy products.The microorganisms used to produce fermented dairy products belong to the taxonomic order Lactobacillales, which includes the families Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. Many of the respective species are characterized by the fact that they produce lactic acid and are therefore known as the lactic acid bacteria (LAB). The transcription of genes encoding the proteins that are involved in cysteine and methionine metabolism in lactic acid bacteria and other Lactobacillales is controlled by both regulator-binding and RNA structural switches. In various Streptococcaceae, the LysR-family transcription regulators MtaR and CmbR have been shown to be involved in activation as well as repression of genes such as cysD, cysK, metA, metC, metE, and metF (e.g., for Lactococcus lactis [25,70] and Streptococcus mutans [36,68]). The transcription regulator HomR was reported to control the expression of metB in S. mutans and Streptococcus thermophilus (69). In addition, three types of RNA structural switches for the regulation of cysteine and methionine metabolism have been reported for low-GC-content Grampositive bacteria: the T box, the S box, and the S MK box (14,22,59,77,80). These sequence elements at the 5= untranslated region of an...

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