The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus . We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential. Supplementary information The online version of this article (doi:10.1038/nbt1034) contains supplementary material, which is available to authorized users.

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The eight IS231 variants characterized so far (IS231 A-F, V and W) display similar transposases with an overall 40% identity. Comparison with all the prokaryotic transposable elements sequenced so far revealed that the IS231 transposases share two conserved regions with those of 35 other insertion sequences of wide origins. These insertion sequences, defining the IS4 family, have a common bipartite organization of their ends and are divided into two similarity groups. Interestingly, the transposase domains conserved within this family display similarities with the well known integrase domain shared by transposases of the IS3 and IS15 families, and integrases of retroelements. This domain is also found in IS30-related elements and Tn7 TnsB protein. Amino acid residues conserved throughout all these prokaryotic and eukaryotic mobile genetic elements define a major transposase/integrase motif, likely to play an important role in the transposition process.

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The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (␤-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.Lactic acid bacteria (LAB) are used worldwide in the preparation of fermented foods, including dairy products. They are also known for the potentially beneficial effects they may exert on the health of humans and animals (see, for example, reference 25). Their "generally recognized as safe" status (1), linked to their metabolic and technological properties, has recently led to their development as potential live-vaccine vehicles. Lactobacillus plantarum NCIMB8826 (17, 33) has been chosen for this purpose in our laboratory on the basis of its capability to persist in the mouse gastrointestinal and urogenital tracts (38). The ability to control the expression level of foreign proteins in LAB may offer certain advantages. However, while several controlled expression systems have been developed for Lactococcus lactis (9, 23), very few inducible promoters are available for lactobacilli: the xylR promoter from Lactobacillus pentosus (29), the ␣-amylase promoter from L. amylovorus (31), and the p-coumarate decarboxylase promoter from L. plantarum (4). One of the most promising lactococcal controlled expression systems is based on the autoregulatory properties of the L. lactis nisin gene cluster (7,23). Nisin is an antimicrobial peptide belonging to the family of lantibiotics (19) and is used as a natural preservative in the food industry (5). Nisin induces the transcription of the genes under control of the nisA and nisF promoters, via a two-component regulatory system (34, 37) consisting of the histidine protein kinase NisK and the response regulator NisR (14,21,22). A transferable nisin-controlled expression (NICE) system (24) based on the combination of the nisA promoter and the nisRK regulatory genes has recently been developed (7,20). It consists of two compatible replicons, a plasmid carrying the nisRK regulato...

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.

The UDP-MurNAc-pentapeptide is transferred to the outer face of the cell membrane by a lipid carrier and incorporated along with UDP-N-acetylglucosamine into the cell wall structure. The synthesis of other types of peptidoglycan precursors was demonstrated a few years ago in the context of several studies concerning vancomycin resistance. Vancomycin and other glycopeptide antibiotics can bind to the DAla-D-Ala terminus of pentapeptide-containing precursors by hydrogen bonding, thereby effectively blocking polymerization and preventing further cross-linking reactions (7, 41). Investigations of the molecular basis of vancomycin resistance started with strains of Enterococcus faecium and Enterococcus faecalis which showed inducible resistance to high levels of vancomycin and teicoplanin, another glycopeptide antibiotic. Examination of enzymes involved in cell wall synthesis in the resistant bacteria indicated that resistance to vancomycin was due to the synthesis of a novel type of peptidoglycan in which the terminal D-alanine residue was replaced by D-lactate, resulting in a drastic reduction of affinity for vancomycin (2,4,12,25,36). Two enzymes designated VanH and VanA are required for the synthesis of this alternative precursor (5). VanH is an ␣-ketoacid dehydrogenase that reduces pyruvate to D-lactate The synthesis of another type of peptidoglycan precursor has been described for Enterococcus gallinarum, which expresses inducible resistance to low levels of vancomycin but is susceptible to teicoplanin. In this case, the modified precursor terminates in D-serine instead of D-lactate (9). This feature results from the presence of another variant D-Ala-D-Ala ligase accepting D-serine (18).The genera Lactobacillus, Leuconostoc, and Pediococcus comprise strains and species constitutively resistant to vancomycin (15,20,33,39,46,49). Recently, peptidoglycan precursors from several of these lactic acid bacteria were analyzed. In Pediococcus pentosaceus and Lactobacillus casei (9, 26), the exclusive presence of a terminal D-lactate has been demonstrated. This presence could result from the action of a ligase which preferentially or exclusively catalyzes the synthesis of a D-Ala-D-Lac depsipeptide, as was suggested by Elisha and Courvalin (19). Analysis of Leuconostoc mesenteroides extracts identified a precursor that also terminates in D-lactate, but with an additional branched L-alanine In this paper, we report that the wild-type strain Lactobacillus plantarum NCIMB8826 is naturally resistant to high levels of vancomycin and teicoplanin and exclusively produces Dlactate-ending peptidoglycan precursors. We describe the construction of a strain defective for both D-and L-LDH, resulting in drastically reduced production of both isomers of lactate. We show that this alteration leads to the synthesis of a new type of precursor ending with D-alanine in addition to the usual muramyl depsipentapeptide observed in the wild-type strain,

We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and ␣-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via ␣-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.Diacetyl has a strong, buttery flavor and is essential at low concentrations in many dairy products, such as butter, buttermilk, and fresh cheeses. It is also considered to be the most important off-flavor in the brewing process and in the wine industry.Diacetyl is a by-product of fermentation by many microorganisms. It is produced chemically by oxidative decarboxylation of the metabolic intermediate ␣-acetolactate (␣-AL). One molecule of ␣-AL is produced from two molecules of pyruvate by the condensing enzyme, ␣-AL synthase (ALS) (14) (Fig. 1). In dairy fermentation, ␣-AL is mainly produced by lactic acid bacteria as a result of the metabolism of citric acid, a minor component of milk (7). The ability to utilize citric acid is found only in some Leuconostoc species and specific variants of Lactococcus lactis, namely, the biovar diacetylactis. Due to the balancing of redox equivalents, sugars are converted via pyruvate to lactic acid, while the more oxidized substrate citric acid is converted into ␣-AL and subsequently into acetoin via ␣-AL decarboxylase (ALDB) (Fig. 1). Specific L. lactis strains isolated from dairy cultures that produce large amounts of ␣-AL from citric acid were shown to lack the ALDB enzyme (8). In dairy fermentation, these mutants are responsible for production of relatively high levels of diacetyl, the direct product of chemical decarboxylation of ␣-AL. New selection methods (4, 6) and deletion of the aldB gene by genetic engineering (15) have made these mutants more readily available.Based on the knowledge of the pathways involved in diacetyl production, several metabolic engineering strategies have been designed to improve diacetyl production by lactic acid bacteria. Since citric acid is only a minor component in milk, most efforts have been directed at converting lactose into diacetyl. Studies based on the overproduction of ALS (als [13] or ilvBN [2]), inactivation of lactate dehydrogenase (LDH) (3, 13), pyruvate formate-lyase (1), or ALDB (15), or a combination of these strategies (13, 15), have resulted in efficient conversion of lactose and glucose into acetoin, especially in the case of LDH inactivation (13).However, diacetyl production from all these engineered strains was low. Attempts to combine both LDH and ALDB inactivation in order to maximize the rerouting towards ␣-AL and diacetyl have so far been unsuccessful. Studies by Lopez de Felipe et al. (11) demonstrated that overproduction of the Streptococcus mutans NADH oxidase (NOX) in L. lactis resulted in a phenotype similar to that of the LDH-deficient strain described by Platteeuw et al. (12). In aerated cultures of L. lactis, more than 80% of the fermented su...