SummaryIn many streptococci, competence for natural DNA transformation is regulated by the Rgg-type regulator ComR and the pheromone ComS, which is sensed intracellularly. We compared the ComRS systems of four model streptococcal species using in vitro and in silico approaches, to determine the mechanism of the ComRS-dependent regulation of competence. In all systems investigated, ComR was shown to be the proximal transcriptional activator of the expression of key competence genes. Efficient binding of ComR to DNA is strictly dependent on the presence of the pheromone (C-terminal ComS octapeptide), in contrast with other streptococcal Rgg-type regulators. The 20 bp palindromic ComR-box is the minimal genetic requirement for binding of ComR, and its sequence directly determines the expression level of genes under its control. Despite the apparent speciesspecific specialization of the ComR-ComS interaction, mutagenesis of ComS residues from Streptococcus thermophilus highlighted an unexpected permissiveness with respect to its biological activity. In agreement, heterologous ComS, and even primary sequence-unrelated, casein-derived octapeptides, were able to induce competence development in S. thermophilus. The lack of stringency of ComS sequence suggests that competence of a specific Streptococcus species may be modulated by other streptococci or by non-specific nutritive oligopeptides present in its environment.

In Lactococcus lactis, which is widely used as a starter in the cheese industry, the first step of aromatic and branched-chain amino acid degradation is a transamination which is catalyzed by two major aminotransferases. We have previously purified and characterized biochemically and genetically the aromatic aminotransferase, AraT. In the present study, we purified and studied the second enzyme, the branched-chain aminotransferase, BcaT. We cloned and sequenced the corresponding gene and used a mutant, along with the luciferase gene as the reporter, to study the role of the enzyme in amino acid metabolism and to reveal the regulation of gene transcription. BcaT catalyzes transamination of the three branched-chain amino acids and methionine and belongs to class IV of the pyridoxal 5-phosphate-dependent aminotransferases. In contrast to most of the previously described bacterial BcaTs, which are hexameric, this enzyme is homodimeric. It is responsible for 90% of the total isoleucine and valine aminotransferase activity of the cell and for 50 and 40% of the activity towards leucine and methionine, respectively. The original role of BcaT was probably biosynthetic since expression of its gene was repressed by free amino acids and especially by isoleucine. However, in dairy strains, which are auxotrophic for branched-chain amino acids, BcaT functions only as a catabolic enzyme that initiates the conversion of major aroma precursors. Since this enzyme is still active under cheese-ripening conditions, it certainly plays a major role in cheese flavor development.

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A novel plasmid named pGdh442 had previously been isolated from a plant Lactococcus lactis strain. This plasmid encodes two interesting properties with applications in the dairy industry: a glutamate dehydrogenase activity that stimulates amino acid conversion to aroma compounds, and cadmium/zinc resistance that can be used as a selectable marker. Moreover, this plasmid can be transferred naturally to other strains, but appears to be incompatible with certain other lactococcal plasmids. During this study, the complete sequence of pGdh442 (68 319 bp) was determined and analysed. This plasmid contains 67 ORFs that include 20 IS elements that may have mediated transfer events between L. lactis and other genera living in the same biotope, such as Streptococcus, Pediococcus and Lactobacillus. Even though it is a low-copy-number plasmid, it is relatively stable due to a theta replication mode and the presence of two genes involved in its maintenance system. However, pGdh442 is incompatible with pSK08-derived protease/lactose plasmids because both possess the same replication and partition system. pGdh442 is not self-transmissible, but can be naturally transmitted via mobilization by conjugative elements carried by the chromosome or by other plasmids, such as the 712-type sex factor, which is widely distributed in L. lactis. In addition to several genes already found on other L. lactis plasmids, such as the oligopeptide transport and utilization genes, pGdh442 also carries several genes not yet identified in L. lactis. Finally, it does not carry genes that would trigger concern over its presence in human food. INTRODUCTIONLactococcus lactis is a lactic acid bacterium (LAB) widely used as a starter culture for the production of various fermented dairy products. Although this bacterium is commonly found in milk, it is also naturally present on plants and on parts of the bodies of cows (Nomura et al., 2006;Salama et al., 1995;Teuber, 1995). Typically, Lactococcus strains possess an abundance of plasmid DNA. Many large L. lactis plasmids are self-transmissible or mobilizable by conjugative elements carried by the chromosome or plasmids of the host strain (Gasson et al., 1995). Through their natural transfer, these plasmids increase the probability of the gene moving between bacteria, and enable strains to acquire a wide variety of new genetic traits. They thus endow their hosts with many traits which offer a selective advantage to colonize specific biotopes. For example, the majority of dairy strains have adapted to growth in milk by acquiring plasmids that encode lactose catabolism (De Vos & Gasson, 1989), casein degradation (Kok, 1990), citrate utilization (Kempler & McKay, 1979) and oligopeptide transport (Yu et al., 1996). In addition, strains isolated from plant and animal environments, which contain a wide variety of cytotoxic compounds, have developed appropriate protection mechanisms (Putman et al., 2000), such as multidrug resistance, nisin resistance and heavy metal resistance (Dougherty et al., 1998;Liu et al.,...

We described a quorum-sensing mechanism in the streptococci genus involving a short hydrophobic peptide (SHP), which acts as a pheromone, and a transcriptional regulator belonging to the Rgg family. The shp/rgg genes, found in nearly all streptococcal genomes and in several copies in some, have been classified into three groups. We used a genetic approach to evaluate the functionality of the SHP/Rgg quorum-sensing mechanism, encoded by three selected shp/rgg loci, in pathogenic and non-pathogenic streptococci. We characterized the mature form of each SHP pheromone by mass-spectrometry. We produced synthetic peptides corresponding to these mature forms, and used them to study functional complementation and cross-talk between these different SHP/Rgg systems. We demonstrate that a SHP pheromone of one system can influence the activity of a different system. Interestingly, this does not seem to be dependent on the SHP/Rgg group and cross-talk between pathogenic and non-pathogenic streptococci is observed.

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Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branchedchain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V max /K m ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted D-2-hydroxyacids but not L-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the Dmandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD ؉ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-Methionine-␥-lyase (MGL) can convert Lmethionine to methanethiol (MTL), ␣-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiolproducing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via ␥-elimination is a key step in the formation of VSCs in B. linens.Due to their low detection threshold and diversity, volatile sulfur compounds (VSCs) are of prime importance in the overall flavor of cheese and make a significant contribution to the typical aromas of different cheeses (12,14,33). VSCs arise primarily from the degradation of L-methionine to methanethiol (MTL) by the cheese microflora. This thiol is a common precursor for a variety of other sulfur-bearing compounds including the auto-oxidation products (11), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and S-methylthioesters, primarily arising from chemical reaction of MTL with acyl coenzyme A (acyl-CoA) (22). Numerous studies have therefore been done to control and/or diversify VSC synthesis during the ripening process by the use of properly selected microorganisms (4, 6, 15, 43). Many cheese microorganisms are capable of producing VSCs from L-methionine. Some of them, such as brevibacteria, especially Brevibacterium linens (17), are known to be very good VSC producers while others, such as lactic acid bacteria (LAB), can produce only limited amounts of VSCs (14).The most direct route for MTL biosynthesis, is the L-methionine ␥-elimination that directly produces MTL, ␣-ketobutyrate, and ammonia from L-methionine. This L-methionine ␥-elimination activity is quite high in B. linens and corynebacteria (17) and is also suspected in several other cheese surface bacteria, such as Micrococcus luteus, Arthrobacter sp., and Staphylococcus equorum (8). In contrast, such activity is quite low in LAB (14). In B. linens, the methionine ␥-elimination is catalyzed by a L-methionine-␥-lyase (MGL), a pyridoxal phosphate (PLP)-dependent enzyme for which L-methionine is the best substrate (16). In contrast, in LAB the reaction is catalyzed by a cystathionine ␤-lyase (CBL) and a cystathionine ␥-lyase (CGL) which are only slightly active towards L-methionine (1, 10, 18). In LAB, another pathway for L-methionine conversion to VSCs also exists but produces limited amounts of MTL (7,35).Coryneform bacteria are generally found on the surface of smear cheeses and give the typical sulfur notes to cheeses such as Limburger, Tilsiter, Livarot, Epoisses, and Munster. To date, B. linens is the only food-grade bacterium from which MGL has been purified and characterized (16,26,31,38,39), but neither its protein sequence nor its gene...