The ␣-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 M leucine, the ␣-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the ␣-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.Synthesis of the three branched-chain amino acids (BCAA), leucine, isoleucine, and valine, has been studied in detail in organisms as diverse as bacteria, fungi, and plants (for reviews, see references 8, 32, 57, and 58). A particular feature of this synthesis is that it is carried out, in part, by the same enzymes for the three amino acids (Fig. 1). However, the relative amounts of the three amino acids in cell proteins are not the same, since leucine, valine, and isoleucine represent 39, 36, and 25%, respectively, of the total BCAA in Escherichia coli (40). Regulation of the BCAA synthesis is therefore necessarily complex. A global regulation model has been established only for E. coli (58). Two levels of regulation, concerning gene transcription and enzyme inhibition, have previously been described.E. coli genes which encode BCAA synthesis enzymes form three clusters and are organized in five transcription units (2). Initiation of transcription of all the genes except ilvC is regulated by nonspecific, pleiotropic regulators responding to amino acid starvation and different changes in the medium (58). In addition to this control, a mechanism of transcriptional attenuation dependent on the synthesis of a leader peptide as described first for the tryptophan operon (33) negatively regulates ilvBN, ilvGMEDA, and leuABCD. These mechanisms allow the expression of the enzymes necessary for BCAA synthesis when needed.Activity of several BCAA-synthesizing enzymes is regulated by retroinhibition (Fig. 1). This control is simple in the case of leucine and isoleucine, since each amino acid inhibits an enzyme specific for an early step of its synthesis (LeuA and IlvA, respectively [53,57]). In contrast, the first enzyme involved in valine synthesis is also required fo...

This study illustrates that a probiotic food containing B. lactis CNCM I-2494 strain reduces visceral hypersensitivity associated with acute stress by normalizing intestinal epithelial barrier via a synergistic interplay with the different probiotic strains and/or metabolites contained in this product.

Live Lactobacillus casei is present in fermented dairy products and has beneficial properties for human health. In the human digestive tract, the resident flora generally prevents the establishment of ingested lactic acid bacteria, the presence of which is therefore transient. The aim of this work was to determine if L. casei DN-114 001 survives during transit and how this bacterium behaves in the digestive environment. We used the human flora-associated (HFA) mouse model. L. casei DN-114 001 was genetically modified by the introduction of erm and lux genes, encoding erythromycin resistance and luciferase, respectively. For this modified strain (DN-240 041), light emission related to luciferase expression could easily be detected in the contents of the digestive tract. When inoculated into the digestive tract of HFA mice, L. casei (DN-240 041) survives but is eliminated with the same kinetics as an inert transit marker, indicating that it does not establish itself. In pure culture of L. casei, luciferase activities were high in the exponential and early stationary growth phases but decreased to become undetectable 1 day after inoculation. Viability was only slightly reduced even after more than 5 days. After transit in HFA mice, luciferase activity was detected even when 5-day-old L. casei cultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 h of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampin. These results suggest that in HFA mice L. casei DN-240 041, and thus probably L. casei DN-114 001, is able to initiate new protein synthesis during its transit with the diet. The beneficial properties of L. casei-fermented milk for human health might be related to this protein synthesis in the digestive tract.Fermented milk containing live lactic acid bacteria shows probiotic effects (8, 35): yogurt with living Lactobacillus bulgaricus and Streptococcus thermophilus is far more efficient to prevent lactose intolerance than the same product with heatkilled bacteria (33). Live Lactobacillus casei reduces diarrhea (26, 27) and appears to modify the digestive microflora (9, 18) and to enhance the immune system during its transit in the digestive tract (DT) (25, 28). The question raised here is how the probiotic bacterium ingested with food survives and adapts to the environmental change from fermented milk to the DT.The animal model we used is, from a bacteriological point of view, close to the human DT: germfree mice associated with a human flora (2, 20). The bacterial genera that are dominant in humans remain dominant in such mice, in contrast with conventional mice in which lactobacilli are dominant (12).To study adaptation of L. casei in the DT, we developed a genetic approach successfully used with other lactic acid bacteria, Lactococcus lactis and S. thermophilus (6, 10, 11). It consists of transcriptional coupling of known promoters of the bacteria with the luciferase genes from ...

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novosynthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.Lactobacillus casei has attracted considerable interest as a probiotic over the last few years (15,19). The strain L. casei DN-114 001 has been shown to alleviate acute diarrhea in healthy children (21,22), to modulate the production of proinflammatory cytokines in Crohn's disease ex vivo (3), and to enhance the immune system during bacterial gastrointestinal (GI) transit (20). However, little is known about the survival of L. casei species and their physiological status in the environment of the GI tract.Probiotics are usually ingested with fermented milk during the late stationary phase of the bacterial cycle. At the onset of the stationary phase, the overall rate of protein synthesis has been shown to drop precipitously to a rate equivalent to approximately 0.2% of the exponential growth rate (13). These changes are concomitant with a decrease in overall mRNA and DNA synthesis (13). The behavior of such metabolically inactive cells in the GI environment was thus investigated with a view to identifying the mechanisms underlying their action on health.To carry out this work, we developed a genetic approach using transcriptional fusion of luciferase genes (luxA and luxB) and selected promoters to study the response of lactic acid bacteria to the GI tract environment (5). The same approach was then extended to a probiotic microorganism, L. casei DN-114 001, to study its response to the GI tract in the humanmicrobiota-associated (HMA) mouse model using transcriptional fusion of the modified L. casei lacTEGF operon promoter and luxA and luxB genes from Photorhabdus luminescens. Resulting data suggest that L. casei can init...