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An industrial strain of Lactobacillus delbrueckii subsp. bulgaricus was grown in a synthetic medium on lactose as carbon substrate, in a pH-regulated fermentor. Growth proceeded in two distinct phases separated by a transient stationary phase. Various experimental approaches were used to identify the cause of this growth arrest. Growth experiments in L. bulgaricus culture supernatant fluids collected at different cultivation times in fermentor, and supplemented or not with various nutritional solutions, enabled us to discard the possibility of a nutritional limitation. Tube cultures of L. bulgaricus in medium supplemented with various lactic acid concentrations showed a potential inhibition by this metabolic end product but confirmed that this inhibition was not responsible for the cessation of growth. It was concluded that at least one inhibitory compound was produced during the growth phase of the strain, and this compound disappeared from the medium in the transient stationary phase, enabling the growth to start again later in the culture. Indeed, the stoichiometric analysis of the culture showed, firstly, that unidentified carbon compounds were produced from lactose during growth, which were probably converted in lactic acid during the transient stationary phase and, secondly, that part of the amino acids consumed gave catabolic end products. Finally, bacteriocin-like compounds were not considered to be responsible for this growth arrest.

-Study of the duplicated glycolytic genes in Lactococcus lactis IL1403. The conversion of sugars into lactic acid is the main metabolic pathway providing energy to lactic acid bacteria. This conversion is also involved in production of different compounds participating to the organoleptic properties of fermented products. The L. lactis knowledge of the genome has given the access to sequences of genes encoding the enzymes involved in the two main metabolic pathways described for the fermentation of glucose in lactic acid bacteria: (1) the homofermentative pathway through glycolysis leading to two lactate molecules per glucose consumed; (2) the heterofermentative pathway through the Pentose Phosphate pathway giving one lactate, one acetate and one CO 2 per molecule of glucose. The research of the genes, corresponding to proteins involved in these metabolic pathways, revealed that some enzymes are encoded by 2 distinct genes. This fact could give to the cell the possibility to produce enzymes with different biochemical properties, or to control their expression according to specific conditions. Two copies of genes potentially encoding glyceraldehyde-3-phosphate dehydrogenase (gap) and enolase (eno) have been identified. Other microorganisms such as E. coli and B. subtilis also possess 2 gap genes sharing up to 60% homology, but having different functions. In L. lactis, gap1 and gap2 genes share around 80% identity at both the nucleotidic and protein level. The analysis of codon usage, the transcription and the effect of genes inactivation shows that gap1 is the only gene involved in glycolysis. The transcription of this essential gene is very high during all phases of growth. Low increase of the level of transcription could be evidenced during growth in glucose, a sugar inducing the Catabolite Repression. Moreover, the presence of potential fixation site for CcpA (Cre box) upstream of initiation transcription box -35 suggests that gap1 transcription is activated by this protein. In contrast, the gap2 gene is dispensable and expressed at a very low level in our experimental conditions. Finally, in opposition to GapB from B. subtilis, the product of L. lactis gap2 might not to be involved in the neoglucogenesis. enoA and enoB genes are coding for proteins sharing 55% identity with known enolase. In opposition to the gap genes, the eno genes does not share significant nucleotidic homologies together. However, enoA presents 87% identity with the enolase genes from sequenced Streptococcus species whereas enoB presents 95% identity with a plasmidic encoded gene isolated from Streptococcus thermophilus. These observations suggest that enoB was transferred from species to other. The analysis of codons bias strongly suggests that EnoA is the

-Growth and energetic parameters of Lactobacillus delbrueckii subsp. bulgaricus were measured during cultures in synthetic medium, at pH regulated at 6.4 and at free pH. These data enabled the actual biomass yield relative to ATP and the maintenance coefficient to be calculated, and the type of control between catabolism and anabolism to be identified. The cultures of L. delbrueckii subsp. bulgaricus at pH 6.4 or at a non-regulated pH were characterized by a concomitant and early decrease in both catabolism and anabolism. At a regulated pH, the growth decrease seems to be due to the accumulation of toxic compounds, acting on anabolic reactions. This inhibition led to a decrease in the catabolic flux, albeit to a lower extent than the anabolic flux. In this condition, the culture was characterized by an excess of energy. On the contrary, at free pH, the lactic acid production led to a pH decrease responsible for the slowing-down of the catabolic flux. At the same time, maintaining the internal pH in the acidified medium was more energy-consuming. The consequence of both the decrease in the glycolytic flux and the increase in the energy consumption led to an energy limitation of growth, as shown by the lower value of the maintenance coefficient.Lactic acid bacteria / Lactobacillus delbrueckii subsp. bulgaricus / energetic analysis / catabolism / anabolism / maintenance coefficient Résumé -Les apports de l'énergétique microbienne à l'analyse des cultures de Lactobacillus delbrueckii subsp. bulgaricus : identification du type de contrôle entre catabolisme et anabolisme. La croissance et les paramètres énergétiques de Lactobacillus delbrueckii subsp. bulgaricus sont quantifiés en cinétique de culture en milieu synthétique, à pH régulé à 6,4 et à pH libre. Ces données permettent d'estimer le rendement ponctuel de biomasse par rapport à l'ATP et le coefficient de maintenance, paramètres dont l'analyse permet d'identifier le type de contrôle reliant le catabolisme à l'anabolisme. Lors des cultures de L. delbrueckii subsp. bulgaricus à pH régulé à 6,4 comme à pH libre, un ralentissement concomitant du catabolisme et de l'anabolisme est observé très précocement en cours de culture. À pH régulé, le ralentissement de croissance semble dû à l'accumulation de produits toxiques, qui affecte en premier lieu l'anabolisme, l'inhibition de l'anabolisme provoquant un rétro-contrôle du flux catabolique, mais dans des proportions moindres que l'anabolisme. La culture est alors en situation d'excédent énergétique. Inversement, à pH libre, la production d'acide lactique provoque une diminution du pH responsable d'un ralentissement du catabolisme. En même temps, l'acidification engendre un coût énergétique supplémentaire pour le * Corresponding author: loubiere@insa-tlse.fr 40 M. Mercade et al.