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Identification of stress proteins in Lactobacillus delbrueckii bulgaricus by two-dimensional electrophoresis. We studied Lactobacillus bulgaricus adaptation to an acid stress. In L. bulgaricus, adapted cells (30 min incubation at pH 4.75) were ?250 fold more tolerant to lethal acid stress (30 min at pH 3.6) than non adapted ones. In order to identify proteins induced during the adaptation, de novo protein synthesis was monitored by 35S methionine labeling of exponentially growing cultures, under standard (pH 6) and adaptive (pH 4.75) conditions. After 2 dimensional (2-D) electrophoresis separation, the protein patterns were compared. At least 30 protein spots showed an increased radioactivity level under acidic conditions. We determined the N-terminal amino acid sequence of 3 highly induced proteins. By comparison to databases, they were identified as the heat shock proteins GroES, GroEL and DnaK. A differential induction was observed for GroES and GroEL, suggesting a post-transcriptionnal regulation mechanism. In this report we established a method for the study of acid stress response in L. bulgaricus. This method will also be useful for further studies on the adaptation of this bacterium to environmental variations. Nous avons ?tudi? l'adaptation de la bact?rie lactique Lactobacillus bulgaricus au stress acide. Nous avons montr? que les bact?ries cultiv?es en milieu "neutre " (pH 6) pr?sentaient une forte l?talit? apr?s transfert dans un milieu acide (choc acide ? pH 3,6). En revanche, les bact?ries pr?alablement adapt?es dans un milieu acide non l?tal (pH 4,75) devenaient ?250 fois plus tol?rantes au choc acide. Apr?s marquage ? la m?thionine 35S et s?paration par ?lectrophor?se bidimensionnelle (2-D), les profils prot?iques obtenus en condition d'adaptation (pH 4,75) et en condition standard (pH 6) sont compar?s. On observe une augmentation significative (> 2) de l'intensit? d'environ 30 spots pendant l'adaptation. La d?termination des s?quences peptidiques N-terminales de 3 prot?ines fortement induites ? pH 4,75 permet d'identifier les prot?ines de choc thermique GroES, GroEL et DnaK. Nous observons des niveaux d'induction diff?rents pour GroES et GroEL, qui pourraient ?tre li?s ? une r?gulation post-transcriptionnelle. Au final, nous avons ?tabli une m?thode d'?tude de la r?ponse au stress acide chez L. bulgaricus. De fa?on plus g?n?rale, cette m?thode permettra d'?tudier l'adaptation de cette bact?rie aux variations environnementales.

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 CO2per 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 main glycolytic enolase. The transcription of these two genes is high during the exponential growth, 2 folds higher in glucose for enoA and similar during glucose or galactose fermentation for enoB. enoA and enoB seem transcribed simultaneously during the growth. These results suggest that both genes may play a significant role in the glycolysis. La conversion des sucres en acide lactique est la principale voie m?tabolique fournissant l'?nergie aux bact?ries lactiques. Cette conversion est ?galement impliqu?e dans la production de diff?rents compos?s participant aux propri?t?s organoleptiques des produits ferment?s. Deux voies m?taboliques ont ?t? d?crites pour la fermentation du glucose chez les bact?ries lactiques : (1) la voie homofermentaire ou glycolyse qui conduit ? la formation de deux mol?cules de lactate par mol?cule de glucose ; (2) la voie h?t?rofermentaire par la voie des pentoses phosphates donnant un lactate, un ac?tate et une mol?cule de CO2par mol?cule de glucose. La recherche des g?nes correspondant aux prot?ines n?cessaires au fonctionnement de ces voies m?taboliques a parfois r?v?l? que certaines enzymes pouvaient ?tre cod?es par deux g?nes distincts. En th?orie, cela donne la possibilit? ? la cellule de produire des enzymes aux propri?t?s biochimiques diff?rentes ou de contr?ler leur expression en fonction de conditions sp?cifiques. Ainsi des g?nes codant de possibles glyc?rald?hyde-3-phosphate d?shydrog?nases et ?nolases sont pr?sents en deux copies chez L. lactis. D'autres micro-organismes comme E. coli ou B. subtilis poss?dent ?galement deux g?nes gap homologues entre eux ? environ 60 % , mais dont la fonction diff?re. Chez L. lactis, les g?nes gap1 et gap2 pr?sentent environ 80 % d'identit? nucl?otidique et prot?ique. L'analyse des biais de codons, de la transcription et de l'effet de l'inactivation de ces g?nes montrent que seule Gap1 est impliqu?e dans la glycolyse. La transcription de ce g?ne essentiel est tr?s forte durant toutes les phases de la croissance. Une faible augmentation de son expression a ?t? mise en ?vidence lors de croissance en glucose, sucre induisant la r?pression catabolique. De plus, la pr?sence d'un site de fixation potentiel de la prot?ine CcpA (boite Cre), en amont de la boite d'initiation de la transcription -35, sugg?re une activation de la transcription de ce g?ne par cette prot?ine. Au contraire, le g?ne gap2 est dispensable et n'est quasiment pas transcrit dans nos conditions exp?rimentales. Enfin, contrairement ? l'enzyme GapB de B. subtilis, le produit du g?ne gap2 de L. lactis ne semble pas ?tre impliqu? sp?cifiquement dans la n?oglucog?n?se. Les g?nes enoA et enoB codent des prot?ines pr?sentant 55 % d'identit? avec l'ensemble des ?nolases caract?ris?es. Ces deux copies, contrairement aux g?nes gap, ne poss?dent qu'une tr?s faible homologie entre elles. Cependant, enoA partage 87 % d'identit? avec les g?nes ?nolases des Streptocoques et enoB environ 95 % avec un g?ne codant une ?nolase plasmidique chez Streptococcus thermophilus. Ces observations sugg?rent qu'enoB aurait ?t? transf?r? d'une esp?ce ? l'autre. L'analyse des biais de codons sugg?re fortement qu'EnoA soit l'?nolase glycolytique principale. La transcription de ces deux g?nes est forte au cours de la phase exponentielle, deux fois plus forte lors d'une fermentation en glucose compar? au galactose pour le g?ne enoA et semblable pour le g?ne enoB. enoA et enoB semblent transcrits simultan?ment au cours de la croissance. Ces diff?rents r?sultats sugg?rent que les deux g?nes pourraient contribuer significativement ? la glycolyse.

Network of regulation of gene transcription of the proteolytic system of Lactococcus lactis . The proteolytic system of lactococci that allows degradation of caseins and proteins of milk is complex. Milk proteins contain all amino acids necessary for growth of lactic acid bacteria. The proteolytic system consists of an extracellularly located proteinase, transport systems for di-tripeptides and oligopeptides and a multitude of intracellular peptidases. Expression of 13 genes was followed by transcriptional fusions in presence of different peptide sources. Transcription of 6 genes is repressed in media containing peptides and that of 4 genes (pepN, pepC, prtP and opp-pepO1 operon) by dipeptides containing one of the 3 branched amino acids (isoleucine, leucine and valine). Repression of gene transcription required that regulatory peptides are translocated into the cell and degraded in amino acids. Cell factors involved in this regulation were identified in derepressed mutants obtained by random mutagenesis by transposition. DtpT, a di-tripeptides transporter and CodY, homologous of the Bacillus subtilis pleiotropic regulator of transcription were the most frequently inactivated proteins. pepC, pepN and opp-pepO1 transcription is not repressed in codY and dtpT mutant. These genes of the proteolytic system belong to a same regulon since their expression is repressed by CodY regulator depending on intracellular concentration of branched amino acids or derivative products of them. Lactococcus lactis poss?de un syst?me prot?olytique complexe pour d?grader les cas?ines, prot?ines majoritaires du lait qui contiennent tous les acides amin?s n?cessaires ? sa croissance. Ce syst?me comprend une prot?ase de paroi extracellulaire, trois syst?mes de transport sp?cifiques des di-tripeptides et des oligopeptides et de nombreuses peptidases localis?es ? l'int?rieur de la cellule. L'influence de la source de peptides sur l'expression de 13 g?nes de ce syst?me a ?t? caract?ris?e gr?ce ? des fusions transcriptionnelles. La transcription de 6 de ces g?nes est r?prim?e en pr?sence d'une source riche en peptides et pour 4 d'entre eux (pepN, pepC, prtP et l'op?ron opp-pepO1) par des dipeptides qui contiennent au moins un des trois acides amin?s branch?s (leucine, isoleucine et valine). Pour permettre la r?pression de la transcription, les dipeptides doivent ?tre transport?s et hydrolys?s en acides amin?s dans la cellule. Des facteurs cellulaires impliqu?s dans la r?gulation de l'expression de ces diff?rents g?nes ont ?t? identifi?s dans des mutants d?r?prim?s, obtenus par mutagen?se al?atoire par transposition. Il s'agit du transporteur des di- et tripeptides (DtpT) et d'une prot?ine homologue ? la prot?ine CodY, qui est un r?gulateur pl?iotrope de la transcription de Bacillus subtilis. Nous avons montr? que les g?nes pepC, pepN et l'op?ron opp-pepO1 constituent un r?gulon dont l'expression est r?prim?e par CodY en fonction de la concentration intracellulaire en acides amin?s branch?s ou d'un produit de leur catabolisme.

The cell responds to environmental changes by triggering or repressing the expression of its genes in order to adapt its metabolism to the new conditions. The measure of promoter activities could thus allow an indirect assessment of the external signals that the cell has integrated and the modification of its metabolic potential. We have developed a method using this idea to evaluate the bacterial metabolism independently of its externalized products. Promoter activity is measured by following the expression of luciferases as reporter genes. Two different luciferase genes were used, luxAB from the bacteria Vibrio harveyi and luc from the eucaryote Photinuspyralis. The activity of the procaryotic and the eucaryotic enzymes is detectable by on-line measure-ment with whole cells when the cells provide the cofactors FMNH and ATP, respectively. This method is very sensitive, allowing the detection of weak promoter activity, or moderate transcription at low cell density. To demonstrate that this method is efficient, we studied promoter activities modulated by the presence of available amino acids with bacterial culture in milk. This allows us to see when the cells are starving, either in pure cultures or in mixed cultures with competing bacteria. As the two luciferases can be detected independently in the same culture, this method should allow the study of the interaction between strains in coculture at the molecular level. ? Inra / Elsevier, Paris.La cellule r?pond aux changements de condition de son milieu en activant ou r?primant de fa?on coordonn?e l'expression de ses g?nes pour adapter son m?tabolisme. La mesure de l'activit? des promoteurs pourrait donc permettre de conna?tre indirectement la perception que la cellule a du milieu et la mani?re dont elle va r?pondre. Nous avons donc con?u une m?thode fond?e sur ce principe pour ?valuer le m?tabolisme cellulaire, ind?pendamment de la mesure de ses produits. La mesure des activit?s promotrices se fait ? l'aide de g?nes rapporteurs ?metteurs de lumi?re, luxAB de Vibrio harveyi, ou luc de Photinus pyralis. La d?tection de l'activit? des luciferases est simple ? mettre en oeuvre et tr?s sensible. De plus, elle peut ?tre r?alis?e dans des milieux complexes comme le lait. La variation de l'activit? de promoteurs r?gul?s par la pr?sence ou l'absence d'acides amin?s permet ainsi de r?v?ler le moment o? le milieu est carence au cours d'une cin?tique de croissance. Cette m?thode peut s'appliquer ? l'?tude de cellules en culture pure, mais aussi en coculture. Il est alors possible d'?tudier le m?tabolisme d'une souche au sein d'un ?cosyst?me complexe pour mieux comprendre les ph?nom?nes de comp?tition ou de symbiose dans les levains. ? Inra / Elsevier, Paris.

The possibility to use modified microorganisms constructed by genetic engineering for food production in the next future will radically change our approach to optimize processes and improve the quality of the corresponding products. The research holding on this domain should allow the construction of strains more resistant to phage attack, producing new mol?cules (e.g. beneficial for health) or modified for their metabolism (growth, production of metabolites, resistance to stress, etc). The construction of such strains can be achieved by genetic engineering, either by modifying a pathway, or by expressing new genes. The engineering of known pathways allowed the development of new process and some of them are technically ready to be applied in the industry. For example, the expression of the anabolic acetolactate synthase, the inactivation of the LDH or of the acetolactate decarboxylase lead to the redirection of pyruvate to diacetyl instead of lactate. This might lead lo a 10-fold increase in diacetyl production, and there are still some possibilities of improvement. Another project of metabolic engineering that is quite advanced. is the modification of proteolysis in Lactococcus lactis, including the degradation of casein, peptides and amino acids. Most genes involved in the assimilation of proteins have been characterized (cell-wall protease. peptides transporters, and peptidases). Isogenic strains with different level of peptidase activities are built in order to improve peptidolysis or change the pattern of its product. In addition to the modification of already existing pathways, the introduction of new pathways in a cell could brighten the metabolic possibilities of bacteria and lead to the construction of new strains, producing new aroma for example. Building a new pathway generally requires the expression of heterologous genes. However. the certitude that a gene is absent supposes that there is an exhaustive knowledge of the bacterial genome, as some genes may be cryptic or uninduced under the conditions tested in the laboratory. Although the important progresses in the research related to lactic acid bacteria in the last years, only few pathways have been well characterized, covering, in terms of genetic information, a small percentage of the real metabolic possibilities. The size of the chromosome of most lactic acid bacteria is usually 1.8-3.4 Mb, about the half of the one of Escherichia coli, Bacil-lus subtilis and of some soil bacteria which have broad metabolic possibilities. On the other side, it is 4-5 fold the size of the smallest known genome, suggesting that the metabolic potentialities of lactic acid bacteria are underestimated. An investigation of data present in databases shows that only 6% of the genome of L. lactis are available. These data cover genes involved in amino acids and base biosynthesis (33%), degradation of peptides (17%). carbon catabolism ( 16%), stress responses ( 13%) and the remaining genes are related to the cell machinery (ribosome, replication, secretion, etc). The real potentialities of these bacteria remain to be estimated and exploited. ? Inra / Elsevier, Paris.La possibilit? d'utiliser prochainement des micro-organismes perfectionn?s par des modifications g?n?tiques construites in vitro changera profond?ment les approches d'am?lioration des proc?d?s et des produits qui en d?coulent. Les recherches actuelles dans le domaine des bact?ries lactiques devraient permettre de cr?er des souches plus r?sistantes aux phages, productrices de mol?cules nouvelles (? effet sant? par exemple) ou bien modifi?es pour leur m?tabolisme (croissance, production de m?tabolites, r?sistance aux stress...). L'ing?nierie m?tabolique permet de construire ce type de souches, soit en modifiant des voies pr?existantes, soit en introduisant l'ensemble des g?nes n?cessaire ? l'expression de nouvelles voies. La modification de voies connues a permis de d?velopper des proc?d?s dont certains sont techniquement pr?ts ? ?tre appliqu?s au niveau industriel comme des souches qui surproduisent environ 10 fois le diac?tyle. Un autre projet d'ing?nierie m?tabolique tr?s avanc? vise ? modifier la proi?olyse, depuis la d?gradation de la cas?ine jusqu'? la lib?ration de peptides non utilis?s et d'acides amin?s, dont certains seraient impliqu?s dans la g?n?ration de compos?s aromatiques. Au-del? de la modification de voies pr?existantes, l'introduction de nouvelles voies dans la cellule pourrait ?largir les possibilit?s de construire des souches ayant des propri?t?s particuli?res, comme la production de nouveaux ar?mes. Il est en g?n?ral admis que l'introduction d'une nouvelle voie n?cessite l'expression de g?nes ?trangers ? l'esp?ce dans l'h?te. Ce postulat suppose cependant une connaissance exhaustive des capacit?s m?taboliques des bact?ries. Malgr? le travail consid?rable r?alis? ces derni?res ann?es sur les bact?ries lactiques, seules quelques voies m?taboliques sont connues. Elles ne repr?sentent, en terme d'information g?n?tique, qu'une fraction minime des capacit?s potentielles de ces bact?ries. Le chromosome des bact?ries lactiques a une taille de 1,8 ? 3,4 Mb, c'est-?-dire environ la moiti? de celui de quelques bact?ries du sol aux capacit?s m?taboliques " ?tendues " et quatre ? cinq fois la taille du plus petit g?nome bact?rien connu. Un recensement r?cent de nos connaissances g?n?tiques chez les lactocoques montre que seulement 6 % des informations ont ?t? d?crypt?es. Les potentialit?s r?elles de ces bact?ries restent donc ? ?tre estim?es... et exploit?es. ? Inra / Elsevier, Paris

Cloning of long DNA segments (> 5 kb) in Bacillus subtilisis often unsuccessful when naturally occuring small (< 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAMβ1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmides does. In addition, replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids ,which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organisms.