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Despite the large number of techniques available for the transformation of bacteria, several species are still resistant to the introduction of foreign DNA. Oenococcus oeni are among the organisms that are particularly refractory to transformation. However, conjugal experiments from Lactococcus lactis to O. oeni with a new plasmid, pGID052, were performed via mobilization with success. This plasmid, a derivative of pORI19, encompasses: (i) the oriT of pIP501, which permitted the transfer to O. oeni, (ii) the replication genes of a native Leuconostoc citreum plasmid. Frequencies of 10(-7) conjugants per recipient were found. The transfer did not affect the structure of this low-copy-number plasmid. Moreover, pGID052 seems segregationally stable and could be used in the future as an expression vector.

International audienceSugar citrate cometabolism in Leuconostoc mesenteroides. Bacteria from the genus Leuconostoc play roles in the dairy industry. The most important functions of this bacteria are their ability to produce CO$_2$ and flavour compounds through lactose heterofermentation and citrate utilization. Although the biotechnological role of the citrate metabolism is very important and widely appreciated, little is known about the genetic properties of Leuconostoc spp. In our laboratory, we cloned the genes responsible for citrate metabolism (clyR mae citCDEFGOP cluster), for D-lactate dehydrogenase (ldhD) and for phosphotransacetylase (pta). In addition we have planned to construct new vectors and we have tried to improve a method to introduce recombinant DNA molecules into Leuconostoc as well. Characterization of the plasmid involved in this study is still in progress. The nucleotide sequence analysis of p22R revealed the presence of C5 cytosine methylase gene typical of type II restriction/modification system. The low transformation efficiency of some strains of Leuconostoc may be due to the presence of that plasmid. The construction of the defective strain for restriction activity could be very useful to genetic manipulations of Leuconostoc in the future.Les Leuconostoc sont utilisés dans l'industrie laitière pour leur capacité à produire du CO$_2$ et des composés d'arôme (diacétyle) grâce au cométabolisme du citrate et du lactose. Les gènes du métabolisme du citrate (clyR mae citCDEFGOP), de la lactate déshydrogénase et de la phosphotransacétylase ont été clonés dans notre laboratoire. Ces différents gènes semblent de bons candidats pour des expériences d'ingénierie métabolique. C'est pourquoi un des aspects de notre travail consiste à développer et améliorer les méthodes de transfert et de manipulation d'ADN de Leuconostoc. En parallèle, la caractérisation du plasmide de 10 kb de L. mesenteroides p22R est en cours. L'analyse de la séquence partielle a révélé la présence d'un gène codant pour une C5 cytosine méthylase. Cette méthylase est typique des systèmes de restriction/modification de type II. Il est possible que la présence de ce plasmide soit responsable de la faible efficacité du transfert de l'ADN chez les Leuconostoc. La construction d'une souche R$^-$M$^+$ devrait faciliter les manipulations ultérieures des bactéries du genre Leuconostoc