Métabolisme sucre-citrate chez Leuconostoc mesenteroides

Abstract

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

Leuconostoc, characteristics, use in dairy technology and prospects in functional foods

“…The acetate kinase directs the flux to the formation of acetate with production of ATP, the additional energy being used for growth, whereas the phosphotransacetylase (PTA) produces acetyl-CoA that is used for biosynthesis or for reoxydation of NADH with production of ethanol by alcohol dehydrogenase. The pta gene has been cloned and appears not to be associated with acetate kinase gene, both genes being as a monocistronic transcript (Bourel et al, 2001).…”

“…As Leuconostoc grows around neutral pH, the dianionic form (Hcit 2À ) of citrate is taken up in symport with a proton. The Arg 425 of the permease is implicated in the transport process of citrate and S-enantiomers of other substrates with a 2-hydroxycarboxylate motif (Bandell & Lolkema, 2000;Bourel et al, 2001). Internally, Hcit 2À is further degraded in acetate and oxaloacetate, which is decarboxylated to pyruvate, thus increasing the pyruvate pool.…”

“…Genes involved in the degradation of citrate are chromosomal and arranged in a clyR-mae-citDEF locus in Ln. mesenteroides (Bourel et al, 2001). The citrate permease (citP) genes from Ln.…”

“…lactis or Ln. mesenteroides (Bourel et al, 2001), the citrate utilization gene cluster (cit CDEFG) from Ln. mesenteroides or (cit MCDEFGRP) from Ln.…”

“…mesenteroides or (cit MCDEFGRP) from Ln. paramesenteroides (Martin, Corrales, de Mendoza, Lopez, & Magni, 1999;Bourel et al, 2001) and the gene of a putative regulatory protein, clyR (Bourel et al, 2001) were cloned. The diacetyl reductase has been purified and the gene cloned and sequenced (Rattray, MylingPetersen, Larsen, & Nilsson, 2003).…”