Study of the duplicated glycolytic genes in Lactococcus lactis IL140

The conversion of sugars into lactic acid is the main metabolic pathway pro viding energy to lactic acid bacteria. This conversion is also involved in production of different compounds participating to the organoleptic propert ies 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 m ain 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 p athway through the Pentose Phosphate pathway giving one lactate, one acetat e and one CO2 per molecule of glucose. The research of the genes, correspon ding to proteins involved in these metabolic pathways, revealed that some e nzymes are encoded by 2 distinct genes. This fact could give to the cell th e 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. col i 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 cod on usage, the transcription and the effect of genes inactivation shows that gap1 is the only gene involved in glycolysis. The transcription of this es sential 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 fi xation 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 exp erimental 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 sign ificant nucleotidic homologies together. However, enoA presents 87% identit y with the enolase genes from sequenced Streptococcus species whereas enoB presents 95% identity with a plasmidic encoded gene isolated from Streptoco ccus thermophilus. These observations suggest that enoB was transferred fro m species to other. The analysis of codons bias strongly suggests that EnoA is the main glycolytic enolase. The transcription of these two genes is hi gh during the exponential growth, 2 folds higher in glucose for enoA and si milar during glucose or galactose fermentation for enoB. enoA and enoB seem transcribed simultaneously during the growth. These results suggest that b oth genes may play a significant role in the glycolysis.