IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
TRANSCRIPTIONAL ANALYSIS OF THE GENES INVOLVED IN DIACETYIL SYNTHETIC ROUTE IN Lactococcus lactis
Autor/es:
G., REPIZO1; N. GARCÍA2; B.MARELLI1, P. LÓPEZ2; CH. MAGNI
Lugar:
tucuman
Reunión:
Simposio; II Simposio Internacional de Bacterias Lácticas; 2006
Resumen:
During glucose fermentation L. lactis produce and release to the medium lactic acid. At acidic external pH, the lactic acid is charged and can easily pass trough the membrane in its protonated form. Once inside the cell the acid dissociates which acidifies the cytoplasm and provokes a strong acidic stress. When external pH diminish until 5 units, L. lactis cells are capable of detecting that unfavorable condition on the medium and unchain a coordinated expression of the operons codifying for the proteins responsible for citrate transport and metabolism. In presence of this compound, the plasmidic citrate permease CitP is in charge of removing lactate form cytosol (through a citrate-2 – lactate-1 antiport). Following, one H+ is consumed per mol of citrate during the decarboxylation of oxalacetate (second reaction of the citrate fermentation pathway), which is favorable for the pH homeostasis. Finally, the pyruvate produced during citrate fermentation is derived to the synthesis of four carbon neutral compounds (4CNC). Although many works related to the overproduction of 4CNC in L. lactis has been published due to the importance that these compounds have for the food industry, there exist few of them that are focused in the regulation of the expression of the genes involved in the synthetic pathway. Taking this into account we decided to study the influence of external pH on regulation of transcription of the genes that codifies for the enzymes belonging to the route of 4CNC production and also to analyze the specificity of stress due to pH on the expression of such genes. In order to achieve that, transcriptional fusions to lacLM gene of the promoter regions corresponding to als, aldB, aldC and butB genes from Lactococcus lactis CRL264 were constructed. Moreover, a transcriptional analysis of these genes using RT-PCR was performed and the transcription start sites of the mentioned genes were mapped. When the promoter strengths were compared we observed that the levels of transcription were higher for Pals, intermediate for PaldC and PaldB, and lower for butBA operon. The expression of these four genes was analyzed in function of pH detecting an induction for Pals and PbutBA promoters. In the case of aldB and aldC promoters an induction was detected only trough transcriptional fusions to lacLM reporter gene. These results could be indicating that the citrate permease, the enzymes responsible for the citrate fermentation and the first enzyme of CNC4 pathway (ALS) are induced coordinately under acidic stress, conforming a mechanism of resistance to acidic conditions in L. lactis.