title: Experimental conditions affect the site of tetrazolium violet reduction in the electron transport chain of Lactococcus lactis.
abstract: The reduction of tetrazolium salts to coloured formazans is often used as an indicator of cell metabolism during microbiology studies, although the reduction mechanisms have never clearly been established in bacteria. The objective of the present study was to identify the reduction mechanisms of tetrazolium violet (TV) in Lactococcus lactis using a mutagenesis approach, under two experimental conditions generally applied in microbiology: a plate test with growing cells, and a liquid test with non-growing (resting) cells. The results showed that in both tests, TV reduction resulted from electron transfer from an intracellular donor (mainly NADH) to TV via the electron transport chain (ETC), but the reduction sites in the ETC depended on experimental conditions. Using the plate test, menaquinones were essential for TV reduction and membrane NADH dehydrogenases (NoxA and/or NoxB) were partly involved in electron transfer to menaquinones. In this case, TV reduction mainly occurred outside the cells and in the outer part of the plasma membrane. During the liquid test, TV was directly reduced by NoxA and/or NoxB, probably in the inner part of the membrane, where NoxA and NoxB are localized. In this case, reduction was directly related to the intracellular NADH pool. Based on these findings, new applications for TV tests are proposed, such as NADH pool determination with the liquid test and the screening of mutants affected in menaquinone biosynthesis with the plate test. Preliminary results using other tetrazolium salts in the plate test showed that the reduction sites depended on the salt, suggesting that similar studies should be carried out with other tetrazolium salts so that the outcome of each test can be interpreted correctly.
authors.affiliation: INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France.
authors.forename: Sybille
authors.initials: S
authors.lastname: Tachon
authors.affiliation: Laboratoire de Génie des Procédés Microbiologiques et Alimentaires, AgroSup Dijon - Université de Bourgogne, INRA, 17 rue Sully, 21065 Dijon, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Michelon
authors.affiliation: INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Chambellon
authors.affiliation: INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France.
authors.forename: Monique
authors.initials: M
authors.lastname: Cantonnet
authors.affiliation: INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Mezange
authors.affiliation: INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France.
authors.forename: Lucy
authors.initials: L
authors.lastname: Henno
authors.affiliation: Laboratoire de Génie des Procédés Microbiologiques et Alimentaires, AgroSup Dijon - Université de Bourgogne, INRA, 17 rue Sully, 21065 Dijon, France.
authors.forename: Rémy
authors.initials: R
authors.lastname: Cachon
authors.affiliation: INRA, UR 477 Biochimie Bactérienne, F-78350 Jouy-en-Josas, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Yvon
mesh_terms: D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D004579:Electron Transport; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D013778:Tetrazolium Salts / Q000737:chemistry / Q000378:metabolism*; D024482:Vitamin K 2 / Q000737:chemistry / Q000378:metabolism
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D013778:Tetrazolium Salts; C531352:tetrazolium violet; D009243:NAD; D024482:Vitamin K 2; D009245:NADH Dehydrogenase