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.

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

“…Reduction of TV to formazan in liquid cultures of PA14, PA14nfxC, and PA14nfxC::mexF harboring the pBBR-mexS construct was quantified as previously described and compared to that in the same strains harboring the pBBR1MCS5 vector control (64). For each strain, 200 l of an overnight culture (adjusted to an optical density of 2 at 600 nm) was added to 25 ml of LB broth containing 25 g ml Ϫ1 gentamicin and TV at a final concentration of 0.01 mg ml Ϫ1 .…”

Two Lactococcus lactis thioredoxin paralogues play different roles in responses to arsenate and oxidative stress

“…Reduction of TV in Lac. lactis has been linked to menaquinones and the membrane-bound NADH dehydrogenases NoxA and NoxB in an NADHdependent manner (Tachon et al, 2009). It may thus be suggested that depletion of TrxA causes an increased availability of NADH, menaquinones or NoxA/NoxB through: (i) shifts in metabolic fluxes, (ii) transcriptional regulation and/or (iii) thiol/disulphide exchange control mechanisms.…”

Redox Potential: Monitoring and Role in Development of Aroma Compounds, Rheological Properties and Survival of Oxygen Sensitive Strains During the Manufacture of Fermented Dairy Products

“…In general, the majority of sulphur aromatic compounds come from methionine [46]. Methanethiol is easily oxidized to dimethyl disulphide and dimethyl trisulphide [47].…”