Mechanisms of int (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride), and ctc (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in escherichia coli k-12

Abstract

The tetrazolium salts INT and CTC are frequently used as indicators of bacterial respiration. Mechanisms of tetrazolium reduction to formazan in eukaryotic cells have been proposed, however those in prokaryotes remain largely unresolved. We undertook studies to determine the sites, and degree of coupling for INT and CTC reduction in the aerobic Escherichia coli K-12 respiratory chain using both intact cells and inverted membrane vesicles. In addition, reduction in whole cells was assayed under anaerobic conditions which elicit different electron transport pathways. Results of experiments with inhibitors of specific electron transport components indicated that both CTC and INT were reduced prior to ubiquinone in the E. coli respiratory chain by the primary aerobic [succinate and NAD(P)H] dehydrogenases. INT was also reduced at ubiquinone and possibly cytochromes b555, 556. Quantitative CTC reduction was more closely correlated with respiration in whole cells than INT, but the reverse was true in inverted membrane vesicles. Both tetrazolium salts were reduced to significant degrees under anaerobic conditions, particularly glucose fermentation. Mid-point reduction potential of CTC was determined to be −200 mV by redox titration. However, it appears that CTC forms a weakly fluorescent, poorly-localized intracellular formazan at redox potentials higher than ca. −200 mV. Inhibition of the aerobic terminal oxidases with KCN or azide strongly increased INT-, but significantly decreased CTC reduction in whole cells, indicating that these agents may not be useful for optimizing CTC-formazan or CTC (+) cell numbers in some bacteria. However, several other chemical agents stimulated CTC and INT reduction. These results suggest strategies for optimizing methods using these tetrazolium salts for assessment of bacterial respiratory activity.