Indigo is one of the oldest natural blue dyes. Microorganisms and their enzymatic activities are deeply involved in the traditional indigo staining procedure. To elucidate the mechanism of the microbial indigo reduction, we directly performed cyclic voltammetry on alkaline fermenting dye suspensions. A pair of characteristic redox peaks of leuco-indigo was observed in a supernatant fluid of the fermenting dye suspension. On the other hand, it was found that the indigo/leuco-indigo redox couple mediated two-way microbially catalyzed oxidation and reduction in a sediment-rich suspension of the fermenting suspension. Acetaldehyde was supposed to be the electron donor and acceptor of the catalytic reactions. In order to verify the bioelectrocatalytic reaction, we isolated indigo-reducing bacterium K2-3′ from the fermenting suspension, and the two-way bioelectrocatalysis was successfully restaged in a model system containing K2-3′ and methyl viologen (as a soluble mediator instead of indigo) as well as acetaldehyde at pH 10.

Leptin is a key regulator of energy intake and expenditure. This peptide hormone is expressed in mouse white adipose tissue, but hardly expressed in 3T3-L1 adipocytes. Using bisulfite sequencing, we found that CpG islands in the leptin promoter are highly methylated in 3T3-L1cells. 5-azacytidine, an inhibitor of DNA methyltransferase, markedly increased leptin expression as pre-adipocytes matured into adipocytes. Remarkably, leptin expression was stimulated by insulin in adipocytes derived from precursor cells exposed to 5-azacytidine, but suppressed by thiazolidinedione and dexamethasone. In contrast, adipocytes derived from untreated precursor cells were unresponsive to both 5-azacytidine and hormonal stimuli, although lipid accumulation was sufficient to boost leptin expression in the absence of demethylation. Taken together, the results suggest that leptin expression in 3T3-L1 cells requires DNA demethylation prior to adipogenesis, transcriptional activation during adipogenesis, and lipid accumulation after adipogenesis.

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In natural indigo dyeing, the water-insoluble indigo included in the composted indigo leaves called sukumo is converted to water-soluble leuco-indigo through the reduction activities of microorganisms under alkaline conditions. To understand the relationship between indigo reduction and microorganisms in indigo-fermentation suspensions, we isolated and identified the microorganisms that reduce indigo and analyzed the microbiota in indigo-fermentation suspensions. Indigo-reducing microorganisms, which were not isolated by means of a conventional indigo carmine-reduction assay method, were isolated by using indigo as a direct substrate and further identified and characterized. We succeeded in isolating bacteria closely related to Corynebacterium glutamicum, Chryseomicrobium aureum, Enterococcus sp. for the first time. Anthraquinone was found to be an effective mediator that facilitated the indigo-reduction activity of the isolated strains. On analysis of the microbiota in indigo-fermentation suspensions, the ratio of indigo-reducing bacteria and others was found to be important for maintaining the indigo-reduction activity.

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