Physiological characterization of Enterococcus faecalis during azoreductase activity

Figures & data

Figure 1.  Reduction of Direct Blue 15, Methyl Red, and tartrazine by E. faecalis whole cells. The units on the y-axis are expressed as dye concentration (µM) per milligram of total protein. (a) Anaerobic reduction. The 10 ml culture inoculated with mid-log phase cells was incubated without shaking at 37°C. (b) Aerobic reduction. The 500 ml flasks containing 50 ml culture were incubated at 37°C and 200 rpm. Due to the high biomass, the ratio of dye concentration to amount to total protein on the y-axis is lower compared with the anaerobic assay. (c) PBSG medium (prolonged lag phase/growth-arrest). The 10 ml culture inoculated with mid-log phase cells (washed in PBSG buffer) was incubated without shaking at 37°C. The controls contained dye only in BHIS or PBSG. Each data point is the mean of triplicates.

Table I.  Kinetics of azo dye reduction by E. faecalis whole cells and cell extract.

	nmol/min/mg
	Methyl Red	Direct Blue 15	Tartrazine
Condition	Mean (SD)	Mean (SD)	Mean (SD)
Whole cell*†	9.83 (1.42)	4.83 (0.11)	6.43 (0.02)
(anaerobic)
Whole cell†	6.17 (0.38)	1.07 (0.08)	1.65 (0)
(aerobic)
PBSG*	8.61 (0.57)	ND*	0.11 (0.01)
(lag phase)
(anaerobic)
Cell extract	9.86 (1.91)	0.21 (0.04)	2.68 (0.03)
(NADH)
(anaerobic)
Cell extract	1.97 (1.17)	0.06 (0.01)	1.3 (0.45)
(NADPH)
(anaerobic)

The azo dye concentration used for all experiments was 20 µM. The whole cell protein content was determined by the Lowry method using BSA as the standard and the protein content for the cell extract was determined at 280 nm. All the experiments were done at 37°C. The data are the mean of triplicates. ND, not detected; SD, standard deviation.

*The amount of cells in the starting culture was ∼10⁷ cfu/ml.

^†BHIS medium.

Figure 2.  The effect of azo dyes on the growth of E. faecalis. (a) Tartrazine, 20 µM and 200 µM. (b) Direct Blue 15, 20 µM, and 200 µM. (c) Methyl Red, 20 µM and 200 µM. The data are a mean of triplicates.

Table II.  Generation time of E. faecalis in the presence of various azo dyes.

	Generation time (min)
	20 µM	200 µM
Azo dyes	Mean (SD)	Mean (SD)
Control (no dye)	56.3 (4.7)
Methyl Red	56.9 (0.4)	60.1 (1.2)
Tartrazine	48.7 (1.1)	40.1 (0.6)
Direct Blue 15	47.1 (2.6)	51.7 (4.0)

The cells were grown in BHIS anaerobically at 37°C. The control had cell growth in BHI without dye. The data are a mean of triplicates. SD, standard deviation.

Figure 3.  Structure of Methyl Red, Direct Blue 15 and tartrazine [5].

Rafii F, Franklin W, Cerniglia CE. Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora. Appl Environ Microbiol 1990; 56: 2146–51

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