Authors: Naoko Tamai, Takahiro Ishii, Yusuke Sato, Hiroko Fujiya, Yasuyuki

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1 Supporting Information Authors: Naoko Tamai, Takahiro Ishii, Yusuke Sato, Hiroko Fujiya, Yasuyuki Muramatsu, Nobuaki Okabe, and Seigo Amachi* Manuscript title: Bromate Reduction by Rhodococcus sp. Br-6 in the Presence of Multiple Redox Mediators Number of pages: 13 Number of Figures: 7 Number of Tables: 2 1

2 Supporting information figure legends Figure S1. Bromate reduction by soil slurry incubated under various conditions. The slurry was incubated with 20 mm acetate and 250 µm bromate under aerobic, anaerobic, and transition conditions. In some cases, 10 mm nitrate or 5 mm chlorate was also added to the anaerobically incubated slurry. Symbols represent the averages of duplicate experiments, and error bars show the range of data. Ranges smaller than the symbols are not displayed. Figure S2. Phylogenetic tree showing the relationship between strain Br-6 and related Rhodococcus species based on 16S rrna gene sequences. The tree was constructed using the neighbor-joining method. Circles and triangles at the branch nodes represent bootstrap percentages (1,000 replicates): filled circles, %; open circles, 70 89%; open triangles, 50 69%. Values < 50% are not shown. The GenBank accession number for each reference strain is shown in parentheses. The scale bar represents the estimated number of substitutions per site. A bromate-reducing bacterium, B8, which was isolated by Davidson et al. 35 is also presented to show a phylogenetic difference from strain Br-6. Figure S3. Bromate reduction by strain Br-6 in the presence of AQDS, MV, and riboflavin. Cells were grown under transition conditions with 20 mm acetate, 250 µm bromate, and 4 µm each of redox mediators. Symbols represent the mean values obtained for triplicate determinations, and bars indicate standard deviations. 2

3 Figure S4. Effect of various concentrations of resazurin (A), resorfin (B), and DCIP (C) on bromate reduction by strain Br-6. Strain Br-6 was grown under transition conditions with 20 mm acetate, 250 µm bromate, and appropriate concentrations of the redox mediator. Symbols represent the mean values obtained for triplicate determinations, and bars indicate standard deviations. Figure S5. Inhibition of DCIP-dependent NADH-oxidizing activity by dicumarol. The reaction mixture (1.5 ml) contained 20 mm Tris-HCl (ph 6.8), 200 µm NADH, 100 µm DCIP, 0 to 50 µm of dicumarol, and an appropriate amount of enzyme. Diaphorase activity was assayed spectrophotometrically at 30 C by monitoring the oxidation of NADH (ε 340 = 6.22 mm -1 cm -1 ). The result of negative control to which no enzyme was added is also shown. Figure S6. (A) Abiotic re-oxidation of DCIPH 2 by ferric iron. DCIP (50 µm) was first reduced to DCIPH 2 by ascorbic acid, and then FeCl 3 6H 2 O (40 to 120 µm) and NTA (0.5 to 1.5 mm) were mixed with DCIPH 2. The re-oxidation of DCIPH 2 was monitored spectrophotometrically at 600 nm. In one case, 50 µm bromate was added instead of ferric iron and NTA to confirm that DCIPH 2 is not re-oxidized directly by bromate. (B) Abiotic reduction of bromate by ferrous iron. Ferrous ethylenediamine sulfate tetrahydrate (100 to 300 µm) was mixed with 50 µm bromate, and bromate reduction was monitored. A representative result from two independent experiments is shown. 3

4 Figure S7. Bromate reduction by an NTA-washed cell suspension of strain Br-6. Cells of strain Br-6 washed twice with 20 mm Tris-HCl (black symbols) or with same buffer containing 335 µm NTA (red symbols) were incubated anaerobically with acetate (10 mm), bromate (50 µm), DCIP (10 µm), and NTA (335 µm) in the absence (squares) or presence (circles) of FeCl 3 6H 2 O (25 µm). Approximately 2.6 mg dry cells were used in the experiment. Symbols represent the averages of duplicate experiments, and error bars show the range of data. 4

5 Bromate (µm) aerobic! anaerobic! transition! anaerobic with NO 3 -! anaerobic with ClO 3 -! days! Fig. S1

6 Williamsia muralis (Y17384) Rhodococcus jostii (AB046357)! Rhodococcus koreensis (AF124343)! Rhodococcus maanshanensis (AB741451)! Rhodococcus globerulus (X80619) Bromate-reducing bacterium B8 (AF442524) Rhodococcus erythropolis (AJ717371) Rhodococcus marinonascens (X80617) Rhodococcus opacus (X80630) Rhodococcus rhodnii (X80621) Rhodococcus ruber (X80625) Rhodococcus pyridinovorans (AF173005) Bromate-reducing bacterium Br-6! Rhodococcus equi (AY741716)! Nocardia asteroides (Z36934) 0.005! Fig. S2

7 30 2 Bromate (µm)! No mediator! AQDS! MV! Riboflavin! 2! 4! 6! 8! days! Fig. S3

8 Bromate (µm) µm! 0.8 µm! 2 µm! 4 µm! 8 µm! 20 µm! 40 µm! 30 A B C Bromate (µm) µm! 0.8 µm! 2 µm! 4 µm! 8 µm! 20 µm! 40 µm! Bromate (µm) µm! 4 µm! 8 µm! 12 µm! 2! 4! 6! 8! days! 2! 4! 6! 8! days! 2! 4! 6! 8! days! Fig. S4

9 1.15! 1.1! Absorbance at 340 nm! 1.05! 1! 0.95! 0.9! No enzyme! 0 µm dicumarol! 10 µm dicumarol! 20 µm dicumarol! 50 µm dicumarol! 0.85! 1! 2! 3! 4! 5! 6! min! Fig. S5

10 25! A B The oxidized form of DCIP (µm)! 2 15! 1 5! 40 µm Fe 3+ /0.5 mm NTA! 80 µm Fe 3+ /1 mm NTA! 120 µm Fe 3+ /1.5 mm NTA! 120 µm Fe 3+ only! 1.5 mm NTA only! 50 µm BrO -! 3 No Fe 3+ and NTA! 2! 4! 6! 8! 1 minutes! Bromate (µm)! µm Fe 2+! 100 µm Fe 2+! 200 µm Fe 2+! 300 µm Fe 2+! 2! 4! 6! 24! hours! Fig. S6

11 Bromate (µm)! ! 2! 3! 4! 5! 6! hours! Fig. S7

12 Table S1. Composition of trace mineral element solution C 6 H 9 NO 6 (nitrilotriacetic acid: NTA) 64 g L -1 FeCl 3 6H 2 O 6.75 g L -1 MnCl 2 4H 2 O 0.5 g L -1 CoCl 2 6H 2 O 0.12 g L -1 CaCl 2 2H 2 O 0.5 g L -1 ZnCl g L -1 CuCl 2 2H 2 O g L -1 H 3 BO g L -1 Na 2 MoO 4 2H 2 O 0.12 g L -1 NaCl 5.0 g L -1 NiCl 2 6H 2 O 0.6 g L -1 Na 2 S 2 O 3 5H 2 O 0.13 g L -1

13 Table S2. Composition of vitamin solution D-Biotin 0.02 g L -1 Folic acid 0.02 g L -1 Pyridoxine-HCl 0.1 g L -1 Thiamine-HCl 0.05 g L -1 Riboflavin 0.05 g L -1 Nicotinic acid 0.05 g L -1 DL-Calcium panthothenate 0.05 g L -1 Vitamin B g L -1 p-aminobenzoic acid 0.05 g L -1 DL-Lipoic acid 0.05 g L -1