Supplementary data:

Identification of 2,4-dihydroxylamino-nitrotoluene produced by TNT degrader Pseudomonas sp. strain TM15 in anoxic environment

Note: Pseudomonas sp. TM15 produced an unidentified metabolite in anoxic environment, so we need to identify the metabolite. The metabolite may be 2,4-dihydroxylamino-6-nitrotoluene (2,4DHANT); however, we were not able to clearly identify this product because no standard chemical of 2,4DHANT was available.

Objectives:

To verify that our target metabolite is 2,4DHANT,

1. We performed IR and LC/MS analyses to characterize our purified compound.

2. We requested Dr. Robin Gerlach (Montana State University, Bozeman, MT), who had a low amount of 2,4DHANT, to analyze our purified sample by HPLC-UV measurement to reveal whether our known product was 2,4DHANT.

Conclusion:

1. IR analysis indicated the unidentified metabolite has hydroxyl group and aromatic ring and LC-MS analysis indicated that the product has m/z 199 of molecular ion.

2. The target metabolite was 2,4DHANT, judging from the results of HPLC-UV measurement that the UV spectrum of unidentified metabolite was identical to that of standard chemical of 2,4DHANT (Fig. 4-C).

Strains

Pseudomonas sp. strain TM15

Procedures:

1. Cells (Pseudomonas sp. TM15; 1 × 109 cells/ml) were inoculated into the M8 minimal medium (200 ml) including 100 mg/l TNT. The culture was incubated in the dark at 30oC for 6 h with shaking (120 rpm).

2. The cells were removed by centrifugation at 5000 × g for 20 sec, and then the culture fluid was filtered with membrane filters (0.2 μm, Toyo Roshi Kaisha Ltd., Tokyo, Japan).

3. The culture fluid (200 ml) was extracted twice with 50 ml of dichloromethane and/or diethyl ether by using 2-step extraction (please see the manuscript about the 2-step extraction), then the extracts were dried over anhydrous sodium sulfate, and excess solvent was removed by rotary evaporation (R-114-EW-3, Sibata Scientific Technology Ltd, Tokyo, Japan).

4. Thin-layer chromatography (TLC) was performed to analyze the TNT metabolites extracted in organic solvents. The samples (20 μl) were spotted on a TLC sheet (10 by 20 cm; Silica gel 60 TLC aluminum sheet, Merck KGaA, Darmatadt, Frankfurt, Germany), and subsequently developed once by the ascending method with a solvent system comprised of ethanol-benzene-hexane (1:2:2 by volume) for a distance of 16 cm (total time 2 h). Spots indicating TNT metabolites were visualized at 254 nm under UV illumination (LPR-33/JM, TAITEC Co. Ltd.), as shown in Fig. 1.

5. To purify 2,4DHANT from various TNT metabolites, spots (A in Fig. 1) were extracted into acetonitrile. Purity of the purified metabolite was immediately assayed by HPLC.

6. (1) To obtain the mass spectra of this compound, the samples were measured by infrared absorption spectrometry (IR; Magna760, Thermo Co. Ltd., Tokyo, Japan) and liquid chromatography-mass spectrometry (LC-MS; API150EX, Applied Biosystems, Foster City, CA) (Fig. 2 and Fig. 3). LC-MS measurements were performed on a Chromolith Performance (4.6 mm × 100 mm; Hitachi Ltd., Tokyo, Japan) with acetonitrile-water (40:60) containing 1% trifluoroacetate as the mobile phase, with a flow rate of 0.4 ml/min using electron ionization mass spectrometry.

(2) To verify the metabolite is 2,4DHANT, we requested Dr. Robin Gerlach, who has 2,4DHANT, to analyze our sample (Fig. 4).

Results:

1

Kubota et al., Kyushu Institute of Technology