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Applied and Environmental Microbiology, December 2006, p. 7468-7476, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01421-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Metabolism of Thioamides by Ralstonia pickettii TA{triangledown}

Anthony G. Dodge,1,3 Jack E. Richman,2,3 Gilbert Johnson,2,3 and Lawrence P. Wackett1,2,3,4*

Department of Microbiology, Immunology, and Cancer Biology,1 Department of Biochemistry, Molecular Biology, and Biophysics,2 BioTechnology Institute,3 Center for Microbial and Plant Genomics, University of Minnesota, St. Paul, Minnesota 551084

Received 20 June 2006/ Accepted 18 September 2006


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ABSTRACT
 
Information on bacterial thioamide metabolism has focused on transformation of the antituberculosis drug ethionamide and related compounds by Mycobacterium tuberculosis. To study this metabolism more generally, a bacterium that grew using thioacetamide as the sole nitrogen source was isolated via enrichment culture. The bacterium was identified as Ralstonia pickettii and designated strain TA. Cells grown on thioacetamide also transformed other thioamide compounds. Transformation of the thioamides tested was dependent on oxygen. During thioamide degradation, sulfur was detected in the medium at the oxidation level of sulfite, further suggesting an oxygenase mechanism. R. pickettii TA did not grow on thiobenzamide as a nitrogen source, but resting cells converted thiobenzamide to benzamide, with thiobenzamide S-oxide and benzonitrile detected as intermediates. Thioacetamide S-oxide was detected as an intermediate during thioacetamide degradation, but the only accumulating metabolite of thioacetamide was identified as 3,5-dimethyl-1,2,4-thiadiazole, a compound shown to derive from spontaneous reaction of thioacetamide and oxygenated thioacetamide species. This dead-end metabolite accounted for only ca. 12% of the metabolized thioacetamide. Neither acetonitrile nor acetamide was detected during thioacetamide degradation, but R. pickettii grew on both compounds as nitrogen and carbon sources. It is proposed that R. pickettii TA degrades thioamides via a mechanism involving consecutive oxygenations of the thioamide sulfur atom.


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INTRODUCTION
 
Amide compounds are very common in biological systems, but thioamides are rare. Correspondingly, reports of amide metabolism are very common, whereas comparatively little has been reported on bacterial thioamide metabolism. Thioamides are found naturally in the copper-chelating compound methanobactin described in Methylosinus trichosporium OB3b (24). The antibiotic sulfinemycin, produced by Streptomyces albus NRRL 3384, has a primary thioamide S-oxide moiety (28). Thioacetamide has applications in leather, textile, paper, rubber, and petroleum industries (36), and 2,6-dichlorothiobenzamide (chlorthiamid) is used as a herbicide (20). Thioamide compounds such as 2-ethyl-4-pyridinecarbothioamide (ethionamide) are important second-line drugs in the treatment of multidrug-resistant Mycobacterium tuberculosis and M. leprae (34, 35). In M. tuberculosis, oxidation of the thioamide sulfur is a necessary step in converting the prodrug ethionamide to its active form (5, 13).

It is currently unclear how bacteria would metabolize thioamides in a manner that supports growth. There are two biochemically logical mechanisms by which thioamides could be metabolized to liberate ammonia and thus support growth as a nitrogen source. First, enzymes could directly hydrolyze the thioamide C-N bond. These reactions have been shown to be catalyzed by some peptidases, but thioamides are typically hydrolyzed slowly compared to structurally analogous amides (4, 7, 8, 29). A second mechanism involves oxygenation of the sulfur atom to generate intermediates that readily hydrolyze or eliminate to form metabolizable species, such as amides or nitriles (9, 13, 32). Metabolites produced by M. tuberculosis cells during ethionamide degradation are consistent with the second metabolic mechanism described above (13). However, this metabolism generates at least one cytotoxic chemical species from ethionamide (5, 13, 34), and the ability of M. tuberculosis to grow during ethionamide degradation has not been reported.

In a recent study, thioamides were provided as test compounds for the online University of Minnesota Biocatalysis and Biodegradation Pathway Prediction System (http://umbbd.msi.umn.edu/predict/) and were predicted to undergo C-N bond cleavage at the thioamide group to liberate ammonia (22). In the same study, thionicotinamide was used as a sole nitrogen source in a bacterial enrichment culture, and growth was observed. Although that study showed that a thioamide could be metabolized to support bacterial growth, the mechanism of nitrogen release was not established.

In the present study, a bacterium was isolated for its ability to grow on thioacetamide as its sole nitrogen source. A pure culture was obtained, determined to be a Ralstonia pickettii strain, and its mechanisms of thioacetamide and thiobenzamide metabolism were elucidated.


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MATERIALS AND METHODS
 
Enrichment culturing.
Enrichment cultures were conducted in a nitrogen-free minimal medium composed of a 0.050 M potassium phosphate buffer (pH 7) supplemented with essential elements as described previously (14). Sodium acetate, D-glucose, and sodium succinate were included at 0.2% (wt/vol) each as carbon sources. For the initial enrichment, thionicotinamide (Sigma-Aldrich, St. Louis, Missouri) was added (2.5 mM) as the sole nitrogen source to a 25-ml aliquot of the minimal medium. Soil (0.5 g) from an ornamental campus garden, which to our knowledge had not been previously treated with thioamide herbicides, was added as the inoculum, and the flask was incubated on a shaker at 28°C. After 14 days, 0.5 ml of the enrichment was used to inoculate another aliquot of minimal medium with thionicotinamide, which was incubated as described above. This process was then repeated three times as growth became evident in the transfer cultures. The fourth transfer culture was streaked for isolated colonies onto 0.25x LB plates and minimal medium plates with 2.5 mM thionicotinamide, prepared as described above with 1.5% agar. Distinct colony types were streaked onto additional plates, and single colonies were used to inoculate aliquots of the enrichment medium. When no single colony type or combination of individual colony types could grow with thionicotinamide as the sole nitrogen source, a fresh aliquot of minimal medium plus 2.5 mM thionicotinamide was inoculated using a sterile loop dragged through an area of confluent growth on a 0.25x LB plate. When growth was evident, 0.25 ml of the mixed culture was used to inoculate a fresh aliquot of minimal medium containing 2.5 mM thioacetamide (Alfa Aesar, Ward Hill, Massachusetts), followed by incubation as described above. The resulting culture was transferred twice and then streaked for isolated colonies onto 0.25x LB plates and minimal medium plates with 5 mM thioacetamide. Individual colony types were tested for the ability to grow with thioacetamide as a sole nitrogen source in the enrichment medium.

Growth of cultures and preparation of resting cells.
Cultures were started in 10- to 25-ml aliquots of minimal medium with 2 mM thioacetamide and 1% glucose by inoculation with a single colony from a freshly grown plate culture. All cultures were incubated on a shaker at 28°C until reaching an optical density at 600 nm (OD600) of 0.4. Cells were harvested by centrifugation (10 min at 3,500 x g), washed once with minimal medium, and then resuspended in minimal medium. For resting cell assays, washed cells were resuspended in minimal medium with 1% glucose, and the OD600 was adjusted and substrate was added as specified. For growth assays, washed cells were added to flasks of media to give an OD600 of 0.01. All treatments were prepared in triplicate. Flasks were incubated until the OD600 was constant. Assays for growth on compounds as carbon and nitrogen sources were conducted with substrates at 15 mM, except as noted.

Strain identification.
Template DNA for 16S rRNA gene PCR amplification was isolated from a single colony of the isolate from a thioacetamide-minimal medium plate by using a boiling preparation method described previously (14). Primers 27F and 1525R were used for amplification (27). Amplified DNA was purified for sequencing by using a QIAquick gel extraction kit (QIAGEN, Valencia, California). Primers 109r, 25f, 357f, 530f, 926f, and 1114f were used for sequencing (27). The assembled sequence was aligned against all DNA sequences deposited in GenBank by using BLAST (1). Additional PCR-based assays for strain identification were conducted as described previously (11).

Biochemical assays for strain identification (11) were conducted using minimal medium with 1 mM sodium nitrate (Sigma-Aldrich) or urea (Fisher Scientific, Fair Lawn, New Jersey) as the sole nitrogen source and 0.4% glucose as the carbon source or else 1% arabinose, phenylacetate (Sigma-Aldrich), mannitol (Fisher Scientific), adipic (hexanedioic) acid, capric (decanoic) acid (Acros Organics, Geel, Belgium), or sodium citrate (Mallinkrodt Specialty Chemicals, Paris, Kentucky) as the sole carbon source and 1 mM ammonium chloride as the nitrogen source.

Metabolism of thioamide compounds.
In addition to thioacetamide and thionicotinamide, the ability of the isolate to transform isothionicotinamide (Alfa Aesar); ethionamide (MP Biomedicals, Inc., Solon, Ohio); thiobenzamide, thioacetanilide, and 2-thiohydantoin (Sigma-Aldrich); and 2-thiouracil and rhodanine (Acros Organics) was evaluated. Stock solutions of the compounds were prepared at 100 mM in absolute ethanol, except thioacetamide, 2-thiohydantoin, 2-thiouracil, and rhodanine, which were prepared at 10 mM in minimal medium and sterilized by filtration through 25-mm syringe filters with 0.2-µm-pore-size Tuffryn membranes (Pall Corp., Ann Arbor, Michigan). All stock solutions were stored at –20°C. Resting cells that had been grown on 2 mM thioacetamide or 1 mM ammonium chloride as the sole nitrogen source were prepared at OD600 of 1.7. Cell suspensions (1-ml aliquots) were incubated in sterile 20-ml scintillation vials with thioamide substrates added to 0.5 mM. Aliquots of the substrates in the medium without cells were also prepared and incubated as controls. After 96 h, 0.1-ml aliquots from each vial were centrifuged, and the supernatants were diluted 1:9 in minimal medium and then analyzed by UV-visible (UV/vis) scans with a Beckman DU 7400 spectrophotometer (Fullerton, California). Assays for the growth of R. pickettii TA on all of the thioamides as nitrogen sources were prepared with 2 mM thioamide and incubated for 14 days.

For assays of thioacetamide and thiobenzamide degradation in the presence or absence of oxygen, 10-ml aliquots of resting cells grown on thioacetamide were prepared as described above. Aerobic treatments were incubated in 125-ml Erlenmeyer flasks, and oxygen-depleted treatments were in 50-ml side-arm Erlenmeyer flasks. Controls without cells were prepared as described above. To create oxygen-depleted culture conditions, the side-arm flasks with sterile medium were connected to an aspirator with sterile silicone tubing. The flasks were sealed with 20.5-mm Suba Seal white rubber septa (Sigma-Aldrich). The medium was sparged, and the headspace was flushed under aspirator vacuum with nitrogen gas flowing first through a 25-mm syringe filter as described above and then through a sterile 20-gauge needle inserted through the septum. After 5 min, the tubing attached to the aspirator was sealed at the side-arm with a pinch clamp and then disconnected from the aspirator. Nitrogen flow into the headspace was continued for a few seconds before the needle was withdrawn. Flasks were incubated on a shaker, and the contents were analyzed by UV/vis scans as described above.

Synthesis of thioamide S-oxides.
Thiobenzamide S-oxide was prepared by the oxidation of thiobenzamide with hydrogen peroxide in pyridine as described previously (42). The yellow crystals obtained were further purified by liquid chromatography on a silica column eluted with acetone. Yellow fractions were pooled and concentrated under vacuum. The resulting crystals were recrystallized twice from acetone. The melting point of the purified crystals was 135 to 137°C (reported melting points were 127 to 128°C [9], 128 to 129°C [25], and 133 to 134°C [46]). The UV spectrum of the compound in minimal medium had a {lambda}max of 330 nm. The extinction coefficient ({varepsilon} = 6,400) determined in 95:5 (vol/vol) dimethyl sulfoxide (DMSO)-water was similar to the published value (12). The 1H-nuclear magnetic resonance (NMR) spectrum was first determined in deuterated chloroform (CDCl3), and the chemical shifts were compared to published values acquired in the same solvent (42). Peaks observed at {delta} = 8.4, 8.1, and 7.8 ppm agreed with reported chemical shifts (42). However, most of the material appeared as a multiplet at {delta} = 7.7 to 7.4 ppm, which was not reported previously (42). Over a period of days, crystals dissolved in CDCl3 changed from a yellow solution to a colorless solution, and changes in the 1H-NMR spectrum were observed, suggesting that the compound was not stable in this solvent. The 1H-NMR spectrum was then determined in deuterated DMSO (DMSO-d6). The observed chemical shifts {delta} = 9.34 (1 H, bs, NH), 8.34 (1 H, bs, NH), and 7.58 to 7.44 (5 H, m, C6H5) ppm agreed with published chemical shifts of thiobenzamide S-oxide in DMSO-d6 (12). This spectrum was reproducible for samples stored several days in DMSO-d6 and therefore appears to be more accurate than the spectrum acquired in CDCl3. The different chemical shifts observed for each NH2 proton suggest that one proton is tightly hydrogen bonded to the S-oxide oxygen. For mass spectrometry (MS) analysis, crystals were dissolved in methanol (1 mg/ml), and the solution was injected into a Finnigan LCQ electrospray ion trap mass spectrometer (Thermo Electron Corp., Waltham, Massachusetts). A molecular ion [(m+H)/z = 154.0] consistent with thiobenzamide S-oxide (monoisotopic mass = 153.0) was detected. Thioacetamide S-oxide was prepared by adding hydrogen peroxide to thioacetamide dissolved in acetone as described previously (32). The infrared spectrum of the resulting crystals in Nujol agreed with the published spectrum for thioacetamide S-oxide (47). Crystals analyzed by MS as described above showed a molecular ion [(m+H)/z = 91.9] consistent with thioacetamide S-oxide (monoisotopic mass = 91.0). During melting-point determination, we observed decomposition beginning at 135°C, which is close to the temperatures reported as melting points (32, 43). The UV spectrum of the compound in minimal medium had a {lambda}max of 290 nm. The extinction coefficient determined in distilled H2O (dH2O) was 9,100, which is similar to reported values (32, 43).

Identification of thioamide degradation products.
Culture supernatants were assayed for sulfate by using a barium chloride-based turbidimetric assay (sensitive to 0.25 mM sulfate) (26). The assay for sulfur dioxide or sulfite was a modification of the standard phenanthroline method using reagents prepared as described previously (2). Culture supernatant aliquots of 0.200 ml were assayed by adding 1 ml of phenanthroline reagent and 0.1 ml of ferric ammonium sulfate reagent. After incubation for 10 min at room temperature, 0.200 ml of ammonium bifluoride reagent was added. After 5 min, the absorbance at 510 nm was read with a spectrophotometer. A sodium sulfite (Acros Organics) solution (~10 mM) was standardized by the iodide/iodate titration method (2) and used to prepare a standard curve (linear to 0.04 mM). Analysis for elemental sulfur was done by using a published colorimetric assay (10) (sensitive to 0.2 mM sulfur). To assay for sulfide production, deep-butt slants of minimal medium with 5 mM thioacetamide, 0.4% glucose, and 0.02% ferrous sulfate (Sigma-Aldrich) were inoculated and observed for the growth and production of black ferrous sulfide. Ammonia in culture supernatants was assayed by using an enzymatic assay kit (Sigma-Aldrich) that measured NADPH consumption during the incorporation of ammonia into {alpha}-ketoglutaric acid by L-glutamate dehydrogenase.

Metabolites of thiobenzamide and thioacetamide were identified by using gas chromatography (GC)-MS. For thiobenzamide degradation, resting cells were prepared at an OD600 of 2.5 with thiobenzamide added to 1 mM. For thioacetamide degradation, cells were prepared at an OD600 of 1.0 with thioacetamide added to 5 mM. Culture aliquots (10 ml) were centrifuged, and the supernatants were filtered and then extracted with 2 ml of methyl-tertiary-butyl ether (MTBE). To assay for acidic metabolites, a second aliquot was prepared in parallel and acidified to pH 3 with 0.1 N HCl before extraction. The MTBE fractions were analyzed with a Hewlett-Packard 6890 GC system and 5973 mass selective detector. For thiobenzamide metabolites, a fused silica Bpx5 (5% phenyl polysilphenylene-siloxane) bonded phase capillary column (30 m by 0.25 mm, 0.25-µm-diameter film; SGE, Austin, Texas) was used. The gas flow was 1 ml/min, and the inlet temperature was 250°C. The GC temperature profile was 5 min at 100°C, a 10°C/min ramp up to 300°C, and then holding for 5 min at 300°C. For thioacetamide metabolites, a Stabilwax-DA bonded polyethylene glycol capillary column (30 m by 0.25 mm, 0.25-µm-diameter film; Restek Corp., Bellefonte, Pennsylvania) was used. Gas flow and inlet temperature were as described above. The GC temperature profile was 3 min at 60°C, a 15°C/min ramp up to 240°C, and then holding for 5 min at 240°C.

High-performance liquid chromatography (HPLC) analysis of thiobenzamide and thioacetamide metabolites was conducted on a Series 1100 liquid chromatograph with a diode-array detector (Hewlett-Packard, Waldbronn, Germany) using a Discovery HS-PEG (polyethylene glycol) column (25 cm by 4.66 mm; particle size, 5 µm; Supelco, Bellefonte, Pennsylvania). The mobile phase was 5% acetonitrile in water. Thiobenzamide metabolites were eluted for 35 min and monitored at 224, 290, and 330 nm. Thioacetamide metabolites were eluted for 10 min and monitored at 200, 262, and 290 nm. Commercial and synthesized standards were used to prepare standard curves for determining the concentrations of observed metabolites. Resting cells were prepared in 25-ml aliquots. The cell density was adjusted to an OD600 of 2.5 for thiobenzamide degradation, and the substrate was added to 0.25 mM. For thioacetamide degradation, the cell density was adjusted to an OD600 of 1.0, and the substrate was added to 1.0 mM. Aliquots of 1 ml were removed at various time points, cells were pelleted with a microcentrifuge, and the supernatants were filtered using 13-mm syringe filters with 0.2-µm-pore-size polytetrafluoroethylene membranes (Pall Corp.). Filtered thiobenzamide supernatants were analyzed undiluted; thioacetamide supernatants were analyzed undiluted and diluted 1:3 with minimal medium.

Participating chemical species in the formation of 3,5-dimethyl-1,2,4-thiadiazole.
To test for spontaneous production of 3,5-dimethyl-1,2,4-thiadiazole, 6.5 mg of thioacetamide S-oxide and 17.3 mg of thioacetamide were stirred together in 10 ml of minimal medium for 40 h. Periodically, 1-ml aliquots were removed and extracted with MTBE, and the MTBE fractions were analyzed by GC-MS as described above. A solution of thioacetamide S-oxide only in minimal medium (4.2 mg/5 ml) was stirred, extracted, and analyzed by GC-MS as described above. Chemical oxidations of thioacetamide and thioacetamide S-oxide were conducted by adding 0.267 g of Oxone (Sigma-Aldrich) (potassium peroxymonosulfate) to 0.02 g of thioacetamide or thioacetamide S-oxide in 10 ml of minimal medium. The solutions were stirred at room temperature, and 1-ml aliquots were removed, extracted with MTBE, and analyzed by GC-MS as described above.

Synthesis and quantitation of 3,5-dimethyl-1,2,4-thiadiazole.
Synthesis and characterization of 3,5-dimethyl-1,2,4-thiadiazole has been described previously (39, 44). For the present study, synthesis was based on a generalized protocol for 3,5-disubstituted 1,2,4-thiadiazoles (39). Acetyl chloride (2.5 ml) was added gradually over 4 min to 4.91 g of thioacetamide dissolved in 100 ml of DMSO. After stirring for 20 min, another aliquot of acetyl chloride was added, and the mixture was stirred for 1 h. To separate and purify the product, the reaction mixture was added to 400 ml of dichloromethane, washed twice with dH2O, and dried over sodium sulfate. The volume was then reduced to 50 ml in a rotary evaporator. The concentrate was washed with 50 ml of dH2O, dried, and then filtered through silica gel. Solvent was distilled out of the mixture at atmospheric pressure, and the product was distilled under aspirator vacuum through a glass helix-packed 6-in. column. A center fraction was collected and determined to contain 90% 3,5-dimethyl-1,2,4-thiadiazole (1.1 g, 22% yield) by GC-MS. A standard curve (R2 = 0.9919) was prepared from the average corrected areas of concentration standards analyzed by GC-MS in triplicate.

16S rRNA gene sequence accession number.
The partial 16S rRNA gene sequence from R. pickettii TA was deposited in GenBank under accession number DQ908951.


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RESULTS
 
Enrichments and isolation of a thioacetamide-metabolizing bacterium.
Enrichments were conducted using thionicotinamide as the sole source of nitrogen. After four transfers, a mixed culture composed of at least five distinct colony types was obtained. None of the colony types were found to grow alone on thionicotinamide, and subsequently the mixed culture was used in another enrichment with thioacetamide as the sole source of nitrogen. After two transfers, a single colony type was isolated that was able to utilize thioacetamide as a sole source of nitrogen for growth. Microscopic observations revealed that the culture was composed of motile gram-negative rod-shaped cells. The pure culture grew to an OD600 of 0.6, with a generation time of 5.9 h, in minimal medium plus 1% glucose and 1 mM thioacetamide as a sole nitrogen source. A BLAST alignment in GenBank (10/06) showed that a 1,493-nucleotide sequence from the isolate's 16S rRNA gene was 99 to 100% identical to several strains of Ralstonia pickettii, 100% identical to Pseudomonas sp. strain BUV1, and 98 to 99% identical to a variety of other Ralstonia spp. and several other cultured and uncultured strains. Biochemical and PCR-based assays were then used to more definitively determine the identity of the strain. The isolate was able to grow on nitrate and urea as sole nitrogen sources and arabinose, phenylacetate, capric acid, citrate, and adipic acid as sole carbon sources but did not grow on mannitol as a carbon source. Conducting the PCR with the R. pickettii-specific primer set Rp-F1/Rp-R1 amplified a 210-bp fragment from DNA of the isolate, but no amplification was observed with primer sets Rp-F1/R38-R1 or Rm-F1/Rm-R1, which are specific for other Ralstonia spp. These results support the identity of the isolate as R. pickettii, based on published results of similar assays that were used to differentiate closely related Ralstonia spp. (11). The isolate was named R. pickettii TA.

Metabolism of thioamide substrates.
The ability of R. pickettii TA to metabolize a broad range of thioamide compounds was tested by taking advantage of the relatively strong thioamide absorbance band at approximately 260 to 270 nm or at longer wavelengths with extended chromophores. Those strong absorbance bands were monitored as evidence for the disappearance of the thioamide functional group. Resting cells were tested for potential metabolic activity following their growth on either thioacetamide or ammonium chloride as their sole nitrogen source. Cells pregrown on thioacetamide catalyzed reactions causing the complete or nearly complete disappearance of the thioamide absorbance bands, as evidenced by UV/vis spectroscopy (Fig. 1, curve A). A remarkable range of compounds was metabolized: alkyl-, phenyl-, pyridinyl-, and cyclic thioamides all showed loss of the absorbance band (Fig. 1, curve A). The loss of thioamide absorbance was indicated to be biologically mediated by parallel incubation conducted with cells pregrown on ammonium chloride, in which the thioamide band was typically undiminished (Fig. 1, curve B). No loss of thioamide absorbance was observed when the substrates were incubated in the medium without cells (Fig. 1, curve C). Only thiohydantoin and rhodanine among the thioamide compounds tested showed significant depletion via cells grown on ammonium salts (Fig. 1), which suggests that the major activity studied here is inducible. Despite the ability to transform all of the thioamide compounds tested, R. pickettii TA utilized only thioacetamide as a source of nitrogen for growth.


Figure 1
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  		FIG. 1. UV/vis spectra of supernatants from R. pickettii TA resting cell suspensions prepared with 0.5 mM concentrations of the pictured compounds and incubated for 96 h. Cells were pregrown on thioacetamide (spectra labeled A) or ammonium chloride (spectra labeled B) as the sole nitrogen source. Controls with the compounds incubated in minimal medium without cells are also shown (spectra labeled C).

Evidence for oxygenase-catalyzed thioamide metabolism.
To determine whether thioamide metabolism by R. pickettii TA might be mediated by a broad-specificity oxygenase(s), subsequent incubations were conducted under aerobic and appreciably anoxic conditions. Cell suspensions incubated under oxygen-depleted conditions showed minimal thioamide transformation, whereas fully aerobic treatments showed nearly complete transformation of both thioacetamide (Fig. 2A) and thiobenzamide (Fig. 2B). Since exclusion of oxygen was not rigorous, it is likely that the small amount of metabolism observed under the nitrogen atmosphere was dependent on trace amounts of oxygen.


Figure 2
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  		FIG. 2. UV/vis spectra of supernatants from R. pickettii TA cell resting cell suspensions that were incubated with 0.5 mM thioacetamide (A) or thiobenzamide (B). Cell suspensions were incubated aerobically (spectra labeled 1) or under oxygen-depleted conditions (spectra labeled 2) for 96 h. Cells in all treatments were pregrown on thioacetamide. Scans of the substrates incubated aerobically in minimal medium without cells are also shown (spectra labeled 3).

Oxygenase-catalyzed reactions with thioamides would be anticipated to occur at the electron-rich sulfur atom and lead to displacement of sulfur oxyanions. Assays for sulfur products released during thioacetamide metabolism were positive for sulfite and negative for sulfate, elemental sulfur, and sulfide. Degradation of 1 mM thioacetamide resulted in the accumulation of 0.3 mM sulfite in the medium. In water, sulfite is in equilibrium with sulfur dioxide gas. Thus, some sulfite loss by volatilization would be expected, and its accumulation would thus be substoichiometric.

Identification of thioamide degradation products.
Thioacetamide degradation was initially demonstrated by UV/vis spectroscopy with R. pickettii TA resting cells (Fig. 1), and similar substrate depletion was also observed during cell growth. In a resting cell suspension, ammonium ion was detected in the medium with a stoichiometry of 3.5 mM ammonia generated from 4.0 mM thioacetamide consumed. The culture filtrate derived from a 48-h resting cell suspension given 0.1 mM thiobenzamide was extracted with MTBE and analyzed by GC-MS. Metabolites were compared to commercial standards of thiobenzamide, benzonitrile, benzamide, and benzoic acid that had average retention times of 17.3, 8.3, 14.0, and 11.5 min, respectively. Analysis of the extract showed two peaks with GC retention times of 8.3 and 14.0 min. The identities of the two peaks were confirmed as benzonitrile and benzamide by MS (data not shown). Analysis of extracts obtained from acidified supernatant did not reveal any additional metabolites. Benzoic acid was not detected in either extraction, although authentic benzoic acid would be extracted and detected under these conditions. An assay for ammonium ion that was conducted using the culture filtrate was negative, confirming that the cells could not liberate ammonia from thiobenzamide or its degradation products.

To determine the order of metabolite production during thiobenzamide degradation, resting cell suspensions of R. pickettii TA cells grown on thioacetamide were incubated with 0.25 mM thiobenzamide, and aliquots were taken at time points and analyzed by reversed-phase HPLC for the presence of thiobenzamide, thiobenzamide S-oxide, benzonitrile, and benzamide. Commercial standards of thiobenzamide, benzonitrile, and benzamide had average elution times of 26.5, 11.2, and 6.7 min, respectively, and a synthesized standard of thiobenzamide S-oxide had an average elution time of 8.1 min. Plotting metabolite concentrations versus time showed conversion of thiobenzamide to benzonitrile and then conversion of benzonitrile to benzamide, which accumulated in the medium and was not degraded further (Fig. 3A). The concentrations of benzonitrile, benzamide, and thiobenzamide at ~20 h nearly equaled the initial thiobenzamide concentration. However, the final amount of benzamide produced from thiobenzamide was substoichiometric (Fig. 3A). This can be explained by the loss of some benzonitrile to volatilization, which was observed when benzonitrile was shaken in minimal medium without cells. Thiobenzamide S-oxide, the predicted first metabolite derived from oxygenase attack on thiobenzamide, could be detected at low levels early in the time course (Fig. 3B). The absorbance spectrum (not shown) of the peaks that eluted at 8.1 min had a {lambda}max at 330 nm, which is characteristic of thiobenzamide S-oxide. It appeared and largely disappeared by 24 h (Fig. 3B), during which time benzonitrile peaked and benzamide was appearing (Fig. 3A). Although the maximum concentration of this metabolite reached only 0.95 µM (Fig. 3B), its appearance and subsequent disappearance during thiobenzamide degradation could be detected above the background level of thiobenzamide S-oxide present as a trace impurity in the thiobenzamide used for the assay, which remained constant at ~0.40 µM (Fig. 3B). The sequential conversion of benzonitrile to benzamide was confirmed by giving resting cell suspensions benzonitrile, which was converted to benzamide. Benzamide was not metabolized further whether generated as an intermediate or provided as a starting material. A synthesized standard of {alpha}-aminophenylmethanesulfonic acid (30), considered to be a potential hydration product of thiobenzamide S,S-dioxide, eluted at 9.5 min and was not detected during thiobenzamide degradation.


Figure 3
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  		FIG. 3. HPLC analysis of metabolites in an R. pickettii TA resting cell suspension incubated with 0.25 mM thiobenzamide ({square}). (A) The concentrations of benzonitrile ({triangleup}) and benzamide ({diamond}) produced are shown. The concentration of thiobenzamide incubated in minimal medium without cells is also shown ({blacksquare}). (B) Thiobenzamide S-oxide ({circ}) was also detected as an intermediate during thiobenzamide degradation and is shown on a more sensitive scale. The concentration of thiobenzamide S-oxide present as an impurity in the thiobenzamide solution incubated without cells is also shown (•).

Analysis (HPLC) of resting cell suspensions given 1 mM thioacetamide revealed low levels of thioacetamide S-oxide (5 µM) that were produced as thioacetamide was degraded. Thioacetamide and thioacetamide S-oxide had average retention times of 4.4 and 3.3 min, respectively (data not shown). Thioacetamide S-oxide, present as an impurity in the thioacetamide uninoculated control, remained constant at 1.5 µM. To identify other metabolites produced during thioacetamide degradation, filtered supernatants of resting cell suspensions given 5 mM thioacetamide were extracted with MTBE and analyzed for metabolites by GC-MS. Standards of thioacetamide, acetonitrile, acetamide, and acetic acid had GC retention times of 15.2, 3.2, 11.1, and 8.5 min, respectively. Thioacetamide S-oxide decomposed after injection and could not be detected. Complete degradation of thioacetamide occurred by 13.5 h. The sole product detected in both regular and acidified samples had a GC retention time of 7.0 min (Fig. 4A). The appearance of this product coincided with thioacetamide degradation, and its concentration peaked when the thioacetamide was completely degraded (Fig. 4A). No change in the concentration of this product was observed after additional incubation (Fig. 4A). Analysis of this peak by MS revealed a molecular ion with m/z = 114 and another fragment of m/z = 73 (Fig. 4B). No matching compound was found in the mass spectrum library. Reports describing chemical oxidation of thioamides have identified 3,5-disubstituted-1,2,4-thiadiazoles as products (39). The predicted thiadiazole resulting from the oxidation of thioacetamide is 3,5-dimethyl-1,2,4-thiadiazole, which has a monoisotopic mass of 114. Mass spectral studies of 3,5-disubstituted-1,2,4-thiadiazoles found that the parent ion fragments by fission of one of three pairs of bonds: 1-2 and 3-4, 3-4 and 5-1, or 2-3 and 4-5 (33). With 3,5-dimethyl-1,2,4-thiadiazole, cleavage of any of the three sets of bonds would generate a species with the molecular formula C2H3SN (monoisotopic mass = 73) and acetonitrile (CH3CN) (monoisotopic mass = 41). This is consistent with the major fragments observed in the mass spectrum (m/z = 114 and 73) (Fig. 4B). Acetonitrile was not detected, which may indicate that it is eliminated as a neutral species. To further verify the identity of this product, a standard of 3,5-dimethyl-1,2,4-thiadiazole was synthesized and found to have a GC retention time of 7.0 min and a mass spectrum showing the same fragments at m/z = 114 and 73 (Fig. 4C). Based on this supporting evidence, the compound with the GC retention time of 7.0 min was identified as 3,5-dimethyl-1,2,4-thiadiazole.


Figure 4
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  		FIG. 4. GC-MS analysis of thioacetamide degradation by R. pickettii TA resting cells. (A) Corrected areas of peaks detected by GC were plotted versus time. The peak at 15.2 min represents thioacetamide ({blacksquare}); the peak at 7.0 min ({triangleup}) was the only degradation product detected. The chromatogram from the 9.5-h time point is shown in the inset box; abundance was determined as the total ion current. (B) The peak at 7.0 min was analyzed by MS. (C) The mass spectrum of the synthesized 3,5-dimethyl-1,2,4-thiadiazole standard is shown for comparison, and the major fragments are identified.

To determine the thioacetamide metabolites that may have contributed to the formation of 3,5-dimethyl-1,2,4-thiadiazole, thioacetamide and thioacetamide S-oxide were evaluated for the ability to form 3,5-dimethyl-1,2,4-thiadiazole spontaneously. 3,5-Dimethyl-1,2,4-thiadiazole was not detected by GC-MS when thioacetamide and thioacetamide S-oxide were stirred together or separately in minimal medium. However, when thioacetamide was oxidized with potassium peroxymonosulfate in minimal medium, no thioacetamide was present after 1 h, and the products detected by GC-MS were 3,5-dimethyl-1,2,4-thiadiazole (18%), acetamide (12%), and acetic acid (70%). When thioacetamide S-oxide was similarly oxidized, none of the 3,5-dimethyl-1,2,4-thiadiazole was present after 1 h, and only trace amounts were detected after 7.5 h. These results suggest that a further oxidation product of thioacetamide S-oxide (possibly the S,S-dioxide) reacts with thioacetamide to produce 3,5-dimethyl-1,2,4-thiadiazole.

Nitrogen-limited growth of R. pickettii TA.
To evaluate the utilization efficiency of thioacetamide as a nitrogen source, the growth of R. pickettii TA on thioacetamide or ammonium chloride at growth-limiting concentrations was compared. The organism grew to higher densities on ammonium chloride when cells were given equimolar amounts of thioacetamide or ammonium chloride (Fig. 5), suggesting that not all of the nitrogen contained in thioacetamide can be assimilated by the cells. When 3,5-dimethyl-1,2,4-thiadiazole was added to cell suspensions and incubated, there was no observed metabolism of this substrate, indicating that this is a dead-end product. Therefore, the formation of one molecule 3,5-dimethyl-1,2,4-thiadiazole sequesters two atoms of nitrogen that cannot be utilized for growth. Using a standard curve prepared from the synthesized 3,5-dimethyl-1,2,4-thiadiazole standard, we calculated that degradation of 5 mM thioacetamide resulted in the accumulation of 0.3 mM 3,5-dimethyl-1,2,4-thiadiazole in the medium, representing 0.6 mM (12%) of the thioacetamide consumed. In a separate similar incubation, 88% of the thioacetamide nitrogen was released as ammonia into the medium, which is consistent with the observed amount of 3,5-dimethyl-1,2,4-thiadiazole formed during thioacetamide degradation.


Figure 5
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  		FIG. 5. Growth of R. pickettii TA cultures on ammonium chloride ({square}) or thioacetamide (Figure 5) as sole nitrogen sources over a range of concentrations. The mean OD600 readings from three replicates of each treatment are shown; error bars show the range of values in each treatment.

Metabolic fate of thioacetamide carbon.
From the results of thiobenzamide degradation by R. pickettii TA (Fig. 3), we predicted that thioacetamide degradation would yield acetonitrile and/or acetamide, from which ammonia could be released via hydrolysis to produce acetate. Acetonitrile was not detected during the chemical oxidation of thioacetamide, but acetic acid and acetamide together made up 82% of the oxidation products. Therefore, if carbon flux during thiocetamide degradation by R. pickettii TA were similar to that observed during chemical oxidation, and if the organism could assimilate acetamide and acetate as carbon sources, growth on thioacetamide as a carbon source should be possible. When R. pickettii TA was evaluated for growth on acetonitrile or acetamide as carbon and nitrogen sources or on sodium acetate as a carbon source, we found that the organism grew to similar cell densities in each treatment (OD600 = 0.26, 0.27, and 0.29, respectively) in 48 h. Growth of the organism on thioacetamide as a carbon source was slow, taking 14 to 21 days to reach maximum cell density, with some apparent clumping of cells. However, the cells did grow to a density of an OD600 of 0.25 on thioacetamide, and the thioacetamide UV absorbance peak at 262 nm diminished during growth. This result indicates that R. pickettii TA can grow by assimilating carbon from thioacetamide.


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DISCUSSION
 
Enrichment on thioacetamide as the sole nitrogen source was anticipated to yield a hydrolytic reaction pathway that would directly liberate ammonia. Thioamides have been tested as substrates for a number of proteases, and the ability of proteases to cleave these substrates is variable. Leucine aminopeptidase did not cleave any of the thiopeptide substrates tested in one study (6), while papain and carboxypeptidase A showed significant hydrolysis of some thiopeptides but slow or no activity on others (4, 7, 8, 17). Metal substitution experiments found that carboxypeptidase A substituted with cadmium or cobalt for zinc was more active with thioamides than amides (7, 31). In light of these observations, it was anticipated that microbes might produce metallo-amidases that would show activity with terminal thioamides and thus grow on them as the sole nitrogen source.

Unexpectedly, it was observed that an isolated bacterium, R. pickettii TA, used a completely different mechanism to grow on thioacetamide as a nitrogen source. The results suggested that R. pickettii TA initiates thioamide degradation by monooxygenation of the sulfur atom. A second monooxygenation is proposed that leads to the release of oxidized sulfur that accumulates in the medium as sulfur dioxide or sulfite (Fig. 6). An additional two-electron oxidation is required to generate sulfite and nitrile or amide products from a thioamide S,S-dioxide. We currently do not know how this oxidation occurs. When thiobenzamide is the substrate, elimination of the oxidized sulfur yields benzonitrile, which is hydrated to produce benzamide. In contrast, the only accumulating product from thioacetamide metabolism was 3,5-dimethyl-1,2,4-thiadiazole (Fig. 4). Thioacetamide S-oxide was found transiently at low levels. The organism could grow on acetonitrile, acetamide, and acetate as carbon sources, and acetamide, acetic acid, and 3,5-dimethyl-1,2,4-thiadiazole were identified as products of the chemical oxidation of thioacetamide. These results support a model in which the oxygenation of thioacetamide leads to elimination of oxidized sulfur and generates intermediates that can be hydrolyzed to liberate ammonia (Fig. 6). In a competing side reaction, some thioacetamide is proposed to spontaneously react with oxidized thioacetamide metabolites to form 2,3-dimethyl-1,2,4-thiadiazole (Fig. 6).


Figure 6
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  		FIG. 6. Proposed pathway of thioacetamide degradation by R. pickettii TA. Thioacetamide S,S-dioxide is depicted in brackets as a putative intermediate. Sulfur was detected in the medium as sulfur dioxide/sulfite, implying that an additional oxidation of the sulfur occurs. Nitrogen was released as ammonia, but the mechanism is unknown.

Several lines of evidence are consistent with the metabolism of thioamides proceeding via oxygenation of the sulfur atom. First, the metabolism was dependent on oxygen in the culture medium (Fig. 2). Second, the detected products benzonitrile and benzamide (Fig. 3A) are consistent with known spontaneous decomposition products formed during the chemical oxidation of thiobenzamide (9). Third, thiobenzamide S-oxide was detected as a transient intermediate in cell incubation mixtures (Fig. 3B). Fourth, sulfur oxyanions were detected in the culture media, a finding consistent with elimination of the thioamide sulfur at a higher oxidation state. Fifth, 3,5-dimethyl-1,2,4-thiadiazole was detected as a dead-end product. When thioacetamide was chemically oxidized, 3,5-dimethyl-1,2,4-thiadiazole was also formed as a product. In the present study, we tried to demonstrate cell-free oxygenase activity of thioamides and were unsuccessful. However, this is consistent with known properties of microbial oxygenases, many of which are unstable in a cell-free system (41).

Flavoprotein monooxygenases constitute a relatively stable class of oxygenase enzymes and have been implicated in the metabolism of thioamides by M. tuberculosis (5, 13, 42). M. tuberculosis cells oxidized the thioamide group in ethionamide to the alcohol derivative (2-ethyl-4-hydroxymethylpyridine) and the S-oxide, nitrile (2-ethyl-4-cyanopyridine), and amide (2-ethyl-4-amidopyridine) derivatives were detected as intermediates (13). EtaA, a flavoprotein monooxygenase purified from that organism, generated the S-oxide and amide derivatives of ethionamide and other thioamide substrates in vitro (42). Studies with mammals and mammalian microsomal fractions have implicated liver microsomal flavoprotein monooxygenases and cytochrome P450 2E1 as the major participants in thioamide oxidation, with thioamide S-oxides detected as metabolic intermediates and amides detected as accumulating products (19, 32, 48). Investigators in the studies mentioned above postulated that thioamide S-oxides are oxygenated again to yield thioamide S,S-dioxides, which are highly reactive and unstable and therefore cannot be isolated (37). Mechanisms have been proposed to explain the formation of either nitriles or amides from thioamide S,S-dioxides via elimination or substitution reactions, respectively (9, 16). One scenario has sulfur leaving as sulfoxylate (HSO2–), which would subsequently oxidize to a more stable species (16). Assays in mammals have detected the sulfur from oxidized thioamides as sulfate (23), while in the present study we detected released sulfur at the oxidation level of sulfite in the medium. The oxidation state of leaving sulfur is unknown. The production of amides from thioamide S-oxides and S,S-dioxides has also been proposed to occur via formation of intramolecular cyclic oxathiirane intermediates, which decompose and release sulfur monoxide or sulfur dioxide (45), although evidence suggests that this is not the major pathway in biological systems (16, 21). S-oxide and S,S-dioxides have also been described in the bacterial oxidation of dibenzthiophene and related molecules, but any similarities between the enzymes involved in dibenzthiophene metabolism and the enzymes involved in thioamide metabolism are currently unknown (18).

To our knowledge, thioamides have not previously been reported as growth substrates for bacteria. In fact, it is the oxygenation of ethionamide and other thioamides that renders these compounds toxic to M. tuberculosis (5, 13, 42). R. pickettii TA grew on thioacetamide but was unable to grow using thiobenzamide as a nitrogen source, presumably because this organism lacks enzymes that can liberate ammonia from benzonitrile or benzamide. This fits with the several known examples of both nitrilases and amidases that are specific for nitrile or amide substrates with either aromatic or aliphatic side chains (3). R. pickettii TA was able to liberate most of the nitrogen from thioacetamide as ammonia and could also grow on the substrate as a carbon source. The release of ammonia from thioacetamide via enzymatic hydrolysis of acetonitrile and/or acetamide generated by the decomposition of thioacetamide S,S-dioxide could not be confirmed because the predicted intermediates were not detected. It is likely that the putative intermediates were metabolized rapidly relative to thioamide sulfur oxidation and elimination and therefore did not accumulate in the medium.

Mammalian studies have also focused on how the oxygenation of thioamides generates cytotoxic intermediates (15, 16, 38, 40). Studies conducted in vitro and in vivo with radiolabeled thioacetamide and thioacetamide S-oxide revealed that metabolites of these substrates could form covalent linkages with proteins and nucleic acids (15, 16, 38, 40). We did not see evidence of cytotoxicity during growth of Ralstonia pickettii TA on thioacetamide as a nitrogen source. Growth of the organism on thioacetamide as a carbon source was slow, and some clumping of the cells was observed. However, the cells eventually grew to densities similar to cells growing on equimolar amounts of acetonitrile, acetamide, or acetate. Covalent binding of thioacetamide metabolites to cellular molecules may occur in Ralstonia pickettii TA, but this phenomenon was not evaluated experimentally.

We are not aware of previous studies reporting biological formation of 3,5-dimethyl-1,2,4-thiadiazole. While the appearance of this product was not predicted, chemical oxidation of thioamides is a known route to the synthesis of 3,5-disubstituted-1,2,4-thiadiazoles (39). The spontaneous dimerization of various thioamide S-oxides in the presence of acid or heat to form 3,5-disubstituted-1,2,4-thiadiazoles has been reported (9, 12).

At present, there is limited information available on thioamide metabolism by prokaryotes other than M. tuberculosis. Soil isolates of the genera Arthrobacter and Bacillus were found to degrade the herbicide 2,6-dichlorothiobenzamide slowly (half-lives of several weeks) to its amide and carboxylic acid derivatives when growing on other substrates (20). Further studies of these and other microbial strains that can metabolize thioamides might reveal alternative mechanisms of metabolism.


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ACKNOWLEDGMENTS
 
This study was supported by biotechnology training grant GM08347 from the National Institutes of Health-National Institute of General and Medical Sciences, grant DE-FG02-01ER63268 from the U.S. Department of Energy, a University of Minnesota Graduate School Doctoral Dissertation Fellowship, and the University of Minnesota Initiative for Renewable Energy and the Environment.

We thank David Sukovich for help with the degradation assays and inorganic ion determinations, Jennifer Seffernick for advice and assistance with HPLC, and Tom Krick (University of Minnesota Center for Mass Spectrometry and Proteomics) for MS characterization of synthesized thioamide S-oxides.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology, and Biophysics, 140 Gortner Laboratory of Biochemistry, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625-5780. E-mail: wacke003{at}umn.edu. Back

{triangledown} Published ahead of print on 22 September 2006. Back


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Applied and Environmental Microbiology, December 2006, p. 7468-7476, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01421-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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