Applied and Environmental Microbiology, October 2004, p . 6315-6319, Vol . 70, No . 10

Metabolism of 2-Mercaptobenzothiazole by Rhodococcus rhodochrous

Nicolas Haroune,¹ Bruno Combourieu,¹ Pascale Besse,¹ Martine Sancelme,¹ Achim Kloepfer,² Thorsten Reemtsma,² Heleen De Wever,³ and Anne-Marie Delort^1*

Laboratoire de Synthèse et Etude de Systèmes à Intérêt Biologique, Université Blaise Pascal, Aubière, France,¹ Department of Water Quality Control, Technical University of Berlin, Berlin, Germany,² Vito, Mol, Belgium³

Received 19 November 2003/ Accepted 22 May 2004

ABSTRACT

2-Mercaptobenzothiazole, which is mainly used in the rubber industry as a vulcanization accelerator, is very toxic and is considered to be recalcitrant . We show here for the first time that it can be biotransformed and partially mineralized by a pure-culture bacterial strain of Rhodococcus rhodochrous . Three metabolites, among four detected, were identified .

2-Mercaptobenzothiazole (MBT) is the most important and most widely used member of the benzothiazole (BT) family . MBT is typically a rubber additive (16, 17), but it has also other applications, such as inhibiting biocorrosion in cooling systems or in paper manufacturing (4) . The annual MBT production in Western Europe is estimated to be excess of 40,000 tons .

Direct discharges of MBT occur in effluents from factories producing and using MBT . Indirect sources of environmental contamination are mainly leachates from landfills where MBT is deposited and from rubber products (18) . MBT can also be found in tannery wastewater (20) . Finally, BTs have been found in urban runoff, in residential and highway road dust, and in urban particulate matter, most probably as a result of vehicle tire wear (18) . The U.S . Environmental Protection Agency estimated that over 500 tons of MBT may be lost annually to the environment (22) . Its toxicity towards microorganisms (3, 6, 7, 9), its allergenicity, resulting in serious dermatoses (11), and its potential mutagenic effects (12) make its presence in the environment of great concern .

Information on the environmental fate of BT in the literature is scarce, and information on its biodegradative pathways is scarcer still . Several studies were first carried out in laboratories or in pilot-scale activated sludge systems in order to remove such a compound (5, 20, 21) . These studies show that MBT is rather recalcitrant and is not completely mineralized when its concentration reaches a certain threshold . The metabolites observed under these conditions were benzothiazolylsulfonate and 2-methylthiobenzothiazole (MTBT) (10, 20), the disulfide derivative of MBT (5) .

Only a few bacterial isolates have been shown to biotransform MBT . Drotar et al . (10) observed MBT-methylating activity in crude extracts of a variety of soil and water isolates, including a Corynebacterium sp., a Pseudomonas sp., and Escherichia coli, yielding the more stable methylated product MTBT, which accumulated in the medium .

This paper reports studies of the biotransformation of MBT by Rhodococcus rhodochrous OBT18, isolated by De Wever et al . (8) from activated sludge from a wastewater treatment plant of an MBT-producing factory (Bayer, Antwerp, Belgium) . This strain was previously shown to degrade BT, 2-hydroxybenzothiazole (OBT) (2, 8), and 2-aminobenzothiazole (ABT) (13) . The main objective of this work was to identify the structure of the metabolites formed in order to establish the metabolic pathway of MBT for this strain . Because BT structures are difficult to analyze (due to the presence of N and S heteroatoms) and the metabolism of BT is almost unknown, various powerful analytical tools must be used, such as two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopies (¹H-¹⁵N gradient heteronuclear multiple-bond correlation [gHMBC], ¹H-¹³C heteronuclear single-quantum coherence [HSQC], ¹H-¹³C heteronuclear multiple-bond correlations [HMBC], and ¹H-¹H total correlation spectroscopy [TOCSY]) and mass spectrometry (MS) methods (electrospray identification [ESI]-MS and tandem MS [MS/MS]) . Because of the sensitivity limits of these techniques (especially 2D NMR), rather high (i.e., millimolar) concentrations of metabolites are needed . For this reason, Rhodococcus cells were grown in a rich medium (Trypticase soy broth) to get large amounts of biomass, and then resting cells were used for the incubations . Also, the experiment was performed with water instead of buffer (K₂HPO₄, 1 g/liter; KH₂PO₄, 1 g/liter; FeCl₃ · 6H₂O, 4 g/liter; and MgSO₄ · 7H₂O, 40 g/liter; pH 6.7) because salts (especially phosphates) are not convenient for some MS analyses . However, the same metabolites were detected when the kinetics of MBT degradation were observed under both conditions . Although growing cells might behave differently in the environment, information about potential metabolites is important, especially because they can accumulate and be toxic .

Trypticase soy broth-grown resting cells of R . rhodochrous were incubated in distilled water with 1.5 mM MBT as previously described (14) . The kinetics of degradation were monitored by in situ ¹H NMR (2) . ¹H NMR signals corresponding to MBT ( ppm, 7.32, 7.44, 7.55, and 7.69) decreased with time (Fig . 1), and new signals in the aromatic region appeared: after 1 h of incubation, two new doublets ( ppm, 6.43 and 5.96) were present (indicating metabolite 1) . These signals increased with time . A second metabolite, giving signals resonating at 6.95, 7.16, and 7.41 ppm, was detected after 3 h (metabolite 2) . These signals disappeared with time . At 2 h, other signals were observed (indicating metabolite 3), resonating at 7.25 and 6.16 ppm as doublets . After 21 h, metabolites 1 to 3 disappeared, and a new metabolite was detected (metabolite 4; indicated by two doublets resonating at 6.68 and 7.09 ppm) and was still present after 128 h (Fig . 1) . This result showed that MBT was biotransformed but not completely mineralized after 128 h . The total organic carbon (TOC) measurement was used to evaluate the formation of CO₂, and the results showed that about 30% of MBT was completely mineralized after 128 h . The time course of this TOC measurement showed that this mineralization process was not stopped after 128 h . Further evidence of MBT mineralization was given by the release of NH⁴⁺ and SO₄^2– . After 48 h of incubation, these ions were found in 0.2 and 0.7 M concentrations, respectively, per mole of MBT . The stoichiometry was not fully balanced, which may be due to the fact that the form in which N and S are released is unknown, but at least it confirms that part of MBT was mineralized .

 
The structures of these unknown metabolites were established by using more sophisticated 2D NMR experiments and MS . In particular, ¹H-¹H TOCSY NMR experiments were performed with a sample obtained after 4 h of incubation to detect protons hidden by the presence of a massive amount of H₂O in 1D NMR spectra . ¹H-¹³C HSQC and ¹H-¹³C HMBC experiments were carried out as previously described (13, 14) . To perform these experiments, for which a higher metabolite concentration is required, a quantitative assay was carried out . R . rhodochrous (55 g [wet weight]) was incubated in each of 11 Erlenmeyer flasks containing 100 ml of water and 1.5 mM MBT . The biodegradation kinetics were monitored by NMR in order to stop the experiment at the times when the maximum concentrations of the different metabolites were achieved (4 h for metabolites 1 to 3, and 128 h for metabolite 4) . The incubation medium was centrifuged, and the supernatant was freeze-dried and dissolved in D₂O . Selected samples were analyzed by liquid chromatography (LC)-ESI-MS according to a method described elsewhere (19) . Structure elucidation was based on product ion spectra generated by collision-induced dissociation in the negative-ion mode .

Metabolite 1.

¹H and ¹³C NMR data for metabolite 1 are consistent with the structure of a cis-dihydrodiol of MBT (Table 1) . LC-MS/MS data for the major compound detected in the sample taken after 3.5 h of incubation are also listed in Table 1 and coincide with this structure . From the molecular anion m/z 200, a loss of water (–18 atomic mass units) is readily observed, as is the case for for all fragment ions generated from metabolite 2 .

 
Metabolite 2.

Both the multiplicity of the ¹H signals and the ¹³C chemical shifts of metabolite 2 (Table 1) are in accordance with an aromatic ring structure substituted in position 5 or 6, similar to those previously observed for 6-OH derivatives of OBT and ABT (2, 13) . To confirm this structure, metabolite 2 was isolated and purified as previously described for 6-OH-OBT (2) . The mass spectrum (chemical ionization, CH₄) of the isolated compound presented a peak at m/z 182 (M+H)⁺, confirming that the metabolite formed was a hydroxylated derivative . The proposed structure was also confirmed by LC-MS/MS experiments performed with the sample taken at 5.5 h . All fragment ions generated from the molecular anion (m/z 182) correspond to those found for MBT (19), except for the presence of 18 additional atomic mass units in the hydroxyl moiety . The fragment anion m/z 58 confirms that the heteroaromatic ring system was unaltered . Finally, the ¹H-¹⁵N gHMBC spectrum of the purified metabolite 2, recorded as previously described (2), showed only one correlation between the endocyclic ¹⁵N (–206 ppm) and the doublet at 7.16 ppm, clearly indicating that the OH substituent was on the benzene ring at position 6 of MBT (Fig . 2 and Table 1) .

 
Metabolite 3.

Unfortunately, no ¹³C data could be obtained for metabolite 3, because this metabolite was lost during the freeze-drying process necessary to get a concentrated sample for the 2D ¹H-¹³C NMR experiment . Nevertheless, ¹H NMR data (Table 1) recall the pattern obtained for the diacid derivative of BT (14) . This hypothetical structure was confirmed by the detection of the anion m/z 230 (M-H)^– when ESI-MS was performed with samples taken after 4 h of incubation and directly injected into the source (Table 1) .

Metabolite 4.

Only partial structural information could be obtained for metabolite 4 (Table 1) . These data showed that this metabolite contains two protons and at least six carbons that are likely to belong to the initial aromatic part of MBT . In addition, ¹H-¹⁵N gHMBC experiments clearly showed the presence of a nitrogen atom resonating at –323 ppm that could correspond to the nitrogen of the thiazole ring . It can be concluded that part of the MBT structure is conserved .

Very few cases of MBT transformation by pure-culture bacterial strains have been reported to date (10); Corynebacterium spp., Pseudomonas spp., and E . coli isolates were shown to transform MBT into MTBT, but this methylated metabolite accumulated in the medium without further transformation . We describe here a new case of MBT transformation by an isolate from the genus Rhodococcus . Both the type of bacteria and the type of metabolism are very different from previously described cases .

In this work, we found the following information about MBT metabolism . First, we showed that, although MBT is known to be extremely recalcitrant, it can be biotransformed into four metabolites and that 30% of MBT was completely mineralized under our experimental conditions . The transformation of MBT clearly results from enzymatic processes, as no degradation of MBT was observed in control experiments in which no cells, either live or heat-killed, were used . Although the experiments reported here were not performed with growing cells, the results may give indications about processes that could occur in the environment . To our knowledge, this is the first report showing that MBT is potentially degradable by a pure culture and that the enzymatic equipment of R . rhodochrous OBT18 could operate under some environmental conditions . Also, it was shown that 6-OH-MBT is less toxic than MBT, according to the standardized Microtox test performed as previously described (15) . For an exposure time of 15 min, the 50% effective concentrations were 9.5 µM for 6-OH-MBT and 1.5 µM for MBT .

Second, the identification of metabolites 1 to 3 allows a more detailed description of the metabolic pathway of MBT in R . rhodochrous (Fig . 3) . These metabolite structures are completely different than those previously described (5, 10, 20, 21) . Metabolite 1, a cis-dihydrodiol derivative of MBT, results from the activity of a hydroxylating dioxygenase (1) . This is the first report of such an enzyme in BT metabolism . Although a catechol structure could not be detected under our experimental conditions, the presence of the diacid derivative of MBT (metabolite 3) indicates the activity of a catechol 1,2-dioxygenase . We have already shown the involvement of such an enzyme in Rhodococcus pyridinivorans PA in the degradation of BT (14) . As the structure of metabolite 4 was not fully identified, we could not integrate it into a plausible metabolic pathway .

 
Finally, the formation of 6-OH-MBT (metabolite 2) shows that the hydroxylation of the benzene ring at position 6 is a general mechanism involved in the biotransformation of BT derivatives . 6-OH-MBT could be formed by the action of a monooxygenase (Fig . 3) . This hydroxylation was previously shown to operate in ABT in R . rhodochrous (13), in BT and OBT in Rhodococcus erythropolis, R . rhodochrous, and R . pyridinivorans PA (2, 14), and on N-[2-benzothiazolyl]-N,N'-dimethylurea (methabenzthiazuron) in Aspergillus niger (15) and Cunninghamella echinulata (23) .

ACKNOWLEDGMENTS

N . Haroune is a recipient of a grant from the French Ministère de l'Education Nationale et de la Recherche .

We thank Gilles Mailhot for the TOC measurements and F . Bonnemoy for the Microtox assays .

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