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Involvement of c-type cytochrome CymA in the electron transfer of anaerobic nitrobenzene reduction by Shewanella oneidensis MR-1
Biochemical Engineering Journal 68 (2012) 227–230
Contents lists available at SciVerse ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Involvement of c-type cytochrome CymA in the electron transfer of anaerobic nitrobenzene reduction by Shewanella oneidensis MR-1 Pei-Jie Cai a,b,d , Xiang Xiao a,c,∗ , Yan-Rong He a , Wen-Wei Li a,b,∗∗ , Lei Yu a,b , Michael Hon-Wah Lam b,d , Han-Qing Yu a,b a
Department of Chemistry, University of Science & Technology of China, Hefei 230026, China Advanced Laboratory for Environmental Research & Technology, USTC-CityU, Suzhou 215123, China c School of Environment, Jiangsu University, Zhenjiang 212013, China d Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong b
a r t i c l e
i n f o
Article history: Received 8 May 2012 Received in revised form 29 June 2012 Accepted 25 July 2012 Available online 2 August 2012 Keywords: Anaerobic processes Biodegradation Nitrobenzene (NB) Waste-water treatment Protein Shewanella oneidensis MR-1
a b s t r a c t Effective bioreduction of nitrobenzene (NB) by electrochemically active bacteria, such as Shewanella and Geobacter, has been demonstrated. However, the mechanism behind such a bioreduction is unclear yet. In this work, the mechanism for anaerobic NB reduction by Shewanella oneidensis MR-1 was investigated at a gene level. The omcA-mtrCAB gene cluster, an important extracellular electron transfer chain in S. oneidensis MR-1 for the reduction of a variety of compounds, was found to be uninvolved in the NB bioreduction. Knockout of cymA, a tetraheme c-type cytochrome in the periplasmic space, led to a 67% loss in NB bioreduction efficiency in comparison with the wild strain, and caused accumulation of an intermediate, phenylhydroxylamine, at the initial stage of NB bioreduction. These results demonstrate that, unlike the previously reported pure extracellular reduction pathways for many compounds, intracellular reactions are involved in NB bioreduction by S. oneidensis MR-1, and CymA plays an important role in the electron transfer of the intracellular NB bioreduction. In addition, the participation of some unknown protein other than CymA in NB reduction is discovered, and the possible pathways of electron transfer in the NB reduction by S. oneidensis MR-1 are proposed. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nitroaromatic compounds are refractory pollutants released into environment almost exclusively from anthropogenic sources, such as incomplete combustion of fossil fuels and synthesis of chemicals [1]. Nitrobenzene (NB) is a classic nitroaromatic compound with relatively simple structure but high health hazards [2]. Thus, effective degradation of NB becomes imperative. Several physical methods, e.g., sonolysis [3,4] and radiolysis [5], and chemical methods, e.g., Fenton reagent [6–8] and bioelectrochemical system [9], have been used to remove NB, but with relatively high costs. Comparatively, biodegradation of NB offers an economically attractive way [10]. In recent years, many pure anaerobic eubacteria [11] or anaerobic bacteria [12,13] have been reported
Abbreviations: NB, nitrobenzene; AN, aniline; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; DMSO, dimethyl sulfoxide. ∗ Corresponding author at: Department of Chemistry, University of Science & Technology of China, Hefei 230026, China. Fax: +86 511 88790955. ∗∗ Corresponding author at: Department of Chemistry, University of Science & Technology of China, Hefei 230026, China. Fax: +86 512 87161362. E-mail addresses: [email protected] (X. Xiao), [email protected] (W.-W. Li). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.07.022
to catalyze NB reduction, and the anaerobic decomposition pathway of NB has been well understood. However, the pathway of such bioreduction process at gene level is still unclear. Shewanella oneidensis MR-1 has been frequently used as a model organism for microbial degradation mechanism investigations [14,15]. NB bioreduction by Shewanella has been previously reported [13]. The omcA-mtrCAB gene cluster of S. oneidensis MR1 is considered as an important anaerobic respiratory pathway to transfer electron from the cytoplasmic membrane to extracellular electron acceptor during bioreduction of many organics [16–19]. But, it is unclear whether this pathway is also responsible for the NB reduction. To date, the specific mechanism of NB bioreduction by S. oneidensis MR-1 is yet to be elucidated. Thus, if NB bioreduction is through extracellular reduction, it would be interesting to find whether the omcA-mtrCAB gene cluster is involved in the electron transfer. If it is not solely through extracellular reduction, then quite likely the CymA in the periplasmic space may play a role [20]. Therefore, this work aims to elucidate the mechanism of NB anaerobic reduction by S. oneidensis MR-1 at gene level. For this purpose, the performance of NB bioreduction by S. oneidensis MR-1 and its mutant strains was evaluated and the intermediate products were analyzed. The experimental results may facilitate a
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better understanding on the anaerobic bioreduction of nitroaromatic compounds. 2. Materials and methods 2.1. Inoculum and culturing medium Table 1 lists the strains used in this work, including S. oneidensis MR-1 (ATCC® number is 700550TM ) and its mutant strains, which were kindly provided by Prof. K.H. Nealson from the University of Southern California [20]. All the strains were validated and cultured in LB nutrient solution at 30 ◦ C until reaching the cell’s stationary phase. Then, the cells were harvested by centrifugation at 7000 × g for 15 min. Phenylhydroxylamine (ALDRICH Co., USA), and azobenzene (Sinopharm Chemical Reagent Co., Shanghai, China) were used as the standards to determine the intermediates of NB bioreduction. 2.2. NB bioreduction tests NB bioreduction tests were conducted with a synthesized medium. The medium contained (per liter): 3.36 g sodium lactate (the sole electron donor), 11.91 g HEPES, 0.3 g NaOH, 1.5 g NH4 Cl, 0.1 g KCl, 0.67 g NaH2 PO4 ·2H2 O, 5.85 g NaCl and 1 ml of trace mineral stock solution [20]. The defined medium of 100 ml was added into each serum vial with N2 aeration for 10 min. Then, the serum vials were sealed with butyl rubber stoppers and autoclaved. After that, 0.1 ml of sterile vitamin solution [21] and 0.1 ml of amino acid solution (filtering through 0.22 m filter membrane) was added into the serum vial. The NB concentration in the serum vial was 185 mg l−1 . Then, the harvested cells were washed with the defined medium for three times and inoculated. The OD600 of the initial cells concentration in the serum vials was 1.0.
chromatography (HPLC, 2695, Waters Co., USA) with a HCC18 column (reversed-phase column, particle size 5 m, 4.6 mm × 250 mm) and a UV-detector (detection wavelength of 265 nm (NB) and 231 nm (AN)), by comparing the retention times with those of the standard chemicals. Samples of 10 l after filtering through 0.45 m filter membrane were injected. H2 O and methanol were used as the eluent with a volume ratio of 40:60 (%) at a flow rate of 1.0 ml min−1 at 25 ◦ C. 3. Results 3.1. Role of omcA-mtrCAB gene cluster in NB bioreduction To find out whether NB bioreduction by S. oneidensis MR-1 was an extracellular reduction process, the NB reduction performances of wild strain and its mutants (omcA/mtrC, mtrB, mtrA; Table 1) were evaluated. Unlike the MO bioreduction process [18], no obvious difference of NB bioreduction was observed for the wild strain of S. oneidensis MR-1 and its mtr-deleted mutants (Fig. 1). This indicates that the electron transfer to NB was not through the Mtr respiratory pathway. Meanwhile, the total NB concentration, calculated from the remaining NB concentration in the defined medium and the equivalent NB concentration from the produced AN at the same time, indicate that approximately 40% of NB disappeared during NB bioreduction by the cymA-deleted mutant strain, whereas the adsorption experimental results show that only
2.3. Absorption tests NB adsorption test by S. oneidensis MR-1 was carried out in serum vials containing 100 ml of the defined medium without sodium lactate (the electron donor). After centrifugation at 7000 × g for 15 min, the supernatant was transferred into a beaker. The cells were re-suspended with 100 ml of fresh medium without sodium lactate. Then, the NB concentrations in the initial medium and the re-suspension solution were determined. 2.4. Analysis The concentrations of NB, aniline (AN) and the degradation intermediates were determined with a high performance liquid
Table 1 S. oneidensis strains used in this work [20]. Open reading frame
Gene product deleted (gene)
SO4591
Tetraheme cytochrome c (cymA), involved in anaerobic respiration (except trimethylamine oxide) Cytochrome c552 nitrite reductase (nrfA), involved with nitrite reduction OM protein precursor (mtrB), involved in metal oxide reduction Periplasmic decaheme cytochrome c (mtrA), involved in metal oxide reduction Decaheme cytochrome c complex (omcA mtrA), involved in metal oxide reduction Diheme cytochrome c (napB), involved in nitrate reduction Wild strain
SO3980 SO1776 SO1777 SO1778/SO1779 SO0845 MR-1
Fig. 1. (A) The changes of AN concentration in NB bioreduction by S. oneidensis MR-1 and its mutants (cymA, omcA/mtrC, mtrB, mtrA) within 24 h under anaerobic conditions; and (B) the changes of NB concentration in NB bioreduction by S. oneidensis MR-1 and its mutants (cymA, omcA/mtrC, mtrB, mtrA) within 24 h under anaerobic conditions.
P.-J. Cai et al. / Biochemical Engineering Journal 68 (2012) 227–230
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Fig. 2. HPLC chromatogram of the NB bioreducation products by S. oneidensis MR-1 and the cymA-deleted mutant strain.
approximately 8% of NB was absorbed by S. oneidensis MR-1. Thus, it implies that part of the NB had entered into the cells and been reduced intracellularly. 3.2. Role of CymA, the key c-type cytochrome in NB bioreduction The role of CymA, a c-type cytochrome distributed in the periplasmic space, was also investigated in NB bioreduction. Compared with the wild strain, knockout of cymA resulted in a significant loss in NB bioreduction by 67% (Fig. 1), indicating that CymA was the major pathway of electron transfer in NB bioreduction by S. oneidensis MR-1. Interestingly, another isolated peak in the HPLC chromatogram, ascribed to phenylhydroxylamine, was also found at the initial stage of NB bioreduction by cymA mutant strain (Fig. 2). But, this peak was not observed for the wild strain and other mutant strains, indicating that NB bioreduction by S. oneidensis MR-1 was a multistep reaction. 4. Discussion The omcA-mtrCAB gene cluster has been recognized as an important anaerobic respiratory pathway to transfer electrons from the cytoplasmic membrane to extracellular electron acceptor [16,17]. Previous studies have demonstrated that the Mtr respiratory pathway could be a major pathway of electron transfer for an extracellular organic pollutants bioreduction [18,19]. In this work, however, no obvious decrease in NB bioreduction by S. oneidensis MR-1 was observed when the Mtr respiratory pathway was blocked (Fig. 1), suggesting that the electron transfer was not through Mtr respiratory pathway. The “disappeared” NB implies that NB partially entered into cells, and the NB bioreduction partially occurred inside the cells. CymA is a c-type cytochrome belonging to the NapC/NirT family in the periplasmic space, and is essential for the anaerobic respiration of S. oneidensis [21]. It is known as an important pathway to transfer electrons from menquionone/menaquinol (MQ/MQH2 ) pool to some electron acceptors, e.g., nitrite, fumarate, and dimethyl sulfoxide (DMSO) [22,23]. However, so far there is no direct evidence to show that CymA is involved in NB bioreduction. Fig. 1 shows that the knockout of cymA led to a significant loss of NB bioreduction ability. This suggests that CymA could be a key c-type cytochrome in the electron transformation chain of NB reduction. In addition, an intermediate product was found at the initial stage of the bioreduction process by a cymA-deleted mutant strain, but disappeared at the middle stage of the bioreduction.
Fig. 3. Proposed mechanisms of anaerobic reduction NB by S. oneidensis MR-1 (the question mark indicates the unknown proteins involved in NB bioreduction by S. oneidensis MR-1; the solid lines indicate the bioreduction pathway of NB; the dotted lines show the electron transfer pathway).
These results further confirm that CymA plays a significant role in the electron transfer during NB bioreduction in periplasmic space. Interestingly, the results also suggest that some unknown proteins in the periplasmic space seem to be also involved in the electron transfer, because the reduction of NB by S. oneidensis MR-1 continued after blocking the CymA pathway. Therefore, it can be inferred that the CymA pathway of Shewanella enables a sufficient supply of electrons and a rapid reduction of NB to phenylhydroxylamine and further to AN. Hence, no accumulation of phenylhydroxylamine occurred for the wild strain. While for the cymA-deleted strain, because the CymA pathway was blocked, only small amount of electrons was transferred through some unknown proteins (as denoted by the question mark in Fig. 3). This insufficient supply of electrons might have led to an incomplete reduction of NB in the multistep reaction and an accumulation of intermediate. Previous studies have shown that several reductases and electron shuttles in other bacteria are recognized to participate in various reduction reactions (e.g., nitrite, DMSO and fumarate) [22,23]. Especially, Bryant and DeLuc [24] found that nitroreductase are involved in the reduction of NB by Enterobacter cloacae. In addition, considering the similarity between NB and nitrite reduction (which has been identified to be nitroreductase catalysis process in Shewanella), it is thus quite possible that NB reduction by S. oneidensis MR-1 is also catalyzed by nitroreductases. These nitroreductases, as downstream proteins of CymA, may contain a series of proteins that efficiently catalyze the entire reactions of NB to phenylhydroxylamine and further to AN. Admittedly, although the present study gives some valuable clues about the electron transfer pathway of NB reduction by Shewanella and proposes a presumed mechanism, the detailed mechanisms of this process are yet to be further elucidated and convinced. Especially, from the results of this study, it is still too early to conclude whether the reduction of NB to phenylhydroxylamine is catalyzed by the periplasmic nitroreductases or directly by some unknown enzymes upstream of CymA. All of these need more experimental evidences and may warrant more future research works.
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5. Conclusions In this study, the mechanism of NB bioreduction by S. oneidensis MR-1 is investigated at a gene level. The omcA-mtrCAB gene cluster was found to be uninvolved in the NB bioreduction, while the electron transfer via CymA in the periplasmic space is observed. The NB mass balance analysis verifies that NB bioreduction is not a pure extracellular reaction. The significant loss in NB-bioreducing ability of the strain with blocked CymA indicates that CymA plays a critical role in the electron transfer chain of NB bioreduction by S. oneidensis MR-1. Nevertheless, some NB reduction capability still remains for the bacterium after being deleted of CymA, suggesting that some unknown protein other than CymA is also involved in this reduction process. In addition, the results suggest that some periplasmic reductases, as downstream proteins of CymA, might directly catalyze the NB reduction. But these mechanisms need further elucidations in future works. Acknowledgements This work was partially supported by the NSFC (20907050) and the NSFC-JST Joint Project (21021140001). The authors wish to thank Prof. K.H. Nealson from University of Southern California for kindly providing the bacteria used in this work. References [1] J.M. Spain, Biodegradation of nitroaromatic compounds, Annu. Rev. Microbiol. 49 (1995) 523–555. [2] H.L. Li, Y. Cheng, H.F. Wang, H.F. Sun, Y.F. Liu, K.X. Liu, S.X. Peng, Inhibition of nitrobenzene-induced DNA and hemolglobin adductions by dietary constituents, Appl. Radiat. Isot. 58 (2003) 1131–1142. [3] H.M. Hung, F.H. Ling, M.R. Hoffmann, Kinetics and mechanism of the enhanced reductive degradation of nitrobenzene by elemental iron in the presence of ultrasound, Environ. Sci. Technol. 34 (2000) 1758–1763. [4] L.K. Weavers, F.H. Ling, M.R. Hoffmann, Aromatic compound degradation in water using a combination of sonolysis and ozonolysis, Environ. Sci. Technol. 32 (1998) 2727–2733. [5] S.J. Zhang, H. Jiang, M.J. Li, H.Q. Yu, H. Yin, Q.R. Li, Kinetics and mechanisms of radiolytic degradation of nitrobenzene in aqueous solutions, Environ. Sci. Technol. 41 (2007) 1977–1982. [6] L. Carlos, D. Fabbri, A.L. Capparelli, A.B. Prevot, E. Pramauro, F.G. Einschlag, Effect of simulated solar light on the autocatalytic degradation of nitrobenzene using Fe3+ and hydrogen peroxide, J. Photochem. Photobiol. A 201 (2009) 32–38. [7] L. Carlos, D. Nichela, J.M. Triszcz, J.I. Felice, F.S.G. Einschlag, Nitration of nitrobenzene in Fenton’s processes, Chemosphere 80 (2010) 340–345.
[8] J. Casado, J. Fornaguera, M.I. Galán, Mineralization of aromatics in water by sunlight-sssisted electro-Fenton technology in a pilot reactor, Environ. Sci. Technol. 39 (2005) 1843–1847. [9] Y. Mu, R.A. Rozendal, K. Rabaey, J. Keller, Nitrobenzene removal in bioelectrochemical systems, Environ. Sci. Technol. 43 (2009) 8690–8695. [10] Ö.S. Kuscu, D.T. Sponza, Effects of nitrobenzene concentration and hydraulic retention time on the treatment of nitrobenzene in sequential anaerobic baffled reactor (ABR)/continuously stirred tank reactor (CSTR) system, Bioresour. Technol. 100 (2007) 2162–2170. [11] A. Oren, P. Gurevich, Y. Henis, Reduction of nitrosubstituted aromatic compounds by the halophilic anaerobic eubacteria Haloanaerobium praemlens and Sporohalobacter marismortul, Appl. Environ. Microbiol. 57 (1991) 3367–3370. [12] A. Preuss, J. Fimpel, G. Diekert, Anaerobic transformation of 2, 4,6trinitrotoluene (TNT), Arch. Microbiol. 159 (1993) 345–353. [13] F.B. Luan, W.D. Burgos, L. Xie, Q. Zhou, Bioreduction of nitrobenzene, natural organic matter, and hematite by Shewanella putrefaciens CN32, Environ. Sci. Technol. 44 (2010) 184–190. [14] N. Tajima, A. Kouzuma, K. Hashimoto, K. Watanabe, Selection of Shewanella oneidensis MR-1 Gene-knockout mutants that asapt to an electrode-respiring condition, Biosci. Biotechnol. Biochem. 75 (2011) 2229–2233. [15] Y.F. Qian, L. Shi, M. Tien, SO2907, a putative TonB-dependent receptor, is involved in siddimilatory iron reduction by Shewanella oneidensis strain MR-1, J. Biol. Chem. 286 (2011) 33973–33980. [16] L. Shi, T.C. Squier, J.M. Zachara, J.K. Fredrickson, Respiration of metal (hydr) oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes, Mol. Microbiol. 65 (2007) 12–20. [17] R.S. Hartshorne, C.L. Reardon, D. Ross, J. Nuester, T.A. Clarke, A.J. Gates, P.C. Mills, J.K. Fredrickson, J.M. Zachara, L. Shi, A.S. Beliaev, M.J. Marshall, M. Tien, S. Brantley, J.N. Butt, D.J. Richardson, Characterization of an electron conduit between bacteria and the extracellular environment, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 22169–22174. [18] P.J. Cai, X. Xiao, Y.R. He, W.W. Li, J. Chu, C. Wu, M.X. He, Z. Zhang, G.P. Sheng, M.H.W. Lam, F. Xu, H.Q. Yu, Anaerobic biodecolorization mechanism of methyl orange by Shewanella oneidensis MR-1, Appl. Microbiol. Biotechnol. 93 (2012) 1769–1776. [19] X. Xiao, C.C. Xu, Y.M. Wu, P.J. Cai, W.W. Li, D.L. Du, H.Q. Yu, Biodecolorization of metal-complex dye naphthol green B by Shewanella oneidensis MR-1 under anaerobic conditions, Bioresour. Technol. (2012) 099, http://dx.doi.org/10.1016/j.biortech.2010.01. [20] O. Bretschger, A. Obraztsova, C.A. Sturm, I.S. Chang, Y.A. Gorby, S.B. Reed, D.E. Culley, C.L. Reardon, S. Barua, M.F. Romine, J. Zhou, A.S. Beliaev, R. Bouhenni, D. Saffarini, F. Mansfeld, B.H. Kim, J.K. Fredrickson, K.H. Nealson, Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants, Appl. Environ. Microbiol. 73 (2007) 7003–7012. [21] C.R. Myers, J.M. Myers, Cloning and sequence of cymA a gene encoding a tetraheme cytochrome c required for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1, J. Bacteriol. 179 (1997) 1143–1152. [22] B. Schuetz, M. Schicklberger, J. Kuermann, A.M. Spormann, J. Gescher, Periplasmic electron transfer via the c-type cytochromes MtrA and FccA of Shewanella oneidensis MR-1, Appl. Environ. Microbiol. 75 (2009) 7789–7796. [23] C. Schwalb, S.K. Chapman, G.A. Reid, The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis, Biochemistry 42 (2003) 9491–9497. [24] C. Bryant, M. DeLuca, Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae, J. Biol. Chem. 266 (1991) 4119–4125.
Contents lists available at SciVerse ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Involvement of c-type cytochrome CymA in the electron transfer of anaerobic nitrobenzene reduction by Shewanella oneidensis MR-1 Pei-Jie Cai a,b,d , Xiang Xiao a,c,∗ , Yan-Rong He a , Wen-Wei Li a,b,∗∗ , Lei Yu a,b , Michael Hon-Wah Lam b,d , Han-Qing Yu a,b a
Department of Chemistry, University of Science & Technology of China, Hefei 230026, China Advanced Laboratory for Environmental Research & Technology, USTC-CityU, Suzhou 215123, China c School of Environment, Jiangsu University, Zhenjiang 212013, China d Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong b
a r t i c l e
i n f o
Article history: Received 8 May 2012 Received in revised form 29 June 2012 Accepted 25 July 2012 Available online 2 August 2012 Keywords: Anaerobic processes Biodegradation Nitrobenzene (NB) Waste-water treatment Protein Shewanella oneidensis MR-1
a b s t r a c t Effective bioreduction of nitrobenzene (NB) by electrochemically active bacteria, such as Shewanella and Geobacter, has been demonstrated. However, the mechanism behind such a bioreduction is unclear yet. In this work, the mechanism for anaerobic NB reduction by Shewanella oneidensis MR-1 was investigated at a gene level. The omcA-mtrCAB gene cluster, an important extracellular electron transfer chain in S. oneidensis MR-1 for the reduction of a variety of compounds, was found to be uninvolved in the NB bioreduction. Knockout of cymA, a tetraheme c-type cytochrome in the periplasmic space, led to a 67% loss in NB bioreduction efficiency in comparison with the wild strain, and caused accumulation of an intermediate, phenylhydroxylamine, at the initial stage of NB bioreduction. These results demonstrate that, unlike the previously reported pure extracellular reduction pathways for many compounds, intracellular reactions are involved in NB bioreduction by S. oneidensis MR-1, and CymA plays an important role in the electron transfer of the intracellular NB bioreduction. In addition, the participation of some unknown protein other than CymA in NB reduction is discovered, and the possible pathways of electron transfer in the NB reduction by S. oneidensis MR-1 are proposed. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Nitroaromatic compounds are refractory pollutants released into environment almost exclusively from anthropogenic sources, such as incomplete combustion of fossil fuels and synthesis of chemicals [1]. Nitrobenzene (NB) is a classic nitroaromatic compound with relatively simple structure but high health hazards [2]. Thus, effective degradation of NB becomes imperative. Several physical methods, e.g., sonolysis [3,4] and radiolysis [5], and chemical methods, e.g., Fenton reagent [6–8] and bioelectrochemical system [9], have been used to remove NB, but with relatively high costs. Comparatively, biodegradation of NB offers an economically attractive way [10]. In recent years, many pure anaerobic eubacteria [11] or anaerobic bacteria [12,13] have been reported
Abbreviations: NB, nitrobenzene; AN, aniline; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; DMSO, dimethyl sulfoxide. ∗ Corresponding author at: Department of Chemistry, University of Science & Technology of China, Hefei 230026, China. Fax: +86 511 88790955. ∗∗ Corresponding author at: Department of Chemistry, University of Science & Technology of China, Hefei 230026, China. Fax: +86 512 87161362. E-mail addresses: [email protected] (X. Xiao), [email protected] (W.-W. Li). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.07.022
to catalyze NB reduction, and the anaerobic decomposition pathway of NB has been well understood. However, the pathway of such bioreduction process at gene level is still unclear. Shewanella oneidensis MR-1 has been frequently used as a model organism for microbial degradation mechanism investigations [14,15]. NB bioreduction by Shewanella has been previously reported [13]. The omcA-mtrCAB gene cluster of S. oneidensis MR1 is considered as an important anaerobic respiratory pathway to transfer electron from the cytoplasmic membrane to extracellular electron acceptor during bioreduction of many organics [16–19]. But, it is unclear whether this pathway is also responsible for the NB reduction. To date, the specific mechanism of NB bioreduction by S. oneidensis MR-1 is yet to be elucidated. Thus, if NB bioreduction is through extracellular reduction, it would be interesting to find whether the omcA-mtrCAB gene cluster is involved in the electron transfer. If it is not solely through extracellular reduction, then quite likely the CymA in the periplasmic space may play a role [20]. Therefore, this work aims to elucidate the mechanism of NB anaerobic reduction by S. oneidensis MR-1 at gene level. For this purpose, the performance of NB bioreduction by S. oneidensis MR-1 and its mutant strains was evaluated and the intermediate products were analyzed. The experimental results may facilitate a
228
P.-J. Cai et al. / Biochemical Engineering Journal 68 (2012) 227–230
better understanding on the anaerobic bioreduction of nitroaromatic compounds. 2. Materials and methods 2.1. Inoculum and culturing medium Table 1 lists the strains used in this work, including S. oneidensis MR-1 (ATCC® number is 700550TM ) and its mutant strains, which were kindly provided by Prof. K.H. Nealson from the University of Southern California [20]. All the strains were validated and cultured in LB nutrient solution at 30 ◦ C until reaching the cell’s stationary phase. Then, the cells were harvested by centrifugation at 7000 × g for 15 min. Phenylhydroxylamine (ALDRICH Co., USA), and azobenzene (Sinopharm Chemical Reagent Co., Shanghai, China) were used as the standards to determine the intermediates of NB bioreduction. 2.2. NB bioreduction tests NB bioreduction tests were conducted with a synthesized medium. The medium contained (per liter): 3.36 g sodium lactate (the sole electron donor), 11.91 g HEPES, 0.3 g NaOH, 1.5 g NH4 Cl, 0.1 g KCl, 0.67 g NaH2 PO4 ·2H2 O, 5.85 g NaCl and 1 ml of trace mineral stock solution [20]. The defined medium of 100 ml was added into each serum vial with N2 aeration for 10 min. Then, the serum vials were sealed with butyl rubber stoppers and autoclaved. After that, 0.1 ml of sterile vitamin solution [21] and 0.1 ml of amino acid solution (filtering through 0.22 m filter membrane) was added into the serum vial. The NB concentration in the serum vial was 185 mg l−1 . Then, the harvested cells were washed with the defined medium for three times and inoculated. The OD600 of the initial cells concentration in the serum vials was 1.0.
chromatography (HPLC, 2695, Waters Co., USA) with a HCC18 column (reversed-phase column, particle size 5 m, 4.6 mm × 250 mm) and a UV-detector (detection wavelength of 265 nm (NB) and 231 nm (AN)), by comparing the retention times with those of the standard chemicals. Samples of 10 l after filtering through 0.45 m filter membrane were injected. H2 O and methanol were used as the eluent with a volume ratio of 40:60 (%) at a flow rate of 1.0 ml min−1 at 25 ◦ C. 3. Results 3.1. Role of omcA-mtrCAB gene cluster in NB bioreduction To find out whether NB bioreduction by S. oneidensis MR-1 was an extracellular reduction process, the NB reduction performances of wild strain and its mutants (omcA/mtrC, mtrB, mtrA; Table 1) were evaluated. Unlike the MO bioreduction process [18], no obvious difference of NB bioreduction was observed for the wild strain of S. oneidensis MR-1 and its mtr-deleted mutants (Fig. 1). This indicates that the electron transfer to NB was not through the Mtr respiratory pathway. Meanwhile, the total NB concentration, calculated from the remaining NB concentration in the defined medium and the equivalent NB concentration from the produced AN at the same time, indicate that approximately 40% of NB disappeared during NB bioreduction by the cymA-deleted mutant strain, whereas the adsorption experimental results show that only
2.3. Absorption tests NB adsorption test by S. oneidensis MR-1 was carried out in serum vials containing 100 ml of the defined medium without sodium lactate (the electron donor). After centrifugation at 7000 × g for 15 min, the supernatant was transferred into a beaker. The cells were re-suspended with 100 ml of fresh medium without sodium lactate. Then, the NB concentrations in the initial medium and the re-suspension solution were determined. 2.4. Analysis The concentrations of NB, aniline (AN) and the degradation intermediates were determined with a high performance liquid
Table 1 S. oneidensis strains used in this work [20]. Open reading frame
Gene product deleted (gene)
SO4591
Tetraheme cytochrome c (cymA), involved in anaerobic respiration (except trimethylamine oxide) Cytochrome c552 nitrite reductase (nrfA), involved with nitrite reduction OM protein precursor (mtrB), involved in metal oxide reduction Periplasmic decaheme cytochrome c (mtrA), involved in metal oxide reduction Decaheme cytochrome c complex (omcA mtrA), involved in metal oxide reduction Diheme cytochrome c (napB), involved in nitrate reduction Wild strain
SO3980 SO1776 SO1777 SO1778/SO1779 SO0845 MR-1
Fig. 1. (A) The changes of AN concentration in NB bioreduction by S. oneidensis MR-1 and its mutants (cymA, omcA/mtrC, mtrB, mtrA) within 24 h under anaerobic conditions; and (B) the changes of NB concentration in NB bioreduction by S. oneidensis MR-1 and its mutants (cymA, omcA/mtrC, mtrB, mtrA) within 24 h under anaerobic conditions.
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Fig. 2. HPLC chromatogram of the NB bioreducation products by S. oneidensis MR-1 and the cymA-deleted mutant strain.
approximately 8% of NB was absorbed by S. oneidensis MR-1. Thus, it implies that part of the NB had entered into the cells and been reduced intracellularly. 3.2. Role of CymA, the key c-type cytochrome in NB bioreduction The role of CymA, a c-type cytochrome distributed in the periplasmic space, was also investigated in NB bioreduction. Compared with the wild strain, knockout of cymA resulted in a significant loss in NB bioreduction by 67% (Fig. 1), indicating that CymA was the major pathway of electron transfer in NB bioreduction by S. oneidensis MR-1. Interestingly, another isolated peak in the HPLC chromatogram, ascribed to phenylhydroxylamine, was also found at the initial stage of NB bioreduction by cymA mutant strain (Fig. 2). But, this peak was not observed for the wild strain and other mutant strains, indicating that NB bioreduction by S. oneidensis MR-1 was a multistep reaction. 4. Discussion The omcA-mtrCAB gene cluster has been recognized as an important anaerobic respiratory pathway to transfer electrons from the cytoplasmic membrane to extracellular electron acceptor [16,17]. Previous studies have demonstrated that the Mtr respiratory pathway could be a major pathway of electron transfer for an extracellular organic pollutants bioreduction [18,19]. In this work, however, no obvious decrease in NB bioreduction by S. oneidensis MR-1 was observed when the Mtr respiratory pathway was blocked (Fig. 1), suggesting that the electron transfer was not through Mtr respiratory pathway. The “disappeared” NB implies that NB partially entered into cells, and the NB bioreduction partially occurred inside the cells. CymA is a c-type cytochrome belonging to the NapC/NirT family in the periplasmic space, and is essential for the anaerobic respiration of S. oneidensis [21]. It is known as an important pathway to transfer electrons from menquionone/menaquinol (MQ/MQH2 ) pool to some electron acceptors, e.g., nitrite, fumarate, and dimethyl sulfoxide (DMSO) [22,23]. However, so far there is no direct evidence to show that CymA is involved in NB bioreduction. Fig. 1 shows that the knockout of cymA led to a significant loss of NB bioreduction ability. This suggests that CymA could be a key c-type cytochrome in the electron transformation chain of NB reduction. In addition, an intermediate product was found at the initial stage of the bioreduction process by a cymA-deleted mutant strain, but disappeared at the middle stage of the bioreduction.
Fig. 3. Proposed mechanisms of anaerobic reduction NB by S. oneidensis MR-1 (the question mark indicates the unknown proteins involved in NB bioreduction by S. oneidensis MR-1; the solid lines indicate the bioreduction pathway of NB; the dotted lines show the electron transfer pathway).
These results further confirm that CymA plays a significant role in the electron transfer during NB bioreduction in periplasmic space. Interestingly, the results also suggest that some unknown proteins in the periplasmic space seem to be also involved in the electron transfer, because the reduction of NB by S. oneidensis MR-1 continued after blocking the CymA pathway. Therefore, it can be inferred that the CymA pathway of Shewanella enables a sufficient supply of electrons and a rapid reduction of NB to phenylhydroxylamine and further to AN. Hence, no accumulation of phenylhydroxylamine occurred for the wild strain. While for the cymA-deleted strain, because the CymA pathway was blocked, only small amount of electrons was transferred through some unknown proteins (as denoted by the question mark in Fig. 3). This insufficient supply of electrons might have led to an incomplete reduction of NB in the multistep reaction and an accumulation of intermediate. Previous studies have shown that several reductases and electron shuttles in other bacteria are recognized to participate in various reduction reactions (e.g., nitrite, DMSO and fumarate) [22,23]. Especially, Bryant and DeLuc [24] found that nitroreductase are involved in the reduction of NB by Enterobacter cloacae. In addition, considering the similarity between NB and nitrite reduction (which has been identified to be nitroreductase catalysis process in Shewanella), it is thus quite possible that NB reduction by S. oneidensis MR-1 is also catalyzed by nitroreductases. These nitroreductases, as downstream proteins of CymA, may contain a series of proteins that efficiently catalyze the entire reactions of NB to phenylhydroxylamine and further to AN. Admittedly, although the present study gives some valuable clues about the electron transfer pathway of NB reduction by Shewanella and proposes a presumed mechanism, the detailed mechanisms of this process are yet to be further elucidated and convinced. Especially, from the results of this study, it is still too early to conclude whether the reduction of NB to phenylhydroxylamine is catalyzed by the periplasmic nitroreductases or directly by some unknown enzymes upstream of CymA. All of these need more experimental evidences and may warrant more future research works.
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5. Conclusions In this study, the mechanism of NB bioreduction by S. oneidensis MR-1 is investigated at a gene level. The omcA-mtrCAB gene cluster was found to be uninvolved in the NB bioreduction, while the electron transfer via CymA in the periplasmic space is observed. The NB mass balance analysis verifies that NB bioreduction is not a pure extracellular reaction. The significant loss in NB-bioreducing ability of the strain with blocked CymA indicates that CymA plays a critical role in the electron transfer chain of NB bioreduction by S. oneidensis MR-1. Nevertheless, some NB reduction capability still remains for the bacterium after being deleted of CymA, suggesting that some unknown protein other than CymA is also involved in this reduction process. In addition, the results suggest that some periplasmic reductases, as downstream proteins of CymA, might directly catalyze the NB reduction. But these mechanisms need further elucidations in future works. Acknowledgements This work was partially supported by the NSFC (20907050) and the NSFC-JST Joint Project (21021140001). The authors wish to thank Prof. K.H. Nealson from University of Southern California for kindly providing the bacteria used in this work. References [1] J.M. Spain, Biodegradation of nitroaromatic compounds, Annu. Rev. Microbiol. 49 (1995) 523–555. [2] H.L. Li, Y. Cheng, H.F. Wang, H.F. Sun, Y.F. Liu, K.X. Liu, S.X. Peng, Inhibition of nitrobenzene-induced DNA and hemolglobin adductions by dietary constituents, Appl. Radiat. Isot. 58 (2003) 1131–1142. [3] H.M. Hung, F.H. Ling, M.R. Hoffmann, Kinetics and mechanism of the enhanced reductive degradation of nitrobenzene by elemental iron in the presence of ultrasound, Environ. Sci. Technol. 34 (2000) 1758–1763. [4] L.K. Weavers, F.H. Ling, M.R. Hoffmann, Aromatic compound degradation in water using a combination of sonolysis and ozonolysis, Environ. Sci. Technol. 32 (1998) 2727–2733. [5] S.J. Zhang, H. Jiang, M.J. Li, H.Q. Yu, H. Yin, Q.R. Li, Kinetics and mechanisms of radiolytic degradation of nitrobenzene in aqueous solutions, Environ. Sci. Technol. 41 (2007) 1977–1982. [6] L. Carlos, D. Fabbri, A.L. Capparelli, A.B. Prevot, E. Pramauro, F.G. Einschlag, Effect of simulated solar light on the autocatalytic degradation of nitrobenzene using Fe3+ and hydrogen peroxide, J. Photochem. Photobiol. A 201 (2009) 32–38. [7] L. Carlos, D. Nichela, J.M. Triszcz, J.I. Felice, F.S.G. Einschlag, Nitration of nitrobenzene in Fenton’s processes, Chemosphere 80 (2010) 340–345.
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