Anaerobic Degradation of Ethylbenzene and ... - Semantic Scholar
J. Molec. Microbiol. Biotechnol. (1999) 1(1): 157-164.
Anaerobic Degradation of Ethylbenzene and Toluene 157 JMMB Article
Anaerobic Degradation of Ethylbenzene and Toluene in Denitrifying Strain EbN1 Proceeds via Independent Substrate-Induced Pathways Kathleen M. Champion1, Karsten Zengler, and Ralf Rabus* Max-Planck-Institut für Marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany
Abstract Denitrifying strain EbN1 utilizes either ethylbenzene or toluene as the sole source of organic carbon under strictly anoxic conditions. When cells were grown on ethylbenzene, 1-phenylethanol and acetophenone were detected in the culture supernatant. However, these two compounds were not observed when cells were grown on benzoate. Growth on ethylbenzene, 1phenylethanol, or acetophenone strictly depended on the presence of CO2, whereas growth on benzoate did not. These results suggest that strain EbN1 degrades ethylbenzene via 1-phenylethanol and acetophenone as intermediates, and that acetophenone is subsequently carboxylated. In suspensions of benzoate-grown cells, induction was required for degradation of ethylbenzene, 1-phenylethanol, and acetophenone. Induction was also required for toluenegrown cells to gain activity to degrade ethylbenzene, and, conversely, for ethylbenzene-grown cells to degrade toluene. In accordance with our findings from these studies, two-dimensional gel electrophoretic analysis of extracts of cells grown on benzoate, acetophenone, ethylbenzene, or toluene showed that a number of substrate-specific proteins were induced in strain EbN1. Growth on toluene or ethylbenzene induced a distinct set of proteins. However, some of the induced proteins in ethylbenzene or acetophenone grown cells were identical. This agrees with the finding that acetophenone is an intermediate in the degradation of ethylbenzene. Introduction Degradation of aromatic hydrocarbons by bacteria has long been considered to be restricted to aerobic organisms because molecular oxygen was regarded as an essential co-substrate for the activation of these chemically inert compounds. However, the isolation of anaerobic bacteria capable of utilizing alkylbenzenes as the sole source of carbon and energy under strictly anoxic conditions demonstrated that anaerobic degradation of alkylbenzenes Received February 17, 1999; revised March 9, 1999; accepted March 11, 1999. 1Present address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A. *For correspondence. Email [email protected]; Tel. +49-421-2028-736; Fax. +49-421-2028-790.
© 1999 Horizon Scientific Press
is indeed possible. Interest in this newly discovered bacterial metabolism arose from the assumption that novel biochemical reactions were required for the degradation of the aromatic hydrocarbons in the absence of molecular oxygen. Among the alkylbenzenes, toluene has been studied most intensively with respect to the types of organisms capable of degrading it anaerobically and the biochemical reactions involved in its initial activation. Several pure cultures of denitrifying (Dolfing et al., 1990; Altenschmidt and Fuchs, 1991; Schocher et al., 1991; Evans et al., 1991; Fries et al., 1994; Rabus and Widdel, 1995a), sulfatereducing (Rabus et al., 1993; Beller et al., 1996) and ferric iron-reducing (Lovley and Lonergan, 1990) bacteria have been reported to degrade toluene anaerobically. Recent studies with permeabilized cells and crude extracts demonstrated that a fumarate-dependent formation of benzylsuccinate is probably the general reaction for anaerobic activation of toluene in denitrifying and sulfatereducing bacteria (Biegert et al., 1996; Beller and Spormann, 1997; Beller and Spormann, 1998; Leuthner et al., 1998; Rabus and Heider, 1998). Evidence from genetic analyses suggests that benzylsuccinate synthase is a novel glycyl-radical enzyme (Coschigano et al., 1998; Leuthner et al., 1998). Benzylsuccinate is believed to be converted to benzoyl-CoA, the central aromatic intermediate, by a ß-oxidation-like reaction sequence (Figure 1). Anaerobic degradation of ethylbenzene was first demonstrated with two denitrifying strains, EbN1 (Rabus and Widdel, 1995a) and EB1 (Ball et al., 1996). A unique characteristic of strain EbN1 is its ability to utilize both toluene and ethylbenzene, as growth substrates. Based on growth experiments and substrate conversion studies with whole cells (Rabus and Widdel, 1995a; Ball et al., 1996), it was suggested that anaerobic degradation of ethylbenzene was initiated by oxidation of the methylene group, yielding 1-phenylethanol, and successively, acetophenone. This has recently been confirmed by studies with cell-free extracts of strain EbN1 (Rabus and Heider, 1998). Further degradation of acetophenone was proposed to require carboxylation followed by ß-oxidation to yield the central intermediate, benzoyl-CoA (Rabus and Widdel, 1995a) (Figure 1). Other alkylbenzenes shown to be degradable under anoxic conditions include xylenes, propylbenzene, pcymene and homologous alkylbenzenes (Harms et al., 1999a; 1999b; Rabus and Widdel, 1995a; Rabus et al., 1996; Rabus et al., 1999). Current knowledge of the organisms and biochemistry involved in anaerobic degradation of aromatic compounds has recently been summarized (Heider and Fuchs, 1997a; 1997b). In the present study, we investigated the ethylbenzeneand toluene-specific degradative pathways in the
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Figure 1. Proposed Pathways of Anaerobic Degradation of Toluene (A) and ethylbenzene (B) in denitrifying strain EbN1. Proposed enzymatic reactions: 1, benzylsuccinate synthase; 2, several enzymatic reactions may be involved to yield benzoyl-CoA; 3, initial oxidation of ethylbenzene; 4, 1phenylethanol-dehydrogenase; 5, acetophenone-carboxylase; 6, CoAligase and subsequent ß-oxidation-like reactions to yield benzoyl-CoA. Modified from Rabus and Heider (1998).
denitrifying strain EbN1. Simultaneous adaptation to different aromatic substrates was studied in cell suspensions, and substrate-specific protein induction was analyzed using comparative two-dimensional gel electrophoresis. The results of our studies demonstrate that the pathways for ethylbenzene and toluene degradation operate independently and are induced by their respective substrate. In addition, we show that 1phenylethanol and acetophenone are specific intermediates of ethylbenzene degradation in strain EbN1 and that further degradation of acetophenone involves a CO2-dependent reaction.
Formation of Intermediates and CO2-Dependence of Growth Based on the previous finding that strain EbN1 utilizes 1phenylethanol or acetophenone as growth substrate, we suggested (i) that anaerobic oxidation of ethylbenzene might proceed via these two compounds and (ii) that further degradation of acetophenone would require a carboxylation reaction (Rabus and Widdel, 1995a) (Figure 1). The first assumption was substantiated by the finding that acetophenone (21 µM) and 1-phenylethanol (3.5 µM) accumulated in culture supernatants during growth of strain EbN1 on ethylbenzene. In contrast, neither acetophenone nor 1-phenylethanol was detected during growth on benzoate. Identification of these compounds was based on retention times and spectra as compared to those of standards (see Experimental procedures). If the second assumption were true, it should also be possible to demonstrate dependence on carbon dioxide. Therefore, we studied whether growth of strain EbN1 on benzoate, acetophenone, or ethylbenzene depends on the presence of carbon dioxide. Growth on benzoate (Figure 2C) did not require carbon dioxide, although initiation of growth was somewhat delayed in carbon dioxide-depleted medium. In contrast, growth on ethylbenzene (Figure 2A), as well as acetophenone (Figure 2B), was only observed in the presence of carbon dioxide. In the absence of carbon dioxide neither growth nor nitrate reduction coupled to ethylbenzene or acetophenone oxidation could be determined. The cells in carbon dioxide-depleted medium were still viable, since introduction of CO2 to the cultures allowed resumption of growth (Figure 2A and B). Simultaneous Adaptation to Utilization of Aromatic Compounds In a previous publication we reported that denitrifying strain EbN1 can use toluene, in addition to ethylbenzene, as the sole source of carbon and energy (Rabus and Widdel, 1995a). In order to determine whether strain EbN1 was simultaneously adapted to growth on both of these alkylbenzenes, we conducted additional experiments. Ethylbenzene-grown cultures of strain EbN1 required two days to reach full growth (OD660 of approximately 0.6) on ethylbenzene, but four days to reach full growth on toluene. In contrast, toluene-grown cultures of strain EbN1 required only two days to reach full growth on toluene, but required four days to grow on ethylbenzene. Since ethylbenzene and toluene were present in the medium at the same equilibrium concentration of 0.3 mM, the observed differences in growth times should not be due to differences in substrate availability. To further investigate the possibility that strain EbN1 employs two distinct metabolic routes for the degradation of ethylbenzene and toluene, we studied the presence of the degradative capacities for these two compounds in cell suspensions. Cells were passaged on ethylbenzene or toluene at least 10 times prior to preparation of cell suspensions. Oxidation of alkylbenzenes was determined by measuring the consumption of nitrate and intermediate formation of nitrite. As expected, all cell suspensions immediately oxidized the substrate that had been used for cultivation. Cells adapted to ethylbenzene required a long lag period (approximately 130 h) before they were able to
Anaerobic Degradation of Ethylbenzene and Toluene 159
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utilize toluene (Figure 3B), whereas cells adapted to toluene oxidized ethylbenzene after a lag period of only ten hours (Figure 3A). To further elucidate the pathway of ethylbenzene metabolism, we tested for the oxidation of hypothetical intermediates using suspensions of cells grown on ethylbenzene. As anticipated, the cell suspensions immediately oxidized ethylbenzene. The hypothetical intermediates in ethylbenzene degradation (1phenylethanol, acetophenone, and benzoate) were oxidized immediately and at the same rate as ethylbenzene (Figure 4). In a complementary experiment, we tested the capacity of benzoate grown cells to oxidize acetophenone, 1-phenylethanol, or ethylbenzene. Oxidation of acetophenone and 1-phenylethanol took place after a short lag (4 h), whereas oxidation of ethylbenzene was initiated after approximately 12 h (data not shown).
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Time [h] Figure 2. Anaerobic Growth of Strain EbN1 Anaerobic growth on ethylbenzene (A), acetophenone, (B), and benzoate (C) in the presence (●) and absence (O) of CO2/HCO3-. At the time indicated by the arrow, bicarbonate was added to culture (■) with CO2-depleted medium at a final concentration of 30 mM. [H] oxidized was calculated from consumed nitrate and intermediately produced nitrite as described in legend to Figure 3.
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Two-Dimensional Gel Electrophoresis Cell suspension studies showed that cells cultured on ethylbenzene or toluene immediately oxidized their respective growth substrates, but required an adaptation phase to oxidize the other alkylbenzene. Furthermore, benzoate-grown cells required an adaptation phase for acetophenone or ethylbenzene utilization. In order to correlate these adaptation phenomena with the presence of proteins specifically induced during the respective degradative processes, we analyzed extracts of strain EbN1 cultured on benzoate (Figure 5A), acetophenone (Figure 5B), toluene (Figure 5C), or ethylbenzene (Figure 5D) by two-dimensional gel electrophoresis. The electrophoresis pattern of benzoate-grown cells served as the basis for comparison, since benzoyl-CoA, a direct metabolic derivative of benzoate, is a central intermediate in the anaerobic breakdown of aromatic compounds. Comparison of representative electrophoresis patterns to the benzoate pattern (i.e., protein pattern of benzoate grown cells) showed that induced proteins were present in cells grown on ethylbenzene, acetophenone, and toluene; the induced proteins are circled in Figure 5. The proteins induced during growth of strain EbN1 on ethylbenzene corresponded to several proteins induced during growth on acetophenone. For example, protein spot E1 in the ethylbenzene pattern migrates to the same position as spot A1 in the acetophenone pattern. Also, spots
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Figure 3. Expression of the Capacity to Degrade Ethylbenzene and Toluene in Cell Suspensions of Denitrifying Strain EbN1 (A) Cells had been grown on ethylbenzene. (B) Cells had been grown on toluene. (O) Control with no organic substrate added. (■) Ethylbenzene was added as sole source of electron donor. (●) Toluene was added as sole source of electron donor. [H] oxidized (i.e., oxidized reducing equivalents) was calculated from consumed nitrate and intermediately produced nitrite according to the following equation: [H] oxidized = 5 • [nitrate added – (nitrate remaining + nitrite remaining)] + 2 • [nitrite remaining] (Rabus and Widdel, 1995a).
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Time [h] Figure 4. Expression of the Capacity to Degrade 1-Phenylethanol, Acetophenone and Benzoate in Suspensions of Ethylbenzene Grown Cells Symbols: (■) ethylbenzene; (●) 1-phenylethanol; (▲) acetophenone; (◆) benzoate; (O) no organic substrate added. [H] oxidized was calculated from consumed nitrate and intermediately produced nitrite as described in legend to Figure 3.
E3 and E2 (Figure 5D) migrate to the same position as spots A3 and A2 (Figure 5B), respectively. The migration of proteins to the same positions in gels suggests the proteins are the same gene product. This observed correspondence in protein patterns supports our proposal that acetophenone is an intermediate in ethylbenzene degradation. Differences in steady state levels of protein between acetophenone- and ethylbenzene-grown cells may reflect combined differences in gene expression and protein modifications. It should be noted that limited solubility of membrane proteins may prevent their appearance in gels, so proteins potentially involved in membrane-associated reactions or transport may not be observed. The proteins induced during growth of strain EbN1 on toluene were distinct from the proteins induced during growth on ethylbenzene or acetophenone. Indeed, the presence of induced, non-overlapping protein spots in the toluene (Figure 5C) and ethylbenzene (Figure 5D) electrophoresis patterns is consistent with our results from growth and cell suspension experiments, which showed that strain EbN1 requires an adaptation period when the growth substrate is changed from toluene to ethylbenzene, or from ethylbenzene to toluene. Protein Microsequencing and Database Searching To further characterize selected proteins induced during growth on ethylbenzene (Figure 5D) or acetophenone (Figure 5B), we determined their N-terminal sequences (Table 1). Sequencing reactions were conducted to obtain sufficient information to query databases or to confirm expected protein identities. Proteins E1 and A1, which accumulate to high steady state levels during growth on ethylbenzene (Figure 5D)
and acetophenone (Figure 5B), respectively, migrate with a molecular mass of about 81 kDa and isoelectric point of about 6.5. Protein A1 was sequenced through 31 cycles, to obtain sufficient sequence for database queries, and protein E1 was sequenced through six cycles of Edman degradation to confirm its correspondence to protein A1. As shown in Table 1, the six residue sequence from protein E1 is identical to the first six residues of protein A1. Therefore, we propose that proteins E1 and A1 are the same gene product. Corresponding positions in electrophoresis patterns and identical N-terminal sequences were also demonstrated for proteins A2 and E2 and proteins A3 and E3. By analogy, we assume that protein spots E7 and A4 represent the same gene product. When proteins migrate with the same molecular mass but slightly different isoelectric point within an electrophoresis pattern, the proteins are most likely charge variants of the same polypeptide, as demonstrated previously (Champion et al., 1994). An example of this among the proteins present in strain EbN1 is the pairs of proteins A2 and A3, E2 and E3, and E5 and E6. For each of these protein pairs, identical N-terminal sequences were found (Table 1), suggesting that both proteins in each pair are the same gene product. By analogy, protein E4 is most likely identical to proteins E2 and E3, though E4 has not been sequenced. Protein A4 accumulates to high levels in acetophenone-grown cells, while the corresponding protein, E7, accumulates to somewhat lower levels in ethylbenzene-grown cells. The N-terminal sequence of protein A4 (Mr ~76 kDa/ pI ~5.6) is identical to the Nterminal sequence of proteins E5 and E6 (Mr ~73 kDa/ pI ~6.0-6.1). The modification leading to this altered mass and isoelectric point is currently unknown, though a Cterminal proteolytic cleavage is possible. This is an interesting finding, since it suggests that proteins E5, E6, E7, and A4 are isoforms of a single polypeptide, and therefore, that proteins E5 and E6 are not actually ethylbenzene-specific. However, we cannot formally eliminate the possibility that proteins A4 and E7 are encoded by a different gene than that encoding E5 and E6. N-terminal sequences (22-35 residues) of proteins A1, A2, A7, E5, and E8, which represent all proteins listed in
Table 1. N-Terminal Sequences of Proteins Induced During Growth of Strain EbN1 on Alkylbenzenes N-terminal sequenceb
Growth substrate
A1 A2 A3 A4 A7
SSLTNQDAINSIDIDVGGTFTDFVLTLDGEXHIAK AIPTLEQKLTWLKPAPASSRELDLAAQI AIPTLE MYTVDIDTGGTMTDALVSDGEQRHAIKVDTT MDVMKMFDLTGQVAVITGAGNGLGYIFAEAMAEAG
Acetophenone Acetophenone Acetophenone Acetophenone Acetophenone
E1 E2 E3 E5 E6 E8
SSLTNQ AIPTLE AIPTL MYTVDIDTGGTMTDALVSDGEQRHAIKVDTT MYTVDIDTGGTMTDALVSDG MEAAGALWRRRMQELARGAGKPH
Ethylbenzene Ethylbenzene Ethylbenzene Ethylbenzene Ethylbenzene Ethylbenzene
Protein spota
a Protein numbers refer to protein spots in Figure 5. b N-terminal sequences were determined by Edman degradation of proteins
from polyvinylidene difluoride membranes. X indicates that an amino acid residue could not be assigned.
Figure 5. Two-Dimensional Gel Electrophoresis Mapping of Strain EbN1 Grown on (A) benzoate, (B) acetophenone, (C) toluene, and (D) ethylbenzene as sole source of carbon. Proteins induced during growth on ethylbenzene, acetophenone, or toluene, but not on benzoate, are labeled with circles. Protein spots with assigned numbers, and which have been subjected to N-terminal sequencing, are listed in Table 1. Proteins were detected by staining with Coomassie blue R250. The protein patterns are oriented with high molecular mass proteins toward the top and low molecular mass proteins toward the bottom. Acidic polypeptides are to the left and basic proteins are to the right. The standard protein solution, used for molecular mass markers in the second dimension, contained the following polypeptides: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa).
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Table 1, were screened against protein and translated nucleotide databases using the BLAST (Altschul et al., 1990) program. None of these N-terminal sequences was found to be identical to any sequence deposited in the current databases. This finding supports the notion that novel proteins are involved in the studied metabolic pathways. Nevertheless, the BLAST searches revealed similarities with uncharacterized proteins of similar mass and isoelectric point. The N-terminal sequence of protein A7 showed similarity (54% identities) to 2-deoxy-Dgluconate-3-dehydrogenase from Archaeoglobus fulgidus (Genbank, AE0010201). The N-terminal sequence of protein E5 was found to be similar (42% identities) to Nmethylhydantoinase A from Aquifex aeolicus (Genbank, AE000703). Hydantoinases are also referred to as dihydropyrimidases, which are involved in the metabolism of pyrimidines (May et al., 1998). Discussion The identification of 1-phenylethanol and acetophenone in supernatants of ethylbenzene utilizing cultures of strain EbN1 confirmed earlier studies with denitrifying strains EbN1 (Rabus and Widdel, 1995a; Rabus and Heider, 1998) and EB1 (Ball et al., 1996) which proposed that these compounds are early intermediates of anaerobic ethylbenzene oxidation. At present, it is not clear whether the initial oxidation of ethylbenzene proceeds directly to 1phenylethanol or via styrene as a preceding intermediate (Heider and Fuchs, 1997b). Growth of strain EbN1 on ethylbenzene, 1phenylethanol, or acetophenone was found to be strictly dependent on the presence of CO2 (Figure 2), supporting our previous assumption (Rabus and Widdel, 1995a) that degradation of acetophenone should involve a carboxylation reaction. Similar results were recently reported for strain EB1 (Ball et al., 1996). Such a reaction would be reminiscent of the anaerobic metabolism of the structurally analogous short-chain aliphatic ketone, acetone, as recently summarized by Ensign et al. (1998). Evidence for a CO2-dependent, anaerobic formation of acetoacetate from acetone was mainly obtained from studies with whole cells (Siegel, 1950; Bonnet-Smits et al., 1988; Platen and Schink, 1989; Janssen and Schink, 1995a; 1995b). Only recently, anaerobic acetone carboxylase (Birks and Kelly, 1997) and acetoacetate decarboxylase (Janssen and Schink, 1995c) activities were demonstrated in cell-free extracts. Anaerobic acetone carboxylase activity was found to have distinct biochemical characteristics, in that it is dependent on ATP, Mg2+ and CoA or Acetyl-CoA, it is inducible by its substrate, acetone, and it is associated with the expression of two polypeptides (Birks and Kelly, 1997). Acetophenone would be the first aromatic ketone known to serve as substrate in an anaerobic carboxylation reaction. Furthermore, we present evidence that ethylbenzene and toluene degradation in the denitrifying strain EbN1 proceeds via two independent metabolic routes, which are specifically induced by the respective alkylbenzene substrate. (i) Experiments with cell suspensions showed that ethylbenzene grown cells could immediately utilize ethylbenzene, but showed a lag prior to toluene degradation. Conversely, toluene-grown cells immediately
utilized toluene, but showed a lag prior to ethylbenzene metabolism. (ii) Studies with two-dimensional gel electrophoresis showed that specific, distinct sets of proteins were induced under ethylbenzene- or toluenemetabolizing conditions. Recent studies with cell-free extracts of strain EbN1 on the initial activation reaction of toluene and ethylbenzene degradation (Rabus and Heider, 1998) further supported the inducibility of the two pathways. Extracts of toluene grown cells catalyzed the fumaratedependent formation of [ 14C]-labeled benzylsuccinate from [ phenyl - 14 C]-toluene. In contrast, extracts from ethylbenzene grown cells could not catalyze this reaction but instead formed [ 14C]-labeled 1-phenylethanol and acetophenone from [ methylene-14C]-ethylbenzene. In previous studies, substrate specific induction of proteins in toluene-metabolizing denitrifying Thauera aromatica, strain K172, was demonstrated with one-dimensional SDSPAGE (Altenschmidt and Fuchs, 1992) and twodimensional gel electrophoresis (Heider et al., 1998). In the present study, application of two-dimensional gel electrophoresis allowed us to demonstrate the substrate inducibility of ethylbenzene and toluene specific pathways on the protein level. Proteins were detected by Coomassie brilliant blue staining with the intent of focusing on the major catabolic enzymes in the cells. Extracts from ethylbenzene and acetophenone grown cells were found to contain several proteins that were absent in extracts from benzoate grown cells. Therefore these proteins can be regarded as being specifically induced during ethylbenzene as well as acetophenone utilization. These substrate-induced proteins were found to be unique, in that their N-terminal sequences were not identical to any protein sequences available in the current databases. For further studies, these N-terminal sequences could prove useful in an attempt to clone genes encoding proteins specific for ethylbenzene or acetophenone degradation. Experimental Procedures Source of Organism The ethylbenzene-degrading, denitrifying bacterial strain EbN1 has been subcultured in our laboratory since its isolation (Rabus and Widdel, 1995a). Anaerobic cultivation of strain EbN1 in ascorbate-reduced defined mineral medium with ethylbenzene or toluene diluted (5 and 2%, respectively, v/v) in an inert carrier phase (2,2,4,4,6,8,8-heptamethylnonane) and 10 mM nitrate under strictly anoxic conditions were carried out as previously described (Rabus and Widdel, 1995a). Anoxic, sterile heptamethylnonane was prepared and stored as described previously (Aeckersberg et al., 1991). Cultures for preparation of cell suspensions and cell extracts for twodimensional gel electrophoresis were carried out in 2 l and 500 ml glass bottles, respectively. Preparation of media and techniques for cultivation under anoxic conditions were performed as described by Widdel and Bak (1992). Growth Experiments For growth experiments in the absence of CO2, the medium was modified with respect to the buffer and preparation. Tris/HCl (40 mM) was used as buffer instead of bicarbonate. After autoclaving, the medium was purged with sterile N2 gas for two hours at 100 °C. The pH of the medium was adjusted to 7.2 with NaOH. The medium was then distributed in aliquots of 30 ml under an atmosphere of N2 into anoxic tubes (50 ml). Media prepared in this way had a CO2 concentration of 63 nM, as determined by gas chromatographic analysis. This concentration is about 1000 times lower than that in normal water. Residual bicarbonate was removed from cultures used for inoculation by repeated resuspension of the centrifuged cells in CO2-depleted Tris/HCl buffer (40 mM, pH 7.2). The cells were kept under an atmosphere of N2 during centrifugation, removal of the supernatant, and resuspension. Bicarbonate (40 mM) was added to certain cultures after 42 h of incubation by means of a N2-flushed syringe. Utilization of ethylbenzene, acetophenone, and benzoate was monitored by measuring
Anaerobic Degradation of Ethylbenzene and Toluene 163
the consumption of nitrate in aliquots (0.2 ml) taken with N2-flushed syringes. Growth was determined by measuring the optical density at 660 nm. Experiments with Cell Suspensions All steps for harvesting cells and preparing cell suspensions were carried out inside an anoxic chamber. Cells were obtained from mid-exponential phase cultures at an optical density of 0.3, measured at 660 nm. The cell density was increased two-fold by centrifuging the cultures and resuspending the cells in half the original volume of nitrate-free, ascorbate-reduced mineral medium without organic substrates. Aliquots of 50 ml were distributed to serum bottles (70 ml). Toluene or ethylbenzene (2%, v/v) were added to 2 ml of anoxic heptamethylnonane in the serum bottles. Anoxic heptamethylnonane without organic substrate was added to the controls. After the serum bottles were sealed with butyl rubber stoppers, the gas phase from the anoxic chamber was replaced by N2/CO2 (90/10, v/v). Nitrate (10 mM) was then added by means of N2-flushed microliter syringes. The cell suspensions were incubated on a rotary shaker (70 rpm) at 28 °C in a horizontal position to facilitate substrate diffusion from the carrier phase into the medium. Any contact between the carrier phase in the medium and the butyl rubber stoppers was avoided. Utilization of ethylbenzene and toluene was monitored by measuring the consumption of nitrate in aliquots (0.5 ml) taken with N2-flushed syringes. Chemical Analysis Nitrate and nitrite were determined by high performance liquid chromatography as previously described (Rabus and Widdel, 1995a). Oxidized reducing equivalents ([H]-oxidized) were calculated from nitrateand nitrite-concentrations as reported (Rabus and Widdel, 1995a). CO2 in the modified medium was determined using a gas chromatograph (GC-8A, Shimadzu, Duisburg, Germany) connected to a thermal conductivity detector on a Porapak Q-column (100 mesh, length 2 m, internal diameter 3 mm). The temperature was constant at 40 °C. N2 was used as carrier gas with a flow rate of 32 ml/min. Aromatic compounds were determined with a high performance liquid chromatographic system (Sykam, Gilching/Munich, Germany), as described (Rabus and Widdel, 1995b), employing two different separation systems. The first separation system consisted of a Spherisorb OD S2 reversed-phase column (250 x 5 mm; Grom, Herrenberg-Kayh, Germany), an eluent composed of 25% acetonitrile, and 0.75 mM H3PO4 in distilled H2O, a flow rate of 0.6 ml/min and a column temperature of 15 °C. Under these conditions acetophenone and 1-phenylethanol were separated at 38.3 min and 20.0 min, respectively. Secondly, a Hypersil APS column (250 x 4 mm; Grom, Herrenberg-Kayh, Germany), an eluent of 100% acetonitrile, a flow rate of 0.5 ml/min and a column temperature of 10 °C was used, which allowed the separation of acetophenone and 1phenylethanol at 4.8 min and 5.4 min, respectively. Identification of both compounds was based on retention times and spectra compared to those of standards. Protein Sample Preparation Prior to protein sample preparation cells from an ethylbenzene-grown stock culture were subcultured for at least 8 passages in medium containing toluene, ethylbenzene, acetophenone, or benzoate as the sole source of carbon. By subculturing on the same substrate, we (i) avoided carry over of ethylbenzene from the stock culture, (ii) ensured that proteins required for ethylbenzene metabolism were absent in cells grown on substrates other than ethylbenzene, and (iii) avoided possible stress responses by allowing the cells to adapt to the specific substrate. To prevent possible changes in protein pattern due to exposure to oxygen, cells were separated from the hydrophobic carrier phase, and harvested by centrifugation under anoxic conditions. Preparation of denatured protein samples for two-dimensional gel electrophoresis was performed as follows. First, logarithmically growing cultures (300 ml) were harvested by centrifugation at 9,000g for 30 min at 4 °C. The cells were then washed with a buffer containing 100 mM TrisHCl, pH 7.5 and 5 mM MgCl2. Wet cell pellets were suspended in 4-5 volumes of a solubilizing solution containing 9 M urea, 4% (v/v) Nonidet P40, 2% (w/v) carrier ampholytes (pH 9-11; Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 2% (v/v) 2-mercaptoethanol. Finally, the cells were lysed by three cycles of freezing and thawing, and treatment with a Dounce device (20-30 strokes). The resulting lysates were then centrifuged at 30,000g for 10 min at 20 °C. The supernatants containing denatured bacterial proteins were collected and stored frozen at -80 °C until analysis. Protein concentration was determined using a modified Bradford assay (Ramagli and Rodriguez, 1985) with bovine serum albumin as the standard. Protein samples were adjusted to the same protein concentration prior to analysis with two-dimensional gel electrophoresis.
Two-Dimensional Gel Electrophoresis A large-scale vertical two-dimensional gel electrophoresis system was used for high resolution separation of proteins essentially as described by O’Farrell (1975). Electrophoresis equipment was purchased from Amersham Pharmacia Biotech and employed as described by Anderson and Anderson (1978a,b). Protein samples were separated in the first dimension by isoelectric focusing in 25.4 cm tube gels (1.5 mm inside diameter) (Anderson and Anderson, 1978a) for a total of 30,000 V-h. The pH gradient (approximate pH range 4-8) was generated with a combination of ampholytes containing 25% (v/v) Biolyte (pH 3-10), 25% (v/v) Servalyte (pH 3-10), and 50% (v/v) Biolyte (pH 5-7), as described previously (Champion et al., 1994). The isoelectric focusing equipment allowed 20 identical tube gels to be cast simultaneously and subjected to electrophoresis at the same time. Carbamylated protein standards (Amersham Pharmacia Biotech) were included periodically with samples to serve as charge standards. The second-dimension separation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was carried out using the DALT system (Amersham Pharmacia Biotech). Slab gels (1.5 x 200 x 250 mm) were poured manually according to the manufacturer’s instructions to yield sets of 20 linear 10-17% T (total gel concentration) gradient gels (Anderson, 1991). Bis-acrylamide (2.6%) was used as the cross-linker. The gels were run essentially according to O’Farrell (1975) with modifications by Anderson and Anderson (1978b). High and low molecular mass standards (Amersham Pharmacia Biotech) were separated in the second dimension to serve as size standards. Upon completion of SDS-PAGE, the gels were fixed and stained overnight with 0.25% (w/v) Coomassie blue R250 in H3PO4 (2.5%) and ethanol (50%, v/v). Gels were destained in several washes of ethanol (20%, v/v) at 1 h intervals, followed by a final wash overnight. Protein Microsequencing Upon completion of two-dimensional gel electrophoresis, proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes using a Semi-Phor Model TE77 semi-dry blotter (Amersham Pharmacia Biotech) as described by Matsudaira (1982). Proteins on PVDF membranes (ProBlott, Applied Biosystems, Foster City, CA, USA) were detected by Coomassie blue R250 staining according to the manufacturer’s instructions. The corresponding protein spots from 3-5 replicate membranes were cut out of membranes using a scalpel and subjected to Edman degradation microsequencing (F. Lottspeich, Top Lab, Martinsried, Germany). Database Search Protein and translated nucleotide databases were screened using the BLAST program (Altschul et al., 1990). Acknowledgements We wish to thank Friedrich Widdel for his constant support. This research was supported by the Max-Planck-Gesellschaft. During preparation of the manuscript, R. Rabus was supported by the Alexander von HumboldtFoundation of Germany. References Aeckersberg, F., Bak, F., and Widdel, F. 1991. Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium. Arch. Microbiol. 156: 5-14. Altenschmidt, U., and Fuchs, G. 1991. Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: Indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate. Arch. Microbiol. 156: 152-158. Altenschmidt, U., and Fuchs, G. 1992. Anaerobic toluene oxidation to benzyl alcohol and benzaldehyde in a denitrifying Pseudomonas strain. J. Bacteriol. 174: 4860-4862. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410. Anderson, N.G., and Anderson, N.L. 1978a. Analytical techniques for cell fractions XXI. Two-dimensional analysis of serum and tissue proteins: Multiple isoelectric focusing. Anal. Biochem. 85: 331-340. Anderson, N.G., and Anderson, N.L. 1978b. Analytical techniques for cell fractions XXII. Two-dimensional analysis of serum and tissue proteins: Multiple gradient-slab electrophoresis. Anal. Biochem. 85: 341-354. Anderson, N.L. 1991. Two-dimensional electrophoresis: Operation of the ISO-DALT system. Large Scale Biology Press, Rockville, M.D. Ball, H.A., Johnson, H.A., Reinhard, M., and Sporman, A.M. 1996. Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1. J. Bacteriol. 178: 5755-5761. Beller, H.R., and Sporman, A.M. 1997. Benzylsuccinate formation as a
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means of anaerobic toluene activation by sulfate-reducing strain PRTOL1. Appl. Environ. Microbiol. 63: 3729-3731. Beller, H.R., and Spormann, A.M. 1998. Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product. J. Bacteriol. 180: 5454-5457. Beller, H.R., Sporman, A.M., Sharma, P.K., Cole, J.R., and Reinhard, M. 1996. Isolation and characterization of a novel toluene-degrading, sulfatereducing bacterium. Appl. Environ. Microbiol. 62: 1188-1196. Biegert, T., Fuchs, G., and Heider, J. 1996. Evidence that anaerobic oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate. Eur. J. Biochem. 238: 661-668. Birks, S.J., and Kelly, D.J. 1997. Assay and properties of acetone carboxylase, a novel enzyme involved in acetone-dependent growth and CO2 fixation in Rhodobacter capsulatus and other photosynthetic and denitrifying bacteria. Microbiol. 143: 755-766. Bonnet-Smits, E.M., Robertson, L.A., Van Dijken, J.P., Senior, E., and Kuenen, J.G. 1988. Carbon dioxide fixation as the initial step in the metabolism of acetone by Thiosphaera pantotropha. J. Gen. Microbiol. 134: 2281-2289. Champion, K.M., Cook, R.J., Tollaksen, S.L., and Giometti, C.S. 1994. Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice. Proc. Natl. Acad. Sci. USA. 91: 11338-11342. Coschigano, P.W., Wehrmann, T.S., and Young, L.Y. 1998. Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free radical. Appl. Environ. Microbiol. 64: 1650-1656. Dolfing, J., Zeyer, J., Binder-Eicher, P., and Schwarzenbach, R.P. 1990. Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. Arch. Microbiol. 154: 336-341. Ensign, S.A., Small, F.J., Allen, J.R., and Sluis, M.K. 1998. New roles of CO2 in the microbial metabolism of aliphatic epoxides and ketones. Arch. Microbiol. 169: 179-187. Evans, P.J., Mand, D.T., Kim, K.S., and Young, L.Y. 1991. Anaerobic degradation of toluene by a denitrifying bacterium. Appl. Environ. Microbiol. 57: 1139-1145. Fries, M.R., Zhou, J., Chee-Sanford, J., and Tiedje, J.M. 1994. Isolation, characterization and distribution of denitrifying toluene degraders from a variety of habitats. Appl. Environ. Microbiol. 60: 2802-2810. Harms, G., Rabus, R., and Widdel, F. 1999a. Anaerobic oxidation of pisopropyltoluene (p–cymene) by two new types of denitrifying bacteria. Arch. Microbiol. (In press). Harms, G., Zengler, K., Rabus, R., Aeckersberg, F., Minz, D., RossellóMora, R., and Widdel, F. 1999b. Anaerobic degradation of o-xylene, mxylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria. Appl. Environ. Microbiol. 65: 999-1004. Heider, J., and Fuchs, G. 1997a. Anaerobic metabolism of aromatic compounds. Eur. J. Biochem. 243: 577-596. Heider, J., and Fuchs, G. 1997b. Microbial anaerobic aromatic metabolism. Anaerobe 3: 1-22. Heider, J., Boll, M., Breese, K., Breinig, S., Ebenau-Jehle, C., Feil, U., Gad’on N., Laempe, D., Leuthner, B., Mohamed, M.E., Schneider, S., Burchhardt, G., and Fuchs, G. 1998. Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica. Arch. Micobiol. 170: 120-121. Janssen, P.H., and Schink, B. 1995a. Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biactus. J. Bacteriol. 177: 277-282. Janssen, P.H., and Schink, B. 1995b. Metabolic pathways and energetics of the acetone-oxidizing, sulfate-reducing bacterium Desulfobacterium cetonicum. Arch. Microbiol. 163: 188-194. Janssen, P.H., and Schink, B. 1995c. 14CO2 exchange with acetoacetate catalyzed by dialyzed cell-free extracts of the bacterial strain BunN grown with acetone and nitrate. Eur. J. Biochem. 228: 677-682. Leuthner, B., Leutwein, C., Schulz, H., Hörth, P., Haehnel, W., Schiltz, E., Schägger, H., and Heider, J. 1998. Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol. Microbiol. 28: 615-628. Lovley, D.R., and Lonergan, D.J. 1990. Anaerobic oxidation of toluene, phenol and p-cresol by the dissimilatory iron-reducing organism, GS-15. Appl. Environ. Microbiol. 56: 1858-1864. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262: 10035-10038. May, O., Habenicht, A., Mattes, R., Syldatk, C., and Sieman, M. 1998. Molecular evolution of hydantoinases. Biol. Chem. 379: 743-747.
O’Farrell, P.H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007-4021. Platen, H., and Schink, B. 1989. Anaerobic degradation of acetone and higher ketones by newly isolated denitrifying bacteria. J. Gen. Microbiol. 135: 883-891. Rabus, R., Fukui, M., Wilkes, H., and Widdel, F. 1996. Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfatereducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil. Appl. Environ. Microbiol. 62: 3605-3613. Rabus, R., and Heider, J. 1998. Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria. Arch. Microbiol. 170: 377-384. Rabus, R., Nordhaus, R., Ludwig, W., and Widdel, F. 1993. Complete oxidation of toluene under strictly anoxic conditions by a new sulfatereducing bacterium. Appl. Environ. Microbiol. 59: 1444-1451. Rabus, R., and Widdel, F. 1995a. Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Arch. Microbiol. 163: 96-103. Rabus, R., and Widdel, F. 1995b. Conversion studies with substrate analogues of toluene in a sulfate-reducing bacterium, strain Tol2. Arch. Microbiol. 164: 448-451. Rabus, R., Wilkes, H., Schramm, A., Harms, G., Behrends, A., Amann, R., and Widdel, F. 1999. Anaerobic utilization of alkylbenzenes and n-alkanes from crude oil in an enrichment culture of denitrifying bacteria affiliating with the ß-subclass of Proteobacteria. Environ. Microbiol. 1: 145-157. Ramagli, L.S., and Rodriguez, L.V. 1985. Quantitation of mircogram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 6: 559-563. Schocher, R.J., Seyfried, B., Vazquez, F., and Zeyer, J. 1991. Anaerobic degradation of toluene by pure cultures of denitrifying bacteria. Arch. Microbiol. 157: 7-12. Siegel, J.M. 1950. The metabolism of acetone by the photosynthetic bacterium Rhodopseudomonas gelatinosa. J. Bacteriol. 60: 595-606. Widdel, F., and Bak, F. 1992. Gram-negative mesophilic sulfate-reducing bacteria. In: The Prokaryotes, 2nd ed. Balows A, Trüper HG, Dworkin M, Harder W, Schleifer K-H (eds) New York: Springer-Verlag, pp. 583-624.
Anaerobic Degradation of Ethylbenzene and Toluene 157 JMMB Article
Anaerobic Degradation of Ethylbenzene and Toluene in Denitrifying Strain EbN1 Proceeds via Independent Substrate-Induced Pathways Kathleen M. Champion1, Karsten Zengler, and Ralf Rabus* Max-Planck-Institut für Marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany
Abstract Denitrifying strain EbN1 utilizes either ethylbenzene or toluene as the sole source of organic carbon under strictly anoxic conditions. When cells were grown on ethylbenzene, 1-phenylethanol and acetophenone were detected in the culture supernatant. However, these two compounds were not observed when cells were grown on benzoate. Growth on ethylbenzene, 1phenylethanol, or acetophenone strictly depended on the presence of CO2, whereas growth on benzoate did not. These results suggest that strain EbN1 degrades ethylbenzene via 1-phenylethanol and acetophenone as intermediates, and that acetophenone is subsequently carboxylated. In suspensions of benzoate-grown cells, induction was required for degradation of ethylbenzene, 1-phenylethanol, and acetophenone. Induction was also required for toluenegrown cells to gain activity to degrade ethylbenzene, and, conversely, for ethylbenzene-grown cells to degrade toluene. In accordance with our findings from these studies, two-dimensional gel electrophoretic analysis of extracts of cells grown on benzoate, acetophenone, ethylbenzene, or toluene showed that a number of substrate-specific proteins were induced in strain EbN1. Growth on toluene or ethylbenzene induced a distinct set of proteins. However, some of the induced proteins in ethylbenzene or acetophenone grown cells were identical. This agrees with the finding that acetophenone is an intermediate in the degradation of ethylbenzene. Introduction Degradation of aromatic hydrocarbons by bacteria has long been considered to be restricted to aerobic organisms because molecular oxygen was regarded as an essential co-substrate for the activation of these chemically inert compounds. However, the isolation of anaerobic bacteria capable of utilizing alkylbenzenes as the sole source of carbon and energy under strictly anoxic conditions demonstrated that anaerobic degradation of alkylbenzenes Received February 17, 1999; revised March 9, 1999; accepted March 11, 1999. 1Present address: Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, U.S.A. *For correspondence. Email [email protected]; Tel. +49-421-2028-736; Fax. +49-421-2028-790.
© 1999 Horizon Scientific Press
is indeed possible. Interest in this newly discovered bacterial metabolism arose from the assumption that novel biochemical reactions were required for the degradation of the aromatic hydrocarbons in the absence of molecular oxygen. Among the alkylbenzenes, toluene has been studied most intensively with respect to the types of organisms capable of degrading it anaerobically and the biochemical reactions involved in its initial activation. Several pure cultures of denitrifying (Dolfing et al., 1990; Altenschmidt and Fuchs, 1991; Schocher et al., 1991; Evans et al., 1991; Fries et al., 1994; Rabus and Widdel, 1995a), sulfatereducing (Rabus et al., 1993; Beller et al., 1996) and ferric iron-reducing (Lovley and Lonergan, 1990) bacteria have been reported to degrade toluene anaerobically. Recent studies with permeabilized cells and crude extracts demonstrated that a fumarate-dependent formation of benzylsuccinate is probably the general reaction for anaerobic activation of toluene in denitrifying and sulfatereducing bacteria (Biegert et al., 1996; Beller and Spormann, 1997; Beller and Spormann, 1998; Leuthner et al., 1998; Rabus and Heider, 1998). Evidence from genetic analyses suggests that benzylsuccinate synthase is a novel glycyl-radical enzyme (Coschigano et al., 1998; Leuthner et al., 1998). Benzylsuccinate is believed to be converted to benzoyl-CoA, the central aromatic intermediate, by a ß-oxidation-like reaction sequence (Figure 1). Anaerobic degradation of ethylbenzene was first demonstrated with two denitrifying strains, EbN1 (Rabus and Widdel, 1995a) and EB1 (Ball et al., 1996). A unique characteristic of strain EbN1 is its ability to utilize both toluene and ethylbenzene, as growth substrates. Based on growth experiments and substrate conversion studies with whole cells (Rabus and Widdel, 1995a; Ball et al., 1996), it was suggested that anaerobic degradation of ethylbenzene was initiated by oxidation of the methylene group, yielding 1-phenylethanol, and successively, acetophenone. This has recently been confirmed by studies with cell-free extracts of strain EbN1 (Rabus and Heider, 1998). Further degradation of acetophenone was proposed to require carboxylation followed by ß-oxidation to yield the central intermediate, benzoyl-CoA (Rabus and Widdel, 1995a) (Figure 1). Other alkylbenzenes shown to be degradable under anoxic conditions include xylenes, propylbenzene, pcymene and homologous alkylbenzenes (Harms et al., 1999a; 1999b; Rabus and Widdel, 1995a; Rabus et al., 1996; Rabus et al., 1999). Current knowledge of the organisms and biochemistry involved in anaerobic degradation of aromatic compounds has recently been summarized (Heider and Fuchs, 1997a; 1997b). In the present study, we investigated the ethylbenzeneand toluene-specific degradative pathways in the
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Figure 1. Proposed Pathways of Anaerobic Degradation of Toluene (A) and ethylbenzene (B) in denitrifying strain EbN1. Proposed enzymatic reactions: 1, benzylsuccinate synthase; 2, several enzymatic reactions may be involved to yield benzoyl-CoA; 3, initial oxidation of ethylbenzene; 4, 1phenylethanol-dehydrogenase; 5, acetophenone-carboxylase; 6, CoAligase and subsequent ß-oxidation-like reactions to yield benzoyl-CoA. Modified from Rabus and Heider (1998).
denitrifying strain EbN1. Simultaneous adaptation to different aromatic substrates was studied in cell suspensions, and substrate-specific protein induction was analyzed using comparative two-dimensional gel electrophoresis. The results of our studies demonstrate that the pathways for ethylbenzene and toluene degradation operate independently and are induced by their respective substrate. In addition, we show that 1phenylethanol and acetophenone are specific intermediates of ethylbenzene degradation in strain EbN1 and that further degradation of acetophenone involves a CO2-dependent reaction.
Formation of Intermediates and CO2-Dependence of Growth Based on the previous finding that strain EbN1 utilizes 1phenylethanol or acetophenone as growth substrate, we suggested (i) that anaerobic oxidation of ethylbenzene might proceed via these two compounds and (ii) that further degradation of acetophenone would require a carboxylation reaction (Rabus and Widdel, 1995a) (Figure 1). The first assumption was substantiated by the finding that acetophenone (21 µM) and 1-phenylethanol (3.5 µM) accumulated in culture supernatants during growth of strain EbN1 on ethylbenzene. In contrast, neither acetophenone nor 1-phenylethanol was detected during growth on benzoate. Identification of these compounds was based on retention times and spectra as compared to those of standards (see Experimental procedures). If the second assumption were true, it should also be possible to demonstrate dependence on carbon dioxide. Therefore, we studied whether growth of strain EbN1 on benzoate, acetophenone, or ethylbenzene depends on the presence of carbon dioxide. Growth on benzoate (Figure 2C) did not require carbon dioxide, although initiation of growth was somewhat delayed in carbon dioxide-depleted medium. In contrast, growth on ethylbenzene (Figure 2A), as well as acetophenone (Figure 2B), was only observed in the presence of carbon dioxide. In the absence of carbon dioxide neither growth nor nitrate reduction coupled to ethylbenzene or acetophenone oxidation could be determined. The cells in carbon dioxide-depleted medium were still viable, since introduction of CO2 to the cultures allowed resumption of growth (Figure 2A and B). Simultaneous Adaptation to Utilization of Aromatic Compounds In a previous publication we reported that denitrifying strain EbN1 can use toluene, in addition to ethylbenzene, as the sole source of carbon and energy (Rabus and Widdel, 1995a). In order to determine whether strain EbN1 was simultaneously adapted to growth on both of these alkylbenzenes, we conducted additional experiments. Ethylbenzene-grown cultures of strain EbN1 required two days to reach full growth (OD660 of approximately 0.6) on ethylbenzene, but four days to reach full growth on toluene. In contrast, toluene-grown cultures of strain EbN1 required only two days to reach full growth on toluene, but required four days to grow on ethylbenzene. Since ethylbenzene and toluene were present in the medium at the same equilibrium concentration of 0.3 mM, the observed differences in growth times should not be due to differences in substrate availability. To further investigate the possibility that strain EbN1 employs two distinct metabolic routes for the degradation of ethylbenzene and toluene, we studied the presence of the degradative capacities for these two compounds in cell suspensions. Cells were passaged on ethylbenzene or toluene at least 10 times prior to preparation of cell suspensions. Oxidation of alkylbenzenes was determined by measuring the consumption of nitrate and intermediate formation of nitrite. As expected, all cell suspensions immediately oxidized the substrate that had been used for cultivation. Cells adapted to ethylbenzene required a long lag period (approximately 130 h) before they were able to
Anaerobic Degradation of Ethylbenzene and Toluene 159
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utilize toluene (Figure 3B), whereas cells adapted to toluene oxidized ethylbenzene after a lag period of only ten hours (Figure 3A). To further elucidate the pathway of ethylbenzene metabolism, we tested for the oxidation of hypothetical intermediates using suspensions of cells grown on ethylbenzene. As anticipated, the cell suspensions immediately oxidized ethylbenzene. The hypothetical intermediates in ethylbenzene degradation (1phenylethanol, acetophenone, and benzoate) were oxidized immediately and at the same rate as ethylbenzene (Figure 4). In a complementary experiment, we tested the capacity of benzoate grown cells to oxidize acetophenone, 1-phenylethanol, or ethylbenzene. Oxidation of acetophenone and 1-phenylethanol took place after a short lag (4 h), whereas oxidation of ethylbenzene was initiated after approximately 12 h (data not shown).
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Time [h] Figure 2. Anaerobic Growth of Strain EbN1 Anaerobic growth on ethylbenzene (A), acetophenone, (B), and benzoate (C) in the presence (●) and absence (O) of CO2/HCO3-. At the time indicated by the arrow, bicarbonate was added to culture (■) with CO2-depleted medium at a final concentration of 30 mM. [H] oxidized was calculated from consumed nitrate and intermediately produced nitrite as described in legend to Figure 3.
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Two-Dimensional Gel Electrophoresis Cell suspension studies showed that cells cultured on ethylbenzene or toluene immediately oxidized their respective growth substrates, but required an adaptation phase to oxidize the other alkylbenzene. Furthermore, benzoate-grown cells required an adaptation phase for acetophenone or ethylbenzene utilization. In order to correlate these adaptation phenomena with the presence of proteins specifically induced during the respective degradative processes, we analyzed extracts of strain EbN1 cultured on benzoate (Figure 5A), acetophenone (Figure 5B), toluene (Figure 5C), or ethylbenzene (Figure 5D) by two-dimensional gel electrophoresis. The electrophoresis pattern of benzoate-grown cells served as the basis for comparison, since benzoyl-CoA, a direct metabolic derivative of benzoate, is a central intermediate in the anaerobic breakdown of aromatic compounds. Comparison of representative electrophoresis patterns to the benzoate pattern (i.e., protein pattern of benzoate grown cells) showed that induced proteins were present in cells grown on ethylbenzene, acetophenone, and toluene; the induced proteins are circled in Figure 5. The proteins induced during growth of strain EbN1 on ethylbenzene corresponded to several proteins induced during growth on acetophenone. For example, protein spot E1 in the ethylbenzene pattern migrates to the same position as spot A1 in the acetophenone pattern. Also, spots
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Figure 3. Expression of the Capacity to Degrade Ethylbenzene and Toluene in Cell Suspensions of Denitrifying Strain EbN1 (A) Cells had been grown on ethylbenzene. (B) Cells had been grown on toluene. (O) Control with no organic substrate added. (■) Ethylbenzene was added as sole source of electron donor. (●) Toluene was added as sole source of electron donor. [H] oxidized (i.e., oxidized reducing equivalents) was calculated from consumed nitrate and intermediately produced nitrite according to the following equation: [H] oxidized = 5 • [nitrate added – (nitrate remaining + nitrite remaining)] + 2 • [nitrite remaining] (Rabus and Widdel, 1995a).
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Time [h] Figure 4. Expression of the Capacity to Degrade 1-Phenylethanol, Acetophenone and Benzoate in Suspensions of Ethylbenzene Grown Cells Symbols: (■) ethylbenzene; (●) 1-phenylethanol; (▲) acetophenone; (◆) benzoate; (O) no organic substrate added. [H] oxidized was calculated from consumed nitrate and intermediately produced nitrite as described in legend to Figure 3.
E3 and E2 (Figure 5D) migrate to the same position as spots A3 and A2 (Figure 5B), respectively. The migration of proteins to the same positions in gels suggests the proteins are the same gene product. This observed correspondence in protein patterns supports our proposal that acetophenone is an intermediate in ethylbenzene degradation. Differences in steady state levels of protein between acetophenone- and ethylbenzene-grown cells may reflect combined differences in gene expression and protein modifications. It should be noted that limited solubility of membrane proteins may prevent their appearance in gels, so proteins potentially involved in membrane-associated reactions or transport may not be observed. The proteins induced during growth of strain EbN1 on toluene were distinct from the proteins induced during growth on ethylbenzene or acetophenone. Indeed, the presence of induced, non-overlapping protein spots in the toluene (Figure 5C) and ethylbenzene (Figure 5D) electrophoresis patterns is consistent with our results from growth and cell suspension experiments, which showed that strain EbN1 requires an adaptation period when the growth substrate is changed from toluene to ethylbenzene, or from ethylbenzene to toluene. Protein Microsequencing and Database Searching To further characterize selected proteins induced during growth on ethylbenzene (Figure 5D) or acetophenone (Figure 5B), we determined their N-terminal sequences (Table 1). Sequencing reactions were conducted to obtain sufficient information to query databases or to confirm expected protein identities. Proteins E1 and A1, which accumulate to high steady state levels during growth on ethylbenzene (Figure 5D)
and acetophenone (Figure 5B), respectively, migrate with a molecular mass of about 81 kDa and isoelectric point of about 6.5. Protein A1 was sequenced through 31 cycles, to obtain sufficient sequence for database queries, and protein E1 was sequenced through six cycles of Edman degradation to confirm its correspondence to protein A1. As shown in Table 1, the six residue sequence from protein E1 is identical to the first six residues of protein A1. Therefore, we propose that proteins E1 and A1 are the same gene product. Corresponding positions in electrophoresis patterns and identical N-terminal sequences were also demonstrated for proteins A2 and E2 and proteins A3 and E3. By analogy, we assume that protein spots E7 and A4 represent the same gene product. When proteins migrate with the same molecular mass but slightly different isoelectric point within an electrophoresis pattern, the proteins are most likely charge variants of the same polypeptide, as demonstrated previously (Champion et al., 1994). An example of this among the proteins present in strain EbN1 is the pairs of proteins A2 and A3, E2 and E3, and E5 and E6. For each of these protein pairs, identical N-terminal sequences were found (Table 1), suggesting that both proteins in each pair are the same gene product. By analogy, protein E4 is most likely identical to proteins E2 and E3, though E4 has not been sequenced. Protein A4 accumulates to high levels in acetophenone-grown cells, while the corresponding protein, E7, accumulates to somewhat lower levels in ethylbenzene-grown cells. The N-terminal sequence of protein A4 (Mr ~76 kDa/ pI ~5.6) is identical to the Nterminal sequence of proteins E5 and E6 (Mr ~73 kDa/ pI ~6.0-6.1). The modification leading to this altered mass and isoelectric point is currently unknown, though a Cterminal proteolytic cleavage is possible. This is an interesting finding, since it suggests that proteins E5, E6, E7, and A4 are isoforms of a single polypeptide, and therefore, that proteins E5 and E6 are not actually ethylbenzene-specific. However, we cannot formally eliminate the possibility that proteins A4 and E7 are encoded by a different gene than that encoding E5 and E6. N-terminal sequences (22-35 residues) of proteins A1, A2, A7, E5, and E8, which represent all proteins listed in
Table 1. N-Terminal Sequences of Proteins Induced During Growth of Strain EbN1 on Alkylbenzenes N-terminal sequenceb
Growth substrate
A1 A2 A3 A4 A7
SSLTNQDAINSIDIDVGGTFTDFVLTLDGEXHIAK AIPTLEQKLTWLKPAPASSRELDLAAQI AIPTLE MYTVDIDTGGTMTDALVSDGEQRHAIKVDTT MDVMKMFDLTGQVAVITGAGNGLGYIFAEAMAEAG
Acetophenone Acetophenone Acetophenone Acetophenone Acetophenone
E1 E2 E3 E5 E6 E8
SSLTNQ AIPTLE AIPTL MYTVDIDTGGTMTDALVSDGEQRHAIKVDTT MYTVDIDTGGTMTDALVSDG MEAAGALWRRRMQELARGAGKPH
Ethylbenzene Ethylbenzene Ethylbenzene Ethylbenzene Ethylbenzene Ethylbenzene
Protein spota
a Protein numbers refer to protein spots in Figure 5. b N-terminal sequences were determined by Edman degradation of proteins
from polyvinylidene difluoride membranes. X indicates that an amino acid residue could not be assigned.
Figure 5. Two-Dimensional Gel Electrophoresis Mapping of Strain EbN1 Grown on (A) benzoate, (B) acetophenone, (C) toluene, and (D) ethylbenzene as sole source of carbon. Proteins induced during growth on ethylbenzene, acetophenone, or toluene, but not on benzoate, are labeled with circles. Protein spots with assigned numbers, and which have been subjected to N-terminal sequencing, are listed in Table 1. Proteins were detected by staining with Coomassie blue R250. The protein patterns are oriented with high molecular mass proteins toward the top and low molecular mass proteins toward the bottom. Acidic polypeptides are to the left and basic proteins are to the right. The standard protein solution, used for molecular mass markers in the second dimension, contained the following polypeptides: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa).
Anaerobic Degradation of Ethylbenzene and Toluene 161
162 Champion et al.
Table 1, were screened against protein and translated nucleotide databases using the BLAST (Altschul et al., 1990) program. None of these N-terminal sequences was found to be identical to any sequence deposited in the current databases. This finding supports the notion that novel proteins are involved in the studied metabolic pathways. Nevertheless, the BLAST searches revealed similarities with uncharacterized proteins of similar mass and isoelectric point. The N-terminal sequence of protein A7 showed similarity (54% identities) to 2-deoxy-Dgluconate-3-dehydrogenase from Archaeoglobus fulgidus (Genbank, AE0010201). The N-terminal sequence of protein E5 was found to be similar (42% identities) to Nmethylhydantoinase A from Aquifex aeolicus (Genbank, AE000703). Hydantoinases are also referred to as dihydropyrimidases, which are involved in the metabolism of pyrimidines (May et al., 1998). Discussion The identification of 1-phenylethanol and acetophenone in supernatants of ethylbenzene utilizing cultures of strain EbN1 confirmed earlier studies with denitrifying strains EbN1 (Rabus and Widdel, 1995a; Rabus and Heider, 1998) and EB1 (Ball et al., 1996) which proposed that these compounds are early intermediates of anaerobic ethylbenzene oxidation. At present, it is not clear whether the initial oxidation of ethylbenzene proceeds directly to 1phenylethanol or via styrene as a preceding intermediate (Heider and Fuchs, 1997b). Growth of strain EbN1 on ethylbenzene, 1phenylethanol, or acetophenone was found to be strictly dependent on the presence of CO2 (Figure 2), supporting our previous assumption (Rabus and Widdel, 1995a) that degradation of acetophenone should involve a carboxylation reaction. Similar results were recently reported for strain EB1 (Ball et al., 1996). Such a reaction would be reminiscent of the anaerobic metabolism of the structurally analogous short-chain aliphatic ketone, acetone, as recently summarized by Ensign et al. (1998). Evidence for a CO2-dependent, anaerobic formation of acetoacetate from acetone was mainly obtained from studies with whole cells (Siegel, 1950; Bonnet-Smits et al., 1988; Platen and Schink, 1989; Janssen and Schink, 1995a; 1995b). Only recently, anaerobic acetone carboxylase (Birks and Kelly, 1997) and acetoacetate decarboxylase (Janssen and Schink, 1995c) activities were demonstrated in cell-free extracts. Anaerobic acetone carboxylase activity was found to have distinct biochemical characteristics, in that it is dependent on ATP, Mg2+ and CoA or Acetyl-CoA, it is inducible by its substrate, acetone, and it is associated with the expression of two polypeptides (Birks and Kelly, 1997). Acetophenone would be the first aromatic ketone known to serve as substrate in an anaerobic carboxylation reaction. Furthermore, we present evidence that ethylbenzene and toluene degradation in the denitrifying strain EbN1 proceeds via two independent metabolic routes, which are specifically induced by the respective alkylbenzene substrate. (i) Experiments with cell suspensions showed that ethylbenzene grown cells could immediately utilize ethylbenzene, but showed a lag prior to toluene degradation. Conversely, toluene-grown cells immediately
utilized toluene, but showed a lag prior to ethylbenzene metabolism. (ii) Studies with two-dimensional gel electrophoresis showed that specific, distinct sets of proteins were induced under ethylbenzene- or toluenemetabolizing conditions. Recent studies with cell-free extracts of strain EbN1 on the initial activation reaction of toluene and ethylbenzene degradation (Rabus and Heider, 1998) further supported the inducibility of the two pathways. Extracts of toluene grown cells catalyzed the fumaratedependent formation of [ 14C]-labeled benzylsuccinate from [ phenyl - 14 C]-toluene. In contrast, extracts from ethylbenzene grown cells could not catalyze this reaction but instead formed [ 14C]-labeled 1-phenylethanol and acetophenone from [ methylene-14C]-ethylbenzene. In previous studies, substrate specific induction of proteins in toluene-metabolizing denitrifying Thauera aromatica, strain K172, was demonstrated with one-dimensional SDSPAGE (Altenschmidt and Fuchs, 1992) and twodimensional gel electrophoresis (Heider et al., 1998). In the present study, application of two-dimensional gel electrophoresis allowed us to demonstrate the substrate inducibility of ethylbenzene and toluene specific pathways on the protein level. Proteins were detected by Coomassie brilliant blue staining with the intent of focusing on the major catabolic enzymes in the cells. Extracts from ethylbenzene and acetophenone grown cells were found to contain several proteins that were absent in extracts from benzoate grown cells. Therefore these proteins can be regarded as being specifically induced during ethylbenzene as well as acetophenone utilization. These substrate-induced proteins were found to be unique, in that their N-terminal sequences were not identical to any protein sequences available in the current databases. For further studies, these N-terminal sequences could prove useful in an attempt to clone genes encoding proteins specific for ethylbenzene or acetophenone degradation. Experimental Procedures Source of Organism The ethylbenzene-degrading, denitrifying bacterial strain EbN1 has been subcultured in our laboratory since its isolation (Rabus and Widdel, 1995a). Anaerobic cultivation of strain EbN1 in ascorbate-reduced defined mineral medium with ethylbenzene or toluene diluted (5 and 2%, respectively, v/v) in an inert carrier phase (2,2,4,4,6,8,8-heptamethylnonane) and 10 mM nitrate under strictly anoxic conditions were carried out as previously described (Rabus and Widdel, 1995a). Anoxic, sterile heptamethylnonane was prepared and stored as described previously (Aeckersberg et al., 1991). Cultures for preparation of cell suspensions and cell extracts for twodimensional gel electrophoresis were carried out in 2 l and 500 ml glass bottles, respectively. Preparation of media and techniques for cultivation under anoxic conditions were performed as described by Widdel and Bak (1992). Growth Experiments For growth experiments in the absence of CO2, the medium was modified with respect to the buffer and preparation. Tris/HCl (40 mM) was used as buffer instead of bicarbonate. After autoclaving, the medium was purged with sterile N2 gas for two hours at 100 °C. The pH of the medium was adjusted to 7.2 with NaOH. The medium was then distributed in aliquots of 30 ml under an atmosphere of N2 into anoxic tubes (50 ml). Media prepared in this way had a CO2 concentration of 63 nM, as determined by gas chromatographic analysis. This concentration is about 1000 times lower than that in normal water. Residual bicarbonate was removed from cultures used for inoculation by repeated resuspension of the centrifuged cells in CO2-depleted Tris/HCl buffer (40 mM, pH 7.2). The cells were kept under an atmosphere of N2 during centrifugation, removal of the supernatant, and resuspension. Bicarbonate (40 mM) was added to certain cultures after 42 h of incubation by means of a N2-flushed syringe. Utilization of ethylbenzene, acetophenone, and benzoate was monitored by measuring
Anaerobic Degradation of Ethylbenzene and Toluene 163
the consumption of nitrate in aliquots (0.2 ml) taken with N2-flushed syringes. Growth was determined by measuring the optical density at 660 nm. Experiments with Cell Suspensions All steps for harvesting cells and preparing cell suspensions were carried out inside an anoxic chamber. Cells were obtained from mid-exponential phase cultures at an optical density of 0.3, measured at 660 nm. The cell density was increased two-fold by centrifuging the cultures and resuspending the cells in half the original volume of nitrate-free, ascorbate-reduced mineral medium without organic substrates. Aliquots of 50 ml were distributed to serum bottles (70 ml). Toluene or ethylbenzene (2%, v/v) were added to 2 ml of anoxic heptamethylnonane in the serum bottles. Anoxic heptamethylnonane without organic substrate was added to the controls. After the serum bottles were sealed with butyl rubber stoppers, the gas phase from the anoxic chamber was replaced by N2/CO2 (90/10, v/v). Nitrate (10 mM) was then added by means of N2-flushed microliter syringes. The cell suspensions were incubated on a rotary shaker (70 rpm) at 28 °C in a horizontal position to facilitate substrate diffusion from the carrier phase into the medium. Any contact between the carrier phase in the medium and the butyl rubber stoppers was avoided. Utilization of ethylbenzene and toluene was monitored by measuring the consumption of nitrate in aliquots (0.5 ml) taken with N2-flushed syringes. Chemical Analysis Nitrate and nitrite were determined by high performance liquid chromatography as previously described (Rabus and Widdel, 1995a). Oxidized reducing equivalents ([H]-oxidized) were calculated from nitrateand nitrite-concentrations as reported (Rabus and Widdel, 1995a). CO2 in the modified medium was determined using a gas chromatograph (GC-8A, Shimadzu, Duisburg, Germany) connected to a thermal conductivity detector on a Porapak Q-column (100 mesh, length 2 m, internal diameter 3 mm). The temperature was constant at 40 °C. N2 was used as carrier gas with a flow rate of 32 ml/min. Aromatic compounds were determined with a high performance liquid chromatographic system (Sykam, Gilching/Munich, Germany), as described (Rabus and Widdel, 1995b), employing two different separation systems. The first separation system consisted of a Spherisorb OD S2 reversed-phase column (250 x 5 mm; Grom, Herrenberg-Kayh, Germany), an eluent composed of 25% acetonitrile, and 0.75 mM H3PO4 in distilled H2O, a flow rate of 0.6 ml/min and a column temperature of 15 °C. Under these conditions acetophenone and 1-phenylethanol were separated at 38.3 min and 20.0 min, respectively. Secondly, a Hypersil APS column (250 x 4 mm; Grom, Herrenberg-Kayh, Germany), an eluent of 100% acetonitrile, a flow rate of 0.5 ml/min and a column temperature of 10 °C was used, which allowed the separation of acetophenone and 1phenylethanol at 4.8 min and 5.4 min, respectively. Identification of both compounds was based on retention times and spectra compared to those of standards. Protein Sample Preparation Prior to protein sample preparation cells from an ethylbenzene-grown stock culture were subcultured for at least 8 passages in medium containing toluene, ethylbenzene, acetophenone, or benzoate as the sole source of carbon. By subculturing on the same substrate, we (i) avoided carry over of ethylbenzene from the stock culture, (ii) ensured that proteins required for ethylbenzene metabolism were absent in cells grown on substrates other than ethylbenzene, and (iii) avoided possible stress responses by allowing the cells to adapt to the specific substrate. To prevent possible changes in protein pattern due to exposure to oxygen, cells were separated from the hydrophobic carrier phase, and harvested by centrifugation under anoxic conditions. Preparation of denatured protein samples for two-dimensional gel electrophoresis was performed as follows. First, logarithmically growing cultures (300 ml) were harvested by centrifugation at 9,000g for 30 min at 4 °C. The cells were then washed with a buffer containing 100 mM TrisHCl, pH 7.5 and 5 mM MgCl2. Wet cell pellets were suspended in 4-5 volumes of a solubilizing solution containing 9 M urea, 4% (v/v) Nonidet P40, 2% (w/v) carrier ampholytes (pH 9-11; Amersham Pharmacia Biotech, Piscataway, NJ, USA), and 2% (v/v) 2-mercaptoethanol. Finally, the cells were lysed by three cycles of freezing and thawing, and treatment with a Dounce device (20-30 strokes). The resulting lysates were then centrifuged at 30,000g for 10 min at 20 °C. The supernatants containing denatured bacterial proteins were collected and stored frozen at -80 °C until analysis. Protein concentration was determined using a modified Bradford assay (Ramagli and Rodriguez, 1985) with bovine serum albumin as the standard. Protein samples were adjusted to the same protein concentration prior to analysis with two-dimensional gel electrophoresis.
Two-Dimensional Gel Electrophoresis A large-scale vertical two-dimensional gel electrophoresis system was used for high resolution separation of proteins essentially as described by O’Farrell (1975). Electrophoresis equipment was purchased from Amersham Pharmacia Biotech and employed as described by Anderson and Anderson (1978a,b). Protein samples were separated in the first dimension by isoelectric focusing in 25.4 cm tube gels (1.5 mm inside diameter) (Anderson and Anderson, 1978a) for a total of 30,000 V-h. The pH gradient (approximate pH range 4-8) was generated with a combination of ampholytes containing 25% (v/v) Biolyte (pH 3-10), 25% (v/v) Servalyte (pH 3-10), and 50% (v/v) Biolyte (pH 5-7), as described previously (Champion et al., 1994). The isoelectric focusing equipment allowed 20 identical tube gels to be cast simultaneously and subjected to electrophoresis at the same time. Carbamylated protein standards (Amersham Pharmacia Biotech) were included periodically with samples to serve as charge standards. The second-dimension separation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was carried out using the DALT system (Amersham Pharmacia Biotech). Slab gels (1.5 x 200 x 250 mm) were poured manually according to the manufacturer’s instructions to yield sets of 20 linear 10-17% T (total gel concentration) gradient gels (Anderson, 1991). Bis-acrylamide (2.6%) was used as the cross-linker. The gels were run essentially according to O’Farrell (1975) with modifications by Anderson and Anderson (1978b). High and low molecular mass standards (Amersham Pharmacia Biotech) were separated in the second dimension to serve as size standards. Upon completion of SDS-PAGE, the gels were fixed and stained overnight with 0.25% (w/v) Coomassie blue R250 in H3PO4 (2.5%) and ethanol (50%, v/v). Gels were destained in several washes of ethanol (20%, v/v) at 1 h intervals, followed by a final wash overnight. Protein Microsequencing Upon completion of two-dimensional gel electrophoresis, proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes using a Semi-Phor Model TE77 semi-dry blotter (Amersham Pharmacia Biotech) as described by Matsudaira (1982). Proteins on PVDF membranes (ProBlott, Applied Biosystems, Foster City, CA, USA) were detected by Coomassie blue R250 staining according to the manufacturer’s instructions. The corresponding protein spots from 3-5 replicate membranes were cut out of membranes using a scalpel and subjected to Edman degradation microsequencing (F. Lottspeich, Top Lab, Martinsried, Germany). Database Search Protein and translated nucleotide databases were screened using the BLAST program (Altschul et al., 1990). Acknowledgements We wish to thank Friedrich Widdel for his constant support. This research was supported by the Max-Planck-Gesellschaft. During preparation of the manuscript, R. Rabus was supported by the Alexander von HumboldtFoundation of Germany. References Aeckersberg, F., Bak, F., and Widdel, F. 1991. Anaerobic oxidation of saturated hydrocarbons to CO2 by a new type of sulfate-reducing bacterium. Arch. Microbiol. 156: 5-14. Altenschmidt, U., and Fuchs, G. 1991. Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: Indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate. Arch. Microbiol. 156: 152-158. Altenschmidt, U., and Fuchs, G. 1992. Anaerobic toluene oxidation to benzyl alcohol and benzaldehyde in a denitrifying Pseudomonas strain. J. Bacteriol. 174: 4860-4862. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410. Anderson, N.G., and Anderson, N.L. 1978a. Analytical techniques for cell fractions XXI. Two-dimensional analysis of serum and tissue proteins: Multiple isoelectric focusing. Anal. Biochem. 85: 331-340. Anderson, N.G., and Anderson, N.L. 1978b. Analytical techniques for cell fractions XXII. Two-dimensional analysis of serum and tissue proteins: Multiple gradient-slab electrophoresis. Anal. Biochem. 85: 341-354. Anderson, N.L. 1991. Two-dimensional electrophoresis: Operation of the ISO-DALT system. Large Scale Biology Press, Rockville, M.D. Ball, H.A., Johnson, H.A., Reinhard, M., and Sporman, A.M. 1996. Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1. J. Bacteriol. 178: 5755-5761. Beller, H.R., and Sporman, A.M. 1997. Benzylsuccinate formation as a
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