J. Molec. Microbiol. Biotechnol. (1999) 1(1): 157-164.
JMMB
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, 1-
phenylethanol, 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 toluene-
grown 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 rrabus@mpi-bremen.de;
Tel. +49-421-2028-736; Fax. +49-421-2028-790.
© 1999 Horizon Scientific Press
Anaerobic Degradation of Ethylbenzene Article
and Toluene 157
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), sulfate-
reducing (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 sulfate-
reducing 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, p-
cymene 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 ethylbenzene-
and toluene-specific degradative pathways in the
158 Champion et al.
o degradation of acetophenone would require a carboxylation
o as well as acetophenone (Figure 2B), was only observed
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, 1-
phenylethanol-dehydrogenase; 5, acetophenone-carboxylase; 6, CoA-
ligase 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 1-
phenylethanol and acetophenone are specific
intermediates of ethylbenzene degradation in strain EbN1
and that further degradation of acetophenone involves a
CO2-dependent reaction.
Results
Formation of Intermediates and CO2-Dependence of
Growth
Based on the previous finding that strain EbN1 utilizes 1-
phenylethanol or acetophenone as growth substrate, we
suggested (i) that anaerobic oxidation of ethylbenzene
might proceed via these two compounds and (ii) that further
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),
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

50 A utilize toluene (Figure 3B), whereas cells adapted to toluene

oxidized ethylbenzene after a lag period of only ten hours
40 (Figure 3A).
H-oxidized [mM]

To further elucidate the pathway of ethylbenzene

30 metabolism, we tested for the oxidation of hypothetical
intermediates using suspensions of cells grown on
20 ethylbenzene. As anticipated, the cell suspensions
immediately oxidized ethylbenzene. The hypothetical
10 intermediates in ethylbenzene degradation (1-
phenylethanol, acetophenone, and benzoate) were
0 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,
50 B 1-phenylethanol, or ethylbenzene. Oxidation of
acetophenone and 1-phenylethanol took place after a short
40 lag (4 h), whereas oxidation of ethylbenzene was initiated
H-oxidized [mM]

after approximately 12 h (data not shown).

30
Two-Dimensional Gel Electrophoresis
20 Cell suspension studies showed that cells cultured on
ethylbenzene or toluene immediately oxidized their
10
respective growth substrates, but required an adaptation
0
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
50 C of proteins specifically induced during the respective
degradative processes, we analyzed extracts of strain
EbN1 cultured on benzoate (Figure 5A), acetophenone
H-oxidized [mM]

40
(Figure 5B), toluene (Figure 5C), or ethylbenzene (Figure
30 5D) by two-dimensional gel electrophoresis. The
electrophoresis pattern of benzoate-grown cells served as
20 the basis for comparison, since benzoyl-CoA, a direct
metabolic derivative of benzoate, is a central intermediate
10 in the anaerobic breakdown of aromatic compounds.
Comparison of representative electrophoresis patterns to
0 the benzoate pattern (i.e., protein pattern of benzoate
0 30 60 90 120 150 grown cells) showed that induced proteins were present in
Time [h] cells grown on ethylbenzene, acetophenone, and toluene;
the induced proteins are circled in Figure 5.
Figure 2. Anaerobic Growth of Strain EbN1 The proteins induced during growth of strain EbN1 on
Anaerobic growth on ethylbenzene (A), acetophenone, (B), and benzoate ethylbenzene corresponded to several proteins induced
(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
during growth on acetophenone. For example, protein spot
medium at a final concentration of 30 mM. [H] oxidized was calculated E1 in the ethylbenzene pattern migrates to the same
from consumed nitrate and intermediately produced nitrite as described in position as spot A1 in the acetophenone pattern. Also, spots
legend to Figure 3.

Figure 3. Expression of the Capacity to

50
A 50
B Degrade Ethylbenzene and Toluene in Cell
Suspensions of Denitrifying Strain EbN1
(A) Cells had been grown on
40 40 ethylbenzene. (B) Cells had been grown
on toluene. (O) Control with no organic
H-oxidized [mM]

H-oxidized [mM]

substrate added. (■) Ethylbenzene was

30 30 added as sole source of electron donor.
(●) Toluene was added as sole source of
electron donor. [H] oxidized (i.e., oxidized
20 20 reducing equivalents) was calculated from
consumed nitrate and intermediately
produced nitrite according to the following
10 10 equation: [H] oxidized = 5 • [nitrate added
– (nitrate remaining + nitrite remaining)] +
2 • [nitrite remaining] (Rabus and Widdel,
0 0 1995a).
0 10 20 30 0 50 100 150 200
Time [h] Time [h]
160 Champion et al.

60 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
50
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
40 E1 is identical to the first six residues of protein A1.
H-oxidized [mM]

Therefore, we propose that proteins E1 and A1 are the

same gene product. Corresponding positions in
30 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.
20
When proteins migrate with the same molecular mass
but slightly different isoelectric point within an
electrophoresis pattern, the proteins are most likely charge
10 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
0 proteins A2 and A3, E2 and E3, and E5 and E6. For each
0 10 20 30 40 of these protein pairs, identical N-terminal sequences were
Time [h] found (Table 1), suggesting that both proteins in each pair
are the same gene product. By analogy, protein E4 is most
Figure 4. Expression of the Capacity to Degrade 1-Phenylethanol,
Acetophenone and Benzoate in Suspensions of Ethylbenzene Grown Cells likely identical to proteins E2 and E3, though E4 has not
Symbols: (■) ethylbenzene; (●) 1-phenylethanol; (▲) acetophenone; (◆) been sequenced.
benzoate; (O) no organic substrate added. [H] oxidized was calculated Protein A4 accumulates to high levels in
from consumed nitrate and intermediately produced nitrite as described in acetophenone-grown cells, while the corresponding
legend to Figure 3.
protein, E7, accumulates to somewhat lower levels in
ethylbenzene-grown cells. The N-terminal sequence of
E3 and E2 (Figure 5D) migrate to the same position as protein A4 (Mr ~76 kDa/ pI ~5.6) is identical to the N-
spots A3 and A2 (Figure 5B), respectively. The migration terminal sequence of proteins E5 and E6 (Mr ~73 kDa/ pI
of proteins to the same positions in gels suggests the ~6.0-6.1). The modification leading to this altered mass
proteins are the same gene product. This observed and isoelectric point is currently unknown, though a C-
correspondence in protein patterns supports our proposal terminal proteolytic cleavage is possible. This is an
that acetophenone is an intermediate in ethylbenzene interesting finding, since it suggests that proteins E5, E6,
degradation. Differences in steady state levels of protein E7, and A4 are isoforms of a single polypeptide, and
between acetophenone- and ethylbenzene-grown cells therefore, that proteins E5 and E6 are not actually
may reflect combined differences in gene expression and ethylbenzene-specific. However, we cannot formally
protein modifications. It should be noted that limited eliminate the possibility that proteins A4 and E7 are
solubility of membrane proteins may prevent their encoded by a different gene than that encoding E5 and
appearance in gels, so proteins potentially involved in E6.
membrane-associated reactions or transport may not be N-terminal sequences (22-35 residues) of proteins A1,
observed. A2, A7, E5, and E8, which represent all proteins listed in
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) Table 1. N-Terminal Sequences of Proteins Induced During Growth of
Strain EbN1 on Alkylbenzenes
electrophoresis patterns is consistent with our results from
growth and cell suspension experiments, which showed Protein N-terminal sequenceb Growth
spota substrate
that strain EbN1 requires an adaptation period when the
growth substrate is changed from toluene to ethylbenzene, A1 SSLTNQDAINSIDIDVGGTFTDFVLTLDGEXHIAK Acetophenone
A2 AIPTLEQKLTWLKPAPASSRELDLAAQI Acetophenone
or from ethylbenzene to toluene. A3 AIPTLE Acetophenone
A4 MYTVDIDTGGTMTDALVSDGEQRHAIKVDTT Acetophenone
A7 MDVMKMFDLTGQVAVITGAGNGLGYIFAEAMAEAG Acetophenone
Protein Microsequencing and Database Searching
To further characterize selected proteins induced during E1 SSLTNQ Ethylbenzene
E2 AIPTLE Ethylbenzene
growth on ethylbenzene (Figure 5D) or acetophenone E3 AIPTL Ethylbenzene
(Figure 5B), we determined their N-terminal sequences E5 MYTVDIDTGGTMTDALVSDGEQRHAIKVDTT Ethylbenzene
E6 MYTVDIDTGGTMTDALVSDG Ethylbenzene
(Table 1). Sequencing reactions were conducted to obtain E8 MEAAGALWRRRMQELARGAGKPH Ethylbenzene
sufficient information to query databases or to confirm a
Protein numbers refer to protein spots in Figure 5.
expected protein identities. b N-terminal sequences were determined by Edman degradation of proteins
Proteins E1 and A1, which accumulate to high steady from polyvinylidene difluoride membranes. X indicates that an amino acid
residue could not be assigned.
state levels during growth on ethylbenzene (Figure 5D)
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
Anaerobic Degradation of Ethylbenzene and Toluene 161

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).
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-D-
gluconate-3-dehydrogenase from Archaeoglobus fulgidus
(Genbank, AE0010201). The N-terminal sequence of
protein E5 was found to be similar (42% identities) to N-
methylhydantoinase 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 1-
phenylethanol or via styrene as a preceding intermediate
(Heider and Fuchs, 1997b).
Growth of strain EbN1 on ethylbenzene, 1-
phenylethanol, 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 toluene-
metabolizing 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 fumarate-
dependent 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 SDS-
PAGE (Altenschmidt and Fuchs, 1992) and two-
dimensional 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 two-
dimensional 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

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 nitrate-
and 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 1-
phenylethanol 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 Tris-
HCl, 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.
Anaerobic Degradation of Ethylbenzene and Toluene 163
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 Humboldt-
Foundation of Germany.
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