Phenol Degradation in the Strictly Anaerobic Iron-Reducing
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2009, p. 3912–3919 Vol. 75, No. 12
0099-2240/09/$08.00⫹0 doi:10.1128/AEM.01525-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Phenol Degradation in the Strictly Anaerobic Iron-Reducing
Bacterium Geobacter metallireducens GS-15䌤†
Kathleen M. Schleinitz,1* Sirko Schmeling,4 Nico Jehmlich,2 Martin von Bergen,2 Hauke Harms,1
Sabine Kleinsteuber,1 Carsten Vogt,3 and Georg Fuchs4
Department of Environmental Microbiology,1 Department of Proteomics,2 and Department of Isotope Biogeochemistry,3
UFZ—Helmholtz Centre for Environmental Research, Leipzig, Germany, and Mikrobiologie, Fakultät Biologie,
Albert-Ludwigs-Universität, Freiburg, Germany4
Received 5 July 2008/Accepted 8 April 2009
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Information on anaerobic phenol metabolism by physiological groups of bacteria other than nitrate reducers
is scarce. We investigated phenol degradation in the strictly anaerobic iron-reducing deltaproteobacterium
Geobacter metallireducens GS-15 using metabolite, transcriptome, proteome, and enzyme analyses. The results
showed that the initial steps of phenol degradation are accomplished by phenylphosphate synthase (encoded
by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) as known from Thauera aromatica, but
they also revealed some distinct differences. The pps-ppc gene cluster identified in the genome is functional, as
shown by transcription analysis. In contrast to T. aromatica, transcription of the pps- and ppc-like genes was
induced not only during growth on phenol, but also during growth on benzoate. In contrast, proteins were
detected only during growth on phenol, suggesting the existence of a posttranscriptional regulation mechanism
for these initial steps. Phenylphosphate synthase and phenylphosphate carboxylase activities were detected in
cell extracts. The carboxylase does not catalyze an isotope exchange reaction between 14CO2 and 4-hydroxy-
benzoate, which is characteristic of the T. aromatica enzyme. Whereas the enzyme of T. aromatica is encoded by
ppcABCD, the pps-ppc gene cluster of G. metallireducens contains only a ppcB homologue. Nearby, but oriented
in the opposite direction, is a ppcD homologue that is transcribed during growth on phenol. Genome analysis
did not reveal obvious homologues of ppcA and ppcC, leaving open the question of whether these genes are
dispensable for phenylphosphate carboxylase activity in G. metallireducens or are quite different from the
Thauera counterparts and located elsewhere in the genome.
Anaerobic phenol degradation is best understood in the is carboxylated by the core enzyme composed of ␣-, -, and
facultatively anaerobic denitrifier Thauera aromatica (DSM6984). ␥-subunits. This reaction requires CO2 (rather than bicarbon-
In this strain, phenol is initially converted to phenylphosphate ate, as known from other carboxylases) and is freely reversible.
by phenylphosphate synthase (Pps) with concomitant hydroly- Both the ␣- and -subunits display significant similarities to
sis of ATP (5, 16, 28) (Fig. 1A). The ␣- and -subunits of Pps UbiD of Escherichia coli, a 3-octaprenyl-4-hydroxybenzoyl de-
resemble the central and N-terminal parts of the phosphoenol- carboxylase involved in ubiquinone biosynthesis. Furthermore,
pyruvate synthase, respectively. The -subunit contains the similarities to other (de)carboxylating enzymes acting on aro-
ATP-binding moiety of the enzyme and is thought to transfer matic substrates were detected. Additionally, the ␣- and -sub-
a diphosphoryl group to a conserved histidine residue in the units share 29% sequence identity (49% similarity) with each
␣-subunit (23). There, orthophosphate is released and the other. In contrast to the other subunits, the ␥-subunit is unique
-phosphate group of ATP is transferred to phenol. Both sub- to the phenylphosphate carboxylase. Conversion of 4-OHB to
units are therefore required for phosphorylation. The ␥-sub- the central intermediate benzoyl-coenzyme A (CoA) is accom-
unit is dispensable. However, its presence stimulates the reac- plished by the activities of 4-OHB–CoA ligase and 4-hydroxy-
tion severalfold (28). benzoyl–CoA reductase (dehydroxylating) (4, 6, 8).
In the next step, phenylphosphate is carboxylated by the In the genome of T. aromatica, the pps and ppc genes, coding
action of phenylphosphate carboxylase (Ppc), yielding 4-hy- for the phenylphosphate synthase and phenylphosphate car-
droxybenzoate (4-OHB) (15, 17, 29). The ␦-subunit of the boxylase, are clustered in one operon (9) (Fig. 1B). The operon
enzyme shows similarities to proteins of the hydrolase/phos- is preceded by its putative regulator gene, which is transcribed
phatase family (29). It was suggested to bind phenylphosphate in the opposite direction. Expression of the pps and ppc genes
and to catalyze its dephosphorylation, a reaction that is exer- is strictly regulated by the presence of phenol. No activities of
gonic and virtually irreversible. The resulting phenolate anion the respective enzymes were detected in cells grown on
4-OHB. A gene cluster with an organization identical to that of
the pps and ppc genes and nearly identical to those of T.
* Corresponding author. Mailing address: Helmholtz Centre for En- aromatica was identified in strain EbN1 (27) and was also
vironmental Research, Department of Environmental Microbiology, found to be induced by phenol (35).
Permoserstr. 15, 04318 Leipzig, Germany. Phone: 49-341-235-1378. A completely different pathway was proposed for the fer-
Fax: 49-341-235-1351. E-mail: kathleen.schleinitz@ufz.de.
† Supplemental material for this article may be found at http://aem
menting bacterium Sedimentibacter hydroxybenzoicus. There,
.asm.org/. an ATP-independent 4-OHB decarboxylase was identified that
䌤
Published ahead of print on 17 April 2009. under in vitro conditions was also found to catalyze the reverse
3912VOL. 75, 2009 PHENOL DEGRADATION IN G. METALLIREDUCENS 3913
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FIG. 1. Enzymes and genes involved in phenol degradation. (A) Phenol degradation pathway in T. aromatica (accession number AJ272115).
The enzymes involved are phenylphosphate synthase (PpsABC) (1), phenylphosphate carboxylase (PpcABCD) (2), 4-OHB–CoA ligase (3),
4-hydroxybenzoyl–CoA reductase (4), benzoyl-CoA reductase (5), and lower-pathway enzymes (designations of G. metallireducens enzymes are in
parentheses) cyclohexadienoyl-CoA hydratase, Dch (BamR); hydroxyenoyl-CoA dehydrogenase, Had (BamQ); and oxoenoyl-CoA hydrolase, Oah
(BamA) (6), plus further enzymes involved in modified -oxidation reactions. (Adapted from reference 5.) (B) Organization of genes encoding
phenylphosphate synthase (PpsABC) and phenylphosphate carboxylase (PpcABCD) in T. aromatica (T. arom.) and strain EbN1 and similar gene
clusters in other strains. Additional ORFs hypothesized to be involved in the anaerobic degradation of aromatic compounds are ORF11 and pdeR,
putative XylR-type transcriptional regulators; ORF7, an ORF resembling ubiX; and ORF8, a carboxylase-like ORF. The remaining ORFs of the
G. metallireducens (G. met.) gene cluster are designated consecutively by letters. The numbers above the T. aromatica ORFs are designations
introduced previously (29). The directions of transcription are indicated by arrows. Similar (groups of) ORFs are indicated by shades and patterns.
(C) Genetic region carrying the predicted phenol degradation gene cluster. It is surrounded by gene clusters for the degradation of benzoate,
p-cresol, and 4-OHB.
reaction, i.e., phenol carboxylation (13, 36). On the basis of this ppc-like ORFs and transcribed in the opposite direction. The
observation, the enzyme was suggested to be responsible for other (Gmet_1279; accession no. YP_384240.1) is located else-
phenol degradation in the strain. The physiological function of where in the genome. The putative phenol degradation gene
the reversible decarboxylase has not yet been elucidated. Stud- cluster is located in a genetic region of the genome of G.
ies of a strictly anaerobic syntrophic consortium suggest that metallireducens that carries the genes for the degradation of
the 4-OHB decarboxylase and the phenol carboxylase activities several other aromatic compounds (Fig. 1C).
are associated with two different enzymes (11). Phenol metab- Based on the resemblance between the two gene clusters
olism in other strictly anaerobic, e.g., iron-reducing or sulfate- and on the observation that 4-OHB is an intermediate of
reducing bacteria has not been studied in detail. phenol mineralization in G. metallireducens, the degradation
The iron-reducing, phenol-degrading deltaproteobacterium pathway has previously been proposed to function similarly to
Geobacter metallireducens GS-15 (20) carries a gene cluster that of T. aromatica (28). If phenol is initially transformed to
bearing significant similarity in sequence and organization to 4-OHB in G. metallireducens, further breakdown could take
that of T. aromatica (Fig. 1B) (accession number NC_007517). place as described for T. aromatica. The activities of a 4-OHB–
The deduced amino acid sequences of the open reading frames CoA ligase and a 4-hydroxybenzoyl–CoA reductase were re-
(ORFs) Gmet_2099 through Gmet_2102, Gmet_2104, and cently shown in cell extracts of the strain (26). A pathway for
Gmet_2105 (accession no. YP_385053.1 through YP_385056.1, the metabolism of benzoyl-CoA, including a novel ATP-inde-
YP_385058.1, and YP_385059.1) display between 50 and 78% pendent benzoyl-CoA reductase (BamB-BamI), as well as a
identity (68 to 88% similarity) to the putative regulator, cyclohexadienoyl-CoA hydratase (BamR), a hydroxyenoyl-
PpsABC, PpcB, and the carboxylase-like ORF8 of T. aromatica. CoA dehydrogenase (BamQ), and an oxoenoyl-CoA hydrolase
Furthermore, the G. metallireducens genome carries two ppcD (BamA), has been described (34).
homologues. One (Gmet_2112; accession. no. YP_385066.1) is In this work, we investigated the initial steps of phenol
located downstream of the gene cluster containing the pps- and degradation in G. metallireducens GS-15. Applying a variety of3914 SCHLEINITZ ET AL. APPL. ENVIRON. MICROBIOL.
methods, including metabolite, transcriptome, proteome, and described previously (14). The results were checked by electrophoresis on a 1.5%
enzyme studies, we were able to characterize the peripheral agarose gel.
RNA extraction and reverse transcriptase PCR. Cells from 4 to 6 ml of culture
pathway and highlight important differences from the systems were harvested by centrifugation (7,300 ⫻ g; 4°C; 10 min), shock frozen, and
known so far. stored at ⫺80°C until further use. Prior to RNA extraction, the cells were
freeze-dried (⫺45°C; 0.05 mbar; 20 min) in a freeze dryer (Christ Alpha 2-4;
Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). RNA
MATERIALS AND METHODS was isolated using the TRIzol reagent (Invitrogen) (10). Contaminating DNA
was removed using the DNAfree kit (Ambion/Applied Biosystems, Frankfurt,
Culture conditions, cell harvest, and preparation of cell extracts. G. metalli-
Germany) according to the manufacturer’s protocol for highly contaminated
reducens GS-15 was maintained in 50-ml serum bottles (Glasgerätebau Ochs
RNA. Complete removal of DNA was verified by PCR amplifying long 16S
GmbH, Bovenden, Germany) on mineral salt medium (21) containing 60 mM
rRNA gene fragments or short fragments of two randomly chosen genes from the
Fe(III) citrate as an energy source and 20 mM acetate as a carbon source. For
set under study. RNA purity was assessed by measuring the A260/A280 and A260/A230
transcriptome and proteome studies, the strain was cultured in 100-ml serum
bottles, and 20 mM acetate, 3 mM 4-OHB, or 0.5 mM phenol was added as a ratios. For RNA integrity evaluation, the 23S/16S ratio of the RNA separated
carbon source. For experiments with [14C]phenol and for enzymatic studies, G. electrophoretically on a 1.5% agarose gel was determined using the GeneTools
metallireducens was cultivated in 2-liter flasks, and 2 mM phenol was added. software (Syngene, Cambridge, United Kingdom). RNA concentrations were mea-
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Growth was monitored by measuring substrate concentrations and by counting sured using the RiboGreen RNA Quantitation Reagent and kit (Molecular Probes/
cells, either directly in a Neubauer counting chamber or after DAPI (4⬘,6⬘- Invitrogen, Karlsruhe, Germany). cDNA was synthesized from 100 ng of total RNA
diamidino-2-phenylindole) staining. Paraformaldehyde fixation, DAPI staining, using the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas, St.
and enumeration were performed according to the method of Alfreider et al. (1). Leon-Rot, Germany) and the random hexamer primers provided. Gene expression
Cells were harvested during exponential growth. was studied by partially amplifying gene fragments from undiluted, 10-fold-diluted,
Phenol transformation by whole cells and TLC analysis of products. 14C- and 100-fold-diluted cDNA according to the method described above. Genomic
labeled substrate and intermediates were detected as described elsewhere (16), DNA of G. metallireducens was used as a positive control.
except for the following slight modifications. Cells from 2 liters of culture (ap- 2-D gel electrophoresis. For proteome analysis, cells from 100 ml culture were
proximately 0.6 g [wet weight]) were harvested by centrifugation (10,500 ⫻ g; harvested by centrifugation (7,300 ⫻ g; 4°C; 10 min). Crude cell extracts were
4°C; 10 min). The pellet was washed twice in culture medium lacking phenol and prepared as described previously (3) and stored at ⫺20°C. Two-dimensional
was finally suspended in buffer containing 5 mM NaHCO3 and 1 mM NaNO3 to (2-D) gel electrophoresis was carried out by isoelectric focusing (IEF) in the first
give a 30-fold-concentrated cell suspension (3 ⫻ 1010 cells/ml; approximately 9 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in
mg cell dry mass/ml). [U-14C]phenol (1 Ci ⫽ 34 kBq; specific radioactivity, 80 the second dimension (chemicals and equipment were from GE Healthcare,
mCi/mmol) was added to a 5-ml cell suspension to a final concentration of 2.5 Uppsala, Sweden, unless stated otherwise) (25). Two hundred micrograms of
M. After 0.2, 0.5, 1, 1.5, 5, 10, 30, and 60 min of incubation, 0.5-ml samples were protein was precipitated by adding 5 volumes of ice-cold acetone for 10 min at
withdrawn and prepared for thin-layer chromatography (TLC) as described ⫺20°C. After centrifugation (14,800 ⫻ g; 4°C; 30 min), the supernatant was
previously (16). Ten microliters of resuspended sample was subjected to TLC removed completely and the resulting pellet was dissolved in 140 l DeStreak
that was carried out for 3 h on 20- by 20-cm aluminum plates covered with a rehydration solution containing 0.5% (vol/vol) immobilized pH gradient (IPG)
0.2-mm silica gel (Kieselgel 60 F254; Merck) and with ethanol-dichloromethane- buffer, pH 4 to 7 or pH 3 to 10. Precipitates were removed by centrifugation (15
water (8:1:1 [vol/vol/vol]) as the solvent. After chromatographic separation, ra- min; 23,100 ⫻ g; 20°C), and the supernatant was loaded on 7-cm IPG strips, pH
dioactive substances were visualized by exposing the TLC plate to a phospho- 4 to 7 or pH 3 to 10. Rehydration and IEF were performed in an Ettan IPGphor
rimager plate (Fujix BAS-IP MP 2040S; Fuji, Japan) for 48 h and scanning it with 3 IEF unit according to the manufacturer’s guidelines. Separation in the second
a Molecular Imager FX scanner (Bio-Rad, Hercules, CA). 14C-labeled phe- dimension was carried out by SDS-PAGE using 20- by 20- by 0.1-cm 12%
nylphosphate and 4-OHB were identified by comigration with internal standards polyacrylamide gels (18). Two strips were loaded on each gel to ensure that
that had been added to the stopping solution (1 mM phenylphosphate; 0.1 mM running conditions were the same for both samples. 2-D gels were stained with
4-OHB). These compounds were visualized under 254-nm UV light. colloidal Coomassie brilliant blue G-250 (24) (Roth, Kassel, Germany) and dried
DNA extraction and PCR. Genomic DNA was extracted using the Nucleospin in a stream of unheated air. Phenol-induced proteins were identified by visual
C⫹T kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s inspection and comparison of protein patterns with the Delta 2D v3.3 software
guidelines. PCR was performed using an MJ Research PTC-200 thermal cycler (Decodon, Greifswald, Germany).
(GMI). Unless stated otherwise, reaction mixtures had a total volume of 12.5 l In-gel digestion of proteins. Protein spots were excised, and the slices were
and contained 6.25 l PCR Master Mix (Promega), 5 pmol of each primer washed twice with 200 l methanol-acetic acid-water (50:5:45 [vol/vol/vol]) for
(Microsynth, Balgach, Switzerland), and 1 to 4 l template cDNA (see below) or 1 h and once with acetonitrile for 5 min. The gel pieces were dried under vacuum.
10 to 50 ng genomic DNA. The cysteine residues were reduced by 30 l 10 mM 1,4-dithiothreitol (30 min;
Primer pairs amplifying approximately 500-bp fragments of the ORFs room temperature) and alkylated by 30 l 100 mM 2-iodocetamide (30 min;
Gmet_2100 (ppsA-like; Gmet_2100f, 3⬘-ACC AGT TCG TCA CTG ACG-5⬘, room temperature). After dehydration with acetonitrile, equilibration with am-
and Gmet_2100r, 3⬘-GAA TTC GAA GTG GTA CTG C-5⬘), Gmet_2101 (ppsB- monium bicarbonate, and another dehydration step with acetonitrile, the pro-
like; Gmet_2101f, 3⬘-CTG GGC GAG CTG ATA AGC-5⬘, and Gmet_2101r, teins were dried under vacuum and proteolytically cleaved by modified porcine
3⬘-GAG TTG GCC ATC TTC TGC-5⬘), Gmet_2102 (ppcB-like; Gmet-2102f, trypsin (Sigma-Aldrich, Steinheim, Germany) overnight at 37°C. Peptides were
3⬘-AAA TGG ATG GAG CTG ACC-5⬘, and Gmet2102r, 3⬘-TCT TCC ATC extracted from the gel pieces twice by the addition of 30 l acetonitrile-formic
AAC TCC TCG-5⬘), Gmet_2104 (ppsC-like; Gmet_2104f, 3⬘-TAG TCC GCA acid-water (50:5:45) and concentrated under vacuum. After the addition of 10 l
ACT GGA TGC A-5⬘, and Gmet_2104r, 3⬘-CGC ATT TTC TCC GCT TCC-5⬘), 3% acetonitrile in 0.1% formic acid, the acidified peptides were subjected to
Gmet_2105 (resembling ORF8 of T. aromatica; Gmet_2105f, 5⬘-TTG GTA GTG liquid chromatography-tandem mass spectrometry (LC–MS-MS).
GCA ATC ACC-3⬘, and Gmet_2105r, 5⬘-ATG TCC ATG ATT GAC TCC-3⬘), LC–MS-MS. Separation of peptides was carried out by reversed-phase
and Gmet_2112 (ppcD-like; Gmet_2112f, 5⬘-AGG GCA TTC TAC GTT TGG- nano-LC (LC1100 series; Agilent Technologies, Palo Alto, CA) (analytic col-
3⬘, and Gmet_2112r, 5⬘-CCA GGC ATT TAA GGA TCC-3⬘) were tested in a umn, Zorbax 300SB-C18, 0.075 mm by 150 mm by 5 m; solvent, initially 0.1%
temperature gradient program involving an initial denaturation step at 94°C for 3 formic acid, acetonitrile concentration increasing from 0% to 55% in 30 min) and
min, followed by 36 cycles of 94°C for 45 s, 50 to 70°C for 45 s, and 72°C for 1 analyzed by MS-MS (LC/MSD Trap XCT mass spectrometer; Agilent Technol-
min, with a final elongation step of 72°C for 6 min. The primer pairs used for the ogies). Protein identification was carried out by database searches using MS-MS
amplification of approximately 500-bp fragments of the genes bamA, bamR, and ion search (Mascot v 2.2.1; Matrix Science, London, United Kingdom) against
rpoB (RNA polymerase, -subunit) have been described previously (34). The the NCBI (National Center for Biotechnology Information, Rockville, MD)
program for the amplification of gene fragments from cDNA involved an initial nonredundant protein database NCBInr 20080221.fasta. Trypsin was selected as
denaturation step at 94°C for 3 min, followed by 36 cycles of 94°C for 45 s, 53°C the enzyme, and up to one missed cleavage site was allowed. Variable modifi-
for 45 s (ORFs Gmet_2104, Gmet_2105, and Gmet_2112) or 60°C for 45 s (all cations, such as carbamidomethyl at cysteines or oxidized methionines, were
other ORFs), and 72°C for 1 min, with a final elongation step of 72°C for 10 min. allowed. The search was restricted to peptides containing peptide charge state 2
16S rRNA gene fragments were amplified using 5 pmol (each) of primers 27F or 3 and was conducted with a peptide tolerance of ⫾1.2 Da and an MS-MS
and 1492R (19), the Taq PCR Master Mix (Qiagen), and a PCR program tolerance of ⫾0.8 Da.VOL. 75, 2009 PHENOL DEGRADATION IN G. METALLIREDUCENS 3915
mobile phase. Samples were prepared by centrifugation. Twenty microliters of
the supernatant was injected; detection was performed at 190 nm.
RESULTS
Intermediates of phenol degradation. A concentrated cell
suspension of G. metallireducens GS-15 was incubated with
trace amounts of [U-14C]phenol (2.5 M) in the presence of
CO2. Intermediates derived from [14C]phenol were detected
by TLC and autoradiography. On the TLC plate, a labeled
product comigrating with phenylphosphate was already formed
after 12 s (Fig. 2), and a less polar product comigrating with
FIG. 2. Detection of labeled products formed from 2.5 M 4-OHB also appeared. Whereas the amount of labeled phe-
[U-14C]phenol by suspensions of whole cells of G. metallireducens.
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nylphosphate did not significantly increase during the course of
Samples were taken after 12 s (lane 1), 30 s (lane 2), 1 min (lane 3), 1.5
the experiment, labeled 4-OHB steadily accumulated. This is
min (lane 4), 5 min (lane 5), 10 min (lane 6), 30 min (lane 7), and 60
min (lane 8) of incubation. Substrate and intermediates were sepa- the first indication of phenylphosphate being an intermediate
rated by TLC and detected by phosphorimaging. Identification was of phenol degradation in G. metallireducens, leading to 4-OHB.
based on comigration with unlabeled internal standards visualized The radioactive phenol spot continuously decreased and was
under UV light: A, phenol; B, phenylphosphate; C, 4-OHB; X and Y, no longer detectable after 5 min. Two more faintly labeled
not identified. Note that the phenol spot is never as intense as the
4-OHB spot because phenol is volatile, in contrast to 4-OHB. Hence, spots appeared early (Fig. 2). Spot Y might represent 4-hy-
the intensities of the nonvolatile-product spots directly reflect their droxybenzoyl-CoA, which comigrated with this spot.
concentrations, whereas the intensity of the phenol spot represents a Expression of pps- and ppc-like ORFs. To test whether the
relative value (i.e., the fraction of phenol that did not evaporate during phenylphosphate synthase- and phenylphosphate carboxylase-
the experiment).
like ORFs of G. metallireducens were induced during growth
on phenol, reverse transcription-PCR studies were carried out.
Total RNA of cells growing exponentially on phenol, benzoate,
Phenylphosphate carboxylase assay. Cell extracts were prepared in 50 mM
MOPS (morpholinepropanesulfonic acid)/KOH, pH 7.0, 15% glycerol, 20 mM or acetate was extracted. A set of seven oligonucleotide pairs
mercaptoethanol, 0.5 mg DNase I/ml, using a French press, followed by ultra- was used for expression analysis. As a control, expression of
centrifugation (1 h;, 100,000 ⫻ g). Enzymatic tests were routinely carried out in the housekeeping gene rpoB, coding for the -subunit of RNA
0.5-ml reaction volumes at 30°C and under strictly anaerobic conditions. The polymerase, was studied. Figure 3A shows that rpoB was ex-
standard assay mixture for the net carboxylation assay contained approximately
1.6 mg protein, 2 mM MgCl2, 20 mM KCl, 20 mM mercaptoethanol, 2 mM
pressed independently of the carbon source utilized by the
sodium phenylphosphate, and 20 mM NaH14CO3 (0.4 Bq nmol⫺1). The isotope strain. For all of the ORFs putatively involved in phenol deg-
exchange assay mixture contained approximately 0.8 mg protein, 4 mM 4-OHB radation, products of the expected sizes were obtained from
(sodium salt), and 40 mM NaH14CO3 (0.8 Bq nmol⫺1). The reactions were cDNA of cells grown on phenol. They were also observed in
started by adding [14C]bicarbonate. Four-hundred-microliter samples were rou-
benzoate-grown cells, but at a lower level. No PCR products
tinely withdrawn after 10 min and added to 40 l of 3 M perchloric acid. 14CO2
that had not been fixed was removed by shaking the samples at room tempera- were observed with cDNA of cells grown on acetate, showing
ture for at least 3 h. After the addition of 3 ml Scintillation Cocktail (EcoScint that they were specifically expressed during growth on the
Plus; Roth, Kassel, Germany), the amount of radioactivity in the nonvolatile, aromatic substrates. As another control, the expression of two
acid-stable product (4-OHB) was determined by liquid scintillation counting in a lower pathway genes, bamR and bamA, was tested. As ex-
Tri-Carb 2100 TR Liquid Scintillation Analyzer (Canberra Packard GmbH,
Dreieich, Germany) for 5 min. The amount of labeled 4-OHB formed was
pected, equal amounts of PCR products were obtained with
calculated from the amount of fixed radioactivity by taking into account the cDNA of benzoate- or phenol-grown cells, but not with cDNA
known specific radioactivity of the total 14CO2 added to the assay mixture. The of cells grown on acetate. In a similar way, the carboxylase-like
detection limit was approximately 5 Bq (⬃6 nmol fixed product). ORF (ORF8 in Fig. 1B; accession no. YP_385059.1) and the
A direct carboxylation of phenol without using ATP was tested in the same
ppcD-like ORF (YP_385066.1), which could possibly play roles
assay mixtures containing phenol instead of phenylphosphate. Residual ATP was
removed by the addition of 5 units hexokinase (Sigma) and 4 mM glucose. in phenol degradation by G. metallireducens, were tested for
Reaction mixtures containing hexokinase were incubated at 30°C for 5 min prior expression. Both were expressed in phenol-grown cells, but not
to starting the carboxylation reaction. in acetate-grown cells (Fig. 3B). These results show that the
Protein concentrations were determined with the Coomassie blue protein ORFs under study were all transcribed in the presence of
assay (7), with bovine serum albumin as the standard.
HPLC. The concentrations of phenol and benzoate were determined by high-
phenol.
pressure liquid chromatography (HPLC) (Shimadzu) using a UV/VIS detector. Translation of the transcripts. To investigate if translation
Separation was performed at 23°C and a flow rate of 0.6 ml min⫺1 using a of the transcripts took place, the soluble protein fraction of
Nucleosil 100 C18 column (3 mm by 250 mm; 5-m size of filling; Knauer GmbH, cells growing exponentially on phenol or benzoate was sepa-
Berlin, Germany) as a stationary phase and a buffer containing 60% (vol/vol)
rated by 2-D SDS-PAGE. Comprehensive analysis of more
NaH2PO4 (131.5 mM, pH 2.8) and 40% (vol/vol) acetonitrile as the liquid phase.
Samples (1 ml) were prepared by the addition of 12.5 l 10 N NaOH, thorough than 70 spots in the regions of interest (delimited by the cal-
mixing, and centrifugation (10 min; 18,900 ⫻ g; 4°C). The supernatant was culated molecular masses and isoelectric points [pIs] of the Pps
diluted 10-fold with distilled water, and 20 l of each sample was injected. The and Ppc subunits) was carried out. Two strongly induced pro-
compounds were detected at 271-nm wavelength. teins (Fig. 4) were identified as YP_385054.1 (PpsA-like) and
Acetate concentrations were determined by HPLC (Shimadzu) using a UV
detector. Separation was performed at 70°C and a flow rate of 0.6 ml min⫺1 using
YP_385056.1 (PpcB-like), respectively (Table 1). Their pIs and
a Nucleogel ION 200 OA column (7.8 mm by 300 mm; 10-m size of filling; apparent molecular masses corresponded well with the calcu-
Macherey-Nagel) as the stationary phase and a buffer of 0.005 N H2SO4 as the lated values. Neither of the two proteins was detected in ex-3916 SCHLEINITZ ET AL. APPL. ENVIRON. MICROBIOL.
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FIG. 3. Reverse transcriptase PCR studies of genes involved in
phenol metabolism in G. metallireducens. PCR products produced with
cDNA (lanes 2 to 10) or, as a control, genomic DNA (lane 1) were
separated electrophoretically and visualized by staining them with
ethidium bromide. The cDNA of cells grown on acetate, benzoate, or
phenol was used as an undiluted template (lanes 2, 5, and 8), diluted
10-fold (lanes 3, 6, and 9), or diluted 100-fold (lanes 4, 7, and 10).
(A) Induction of rpoB (RNA polymerase subunit ) (housekeeping
gene); the subunits of phenylphosphate synthase, ppsA, ppsB, and
ppsC; ppcB, phenylphosphate carboxylase subunit ; bamR, cyclohexa-
dienoyl-CoA hydratase; and bamA, oxoenoyl-CoA hydrolase. (B) In-
duction of genes possibly involved in phenol metabolism of G. metal-
lireducens. ORF8, carboxylase-like ORF YP_385056.1; ppcD, HAD FIG. 4. 2-D gel electrophoresis (IEF/SDS-PAGE) of protein ex-
phosphatase-like ORF YP_385066.1. Lane M is a 100-bp ladder; the tracts of G. metallireducens. Proteins were extracted from cells growing
fragment length of the prominent band is 500 bp. exponentially on phenol (A) or benzoate (B). The numbered proteins
were identified by LC–MS-MS. 1, phenylphosphate synthase subunit ␣;
2, phenylphosphate carboxylase subunit ; 3, 4Fe-4S ferredoxin, an
iron-sulfur binding protein, BamI; 4, electron transfer flavoprotein
tracts of benzoate-grown cells. The PpsB-like protein (calcu- subunit ; 5, enoyl-CoA hydratase/isomerase. The complete results of
lated mass, 41.9 kDa; calculated pI, 5.46) and the PpsC-like the proteome analysis are shown in Table S1 and Fig. S1 in the
protein (mass, 16.4 kDa; pI, 5.33), as well as other proteins supplemental material.
possibly involved in the upper pathway of phenol degradation
in G. metallireducens (ORF8 [mass, 22 kDa; pI, 6.66] and PpcD
[mass, 25 kDa; pI, 4.89]), were not detected. As a control, some
of the lower-pathway proteins were identified (Table 1). They discrepancy between the transcription of the genes involved in
were found in extracts of phenol- as well as benzoate-grown phenol carboxylation to 4-OHB and the translation of the
cells. Most of the other induced proteins (see Table S1 and Fig. respective transcripts was unexpected and is discussed below.
S1 in the supplemental material) were related to energy and Phenylphosphate carboxylase activity. The phenylphos-
nucleotide metabolism, protein synthesis, heat shock, and phate-carboxylating activity was tested in vitro with extracts
transport and might help the cell to cope with the toxic nature of cells grown on phenol, using 14CO2 and phenylphosphate
of phenol. Noteworthy was the expression of Bam-like, so-far- as substrates. In control reactions, phenylphosphate was
uncharacterized proteins. In protein spots 12/13, 37, 40/41/42, omitted. Table 2 shows that cell extracts were able to car-
and 47 (see Fig. S1 in the supplemental material), four proteins boxylate phenylphosphate. The specific activity was compa-
were detected that belong to one gene cluster (YP_386227.1 rable to that reported for T. aromatica (10 nmol/min 䡠 mg)
through YP_386240.1). The gene products of five reading (29). When phenol instead of phenylphosphate was tested,
frames of this cluster (YP_386236.1 through YP_386240.1) only a small amount of radioactivity (⬍10% compared to
display between 59 and 80% identity to BamO, BamP, BamS, phenylphosphate) was incorporated from 14CO2 into acid-
BamT, and BamN, respectively. However, the gene order dif- stable products, most likely 4-OHB. This small 14CO2 incor-
fers from that of the bam genes. poration was completely abolished when hexokinase plus
Proteome investigation showed that the gene cluster under glucose were added to the assay to trap residual ATP in cell
study is translated. In contrast to the results of the transcrip- extracts. This result clearly indicated that phenylphosphate
tion analysis, where the mRNAs of the pps- and ppc-like genes rather than phenol was carboxylated.
were found in both benzoate- and phenol-grown cells, a clear Surprisingly, extracts hardly catalyzed an isotope exchange
difference in the expression of the proteins was observed be- reaction between 14CO2 and the carboxy group of 4-OHB. The
tween benzoate- and phenol-grown cells. Translation was in- observed rate was near the detection limit of the assay. This
duced only during growth on phenol but, within the detection exchange reaction is a partial reaction of phenylphosphate
limits of the method, not during growth on benzoate. The carboxylase catalysis in T. aromatica (29), and the isotope ex-VOL. 75, 2009 PHENOL DEGRADATION IN G. METALLIREDUCENS 3917
TABLE 1. Proteins induced during growth on phenol in G. metallireducens GS-15 (Fig. 4)a
Mowse No. of Sequence
Protein Spot label Annotation Accession no. Mr pI
scoreb peptides coverage (%)
PpsA 1 Hypothetical protein Gmet_2100 YP_385054.1 132 3 69,931 5.19 4
PpcB 2 Carboxylyase-related protein YP_385056.1 661 16 57,859 6.22 29
BamI 3 4Fe-4S ferredoxin; iron-sulfur binding protein YP_385033.1 155 6 23,717 5.46 25
BamO 4 Electron transfer flavoprotein -subunit YP_385107.1 211 7 27,682 5.65 30
BamR 5 Enoyl-CoA hydratase/isomerase YP_385104.1 155 3 27,120 5.58 10
a
Proteins were defined as unambiguously identified when at least three peptides were present, the MS-MS Mowse score was ⬎100 and the Mascot-defined
significance was ⬎95. The complete results of the proteome analysis are included in Table S1 and Fig. S1 in the supplemental material.
b
Molecular weight search score. Scoring was based on peptide frequency distribution from the OWL nonredundant protein database.
change rate is 10-fold higher than the net phenylphosphate ORF8 and the ppcD homologue located nearby are transcribed
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carboxylation rate. during growth on phenol. Enzyme studies were conducted to
clarify whether an active phenylphosphate carboxylase was
DISCUSSION synthesized in G. metallireducens. They showed that the
respective enzyme is present in cell extracts of the strain and
It was the aim of this work to characterize the initial steps of that its activity is comparable to that observed in T. aro-
anaerobic phenol metabolism in G. metallireducens and to test, matica (Table 2).
in particular, if the pps- and ppc-like ORFs detected in its From our results, it can be concluded that phenol degrada-
genome are functional. Metabolite, transcriptome, proteome, tion in G. metallireducens is accomplished via the same path-
and enzyme studies revealed that the phenol degradation path- way as described for T. aromatica, i.e., initial activation of
way in this strictly anaerobic iron-reducing deltaproteobacte- phenol to phenylphosphate and subsequent carboxylation to
rium parallels that known from the facultative nitrate-reducing 4-OHB (Fig. 1A). In the following steps, 4-OHB can be trans-
betaproteobacterium T. aromatica. formed to the central intermediate benzoyl-CoA by enzymes
Phenylphosphate was identified as the first intermediate of described previously (26) and further broken down to acetyl-
phenol degradation in the strain, appearing shortly after the CoA and CO2.
onset of phenol consumption and accumulating transiently However, the question remains as to what the architecture
(Fig. 2). Formation of a functional phenylphosphate synthase of the phenylphosphate carboxylase in G. metallireducens is. In
catalyzing the conversion of phenol to phenylphosphate in G. T. aromatica, all four subunits, PpcABCD, are required for
metallireducens is likely. Homologues of the three genes known formation of an active phenylphosphate carboxylase. The
to encode the subunits of the respective enzyme in T. aro- ppcAC genes lacking in the gene cluster studied here could be
matica (ppsABC) are present in G. metallireducens and are replaced by genes located elsewhere in the genome of G.
transcribed; translation of ppsA in the presence of phenol was metallireducens. However, no obvious candidate for a ppcC
proven (Fig. 3 and 4). homologue was identified in BLAST searches using different
Another metabolite that accumulated in the cell suspension algorithms. ORF Gmet_0993 could be a candidate for a ppcA
experiment was 4-OHB. This is in accordance with previous homologue, as it shows 25% identity (42% similarity) to the
studies that detected 4-OHB as a metabolite of phenol break- deduced amino acid sequence of ppcA. Alternatively, the com-
down in G. metallireducens (20). The gene cluster studied here position of the G. metallireducens enzyme could differ from
carries only a homologue for the -subunit of phenylphosphate that of the phenylphosphate carboxylase of T. aromatica. As a
carboxylase. This gene, ppcB, is transcribed and translated consequence, the phenylphosphate carboxylase of G. metalli-
during growth on phenol (Fig. 3 and 4). The carboxylase-like reducens may be expected to display characteristics distinguish-
ing it from Ppc of T. aromatica. Indeed, one distinguishing
catalytic feature of the Geobacter carboxylase was the lack of
TABLE 2. Phenylphosphate carboxylase activities in cell extracts of the 14CO2 isotope exchange reaction.
G. metallireducens GS-15a In T. aromatica the two large subunits of phenylphosphate
Assay
Fixed Sp act carboxylase, PpcA and PpcB, both resemble UbiD, a carbox-
radioactivity (Bq) (nmol/min 䡠 mg) ylase subunit involved in ubiquinone biosynthesis. However, in
Control (no phenylphosphate) 3.5 0 other (de)carboxylases, only one of the large subunits is UbiD-
Carboxylation of phenylphosphate 102.8 11.3 like, and the other more closely resembles UbiX, an isoenzyme
Carboxylation of phenol 7.5 0.5 of UbiD (22). In G. metallireducens, an ubiX-like ORF is lo-
(no hexokinase trap)
Carboxylation of phenol 3.0 0
cated immediately downstream of ppsC (Fig. 1). It resembles
(plus hexokinase trap) ORF8 of T. aromatica, an ORF that has not yet been charac-
14
CO2 isotope exchange reaction 4.1 ⬍1 terized (9). The ubiX-like ORF is transcribed alongside the pps
a
Assays were performed at 30°C under strictly anaerobic conditions according
and ppc genes (Fig. 3B). It is noteworthy that a similar ORF is
to the method of Schühle and Fuchs (29). The standard assay mixture for the net also part of a pps-ppc-like gene cluster in Geobacter sp. strain
carboxylation contained 20 mM NaH14CO3. The phenol carboxylation control FRC-32 (Fig. 1). The organization of this gene cluster is iden-
assay contained in addition 4 mM glucose and 5 U hexokinase and was prein-
cubated at 30°C for 5 min before the carboxylation reaction was started. The tical to that in G. metallireducens, whereas the regions flanking
isotope exchange reaction mixture contained 40 mM NaH14CO3. it differ from both G. metallireducens and T. aromatica. This3918 SCHLEINITZ ET AL. APPL. ENVIRON. MICROBIOL.
implies that the ORF8 (ubiX)-like ORF could play an impor- act in trans (31), it is so far unknown which of the reading
tant role in phenol degradation and might be involved in the frames is active in the regulation of phenol degradation in G.
formation of a functional phenylphosphate carboxylase. metallireducens.
The unidirectionality of the phenylphosphate carboxylase Comparison of the regulation of phenol metabolism on the
reaction in T. aromatica is thought to be brought about by the transcriptional and translational level reveals a pattern similar
␦-subunit exerting phosphatase activity on the substrate. The to that described previously (26). There, the genes for the
gene region studied here carries three ORFs sharing significant peripheral pathway of p-cresol metabolism were transcribed
sequence similarities with kinase/phosphatase genes, ORF B, during growth on benzoate, despite lacking a function in ben-
ORF D, and ppcD (Fig. 1B). The ORF B product resembles zoate breakdown. The respective proteins were observed only
butyrate and acetate kinases, including PcmL (accession no. during growth on p-cresol. Therefore, it was suggested that
YP_385082.1), encoded within the p-cresol catabolic gene clus- posttranscriptional regulatory elements inhibit the synthesis of
ter further downstream. No function in p-cresol metabolism the respective proteins. In our study, we also observed tran-
has been assigned to PcmL. Given the substrate range of this scription of the genes encoding the initial steps of phenol
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family of enzymes, it seems unlikely that the gene product of metabolism during growth of G. metallireducens on benzoate,
ORF B is involved in the metabolism of an aromatic substrate. though at a reduced level. Translation of the mRNA took place
The deduced amino acid sequence of ORF D resembles that of during growth on phenol, but the respective proteins were not
proteins belonging to the HDc superfamily of metal-dependent observed during growth on benzoate. These findings again
phosphohydrolases. Members of this protein family are in- indicate the existence of a posttranscriptional regulation mech-
volved in nucleic acid and protein metabolism and signal trans- anism and raise the question of a general mechanism for post-
duction (2). The PpcD protein in T. aromatica, on the other transcriptional regulation of peripheral pathways for the
hand, belongs to the HAD superfamily of hydrolases. An ORF breakdown of aromatic compounds in G. metallireducens.
putatively encoding an HAD superfamily hydrolase and dis- Whether this regulatory mechanism is operative on the trans-
playing 45% similarity to ppcD is located further downstream lational or on the protein level (e.g., influencing protein sta-
(Fig. 1B) and is transcribed in the presence of phenol (Fig. 3B). bility) is unknown.
Whether this ORF, ORF D, or the ppcD homologue present Previously it was thought that gene expression in bacteria is
elsewhere in the genome of G. metallireducens (Gmet_1279; mainly regulated on the transcriptional level. However, during
accession no. YP_384240.1) takes over the role of PpcD is not the last few decades, many different posttranscriptional control
known. Clearly, further studies are needed to identify the pro- mechanisms have been identified in prokaryotes (12, 30, 33).
tein exerting phosphatase activity and to clarify if the phe- Whether a similar mechanism is operative in the posttranscrip-
nylphosphate carboxylase in G. metallireducens requires a tional regulation of catabolic pathways for aromatic com-
PpcC analogue, the small subunit unique to the carboxylase of pounds in G. metallireducens requires further systematic anal-
T. aromatica. yses.
Catabolic pathways are often regulated on the transcrip-
ACKNOWLEDGMENTS
tional level. This is also the case with the genes for phenol
degradation in G. metallireducens that were specifically tran- We are grateful to Matthias Boll and Simon Wischgoll for the gift of
scribed in the presence of aromatic substrates, but not during G. metallireducens strain GS-15. We thank Christine Schumann,
Michaela Risch, and Ute Lohse for technical support.
growth on acetate. The pps-ppc gene cluster is preceded by an This work was supported by Deutsche Forschungsgemeinschaft and
ORF whose product shows 69 and 67% identity to the putative Fonds der Chemischen Industrie.
regulators of phenol degradation in strain EbN1 and T. aro-
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