Identification of Enzymes Involved in Anaerobic Benzene Degradation by a Strictly Anaerobic Ironreducing Enrichment Culture

Environmental Microbiology (2010) 12(10), 2783–2796 doi:10.1111/j.1462-2920.2010.02248.x

Identiﬁcation of enzymes involved in anaerobic benzene degradation by a strictly anaerobic

iron-reducing enrichment cultureemi_2248 2783..2796

Nidal Abu Laban,1 Draženka Selesi,1 Thomas Rattei,2 sequences suggest that the initial activation reaction Patrick Tischler2 and Rainer U. Meckenstock1* in anaerobic benzene degradation is probably a direct 1Helmholtz Zentrum München – German Research carboxylation of benzene to benzoate catalysed by Center for Environmental Health, Institute of putative anaerobic benzene carboxylase (Abc). The Groundwater Ecology, Ingolstädter Landstraße 1, putative Abc probably consists of several subunits, D-85764, Neuherberg, Germany. two of which are encoded by ORFs 137 and 138, and 2Chair for Genome-oriented Bioinformatics, Technische belongs to a family of carboxylases including phe- Universität München, Life and Food Science Center nylphosphate carboxylase (Ppc) and 3-octaprenyl-4- Weihenstephan, Am Forum 1, D-85354 hydroxybenzoate carboxy-lyase (UbiD/UbiX). Freising-Weihenstephan, Germany. Introduction Summary The aromatic hydrocarbon benzene is a compo- Anaerobic benzene degradation was studied with a nent of crude oil and gasoline and is intensively used highly enriched iron-reducing culture (BF) composed in the chemical industry. Due to its high solubility of mainly Peptococcaceae-related Gram-positive (1.78 g l-1 in water at 25°C), chemical stability -1 microorganisms. The proteomes of benzene-, phenol- (DGf° = +124.4 KJ mol ), and impact on human health, and benzoate-grown cells of culture BF were com- benzene is considered as one of the most hazardous pared by SDS-PAGE. A specific benzene-expressed pollutants of groundwater and sediments (Johnson protein band of 60 kDa, which could not be observed et al., 2003). Therefore, it is of great interest to under- during growth on phenol or benzoate, was subjected stand the fate and the biodegradability of benzene in the to N-terminal sequence analysis. The first 31 amino environment. acids revealed that the protein was encoded by ORF For aerobic degradation of benzene well studied 138 in the shotgun sequenced metagenome of culture pathways are known (Gibson et al., 1968). Furthermore, BF. ORF 138 showed 43% sequence identity to phe- enrichment cultures and microcosm studies indicated nylphosphate carboxylase subunit PpcA of Aroma- that benzene can be effectively degraded in the absence toleum aromaticum strain EbN1. A LC/ESI-MS/MS- of molecular oxygen under iron-reducing (Lovley et al., based shotgun proteomic analysis revealed other 1996; Anderson et al., 1998; Rooney-Varga et al., 1999; specifically benzene-expressed proteins with encod- Kunapuli et al., 2008), sulfate-reducing (Lovley et al., ing genes located adjacent to ORF 138 on the metage- 1995; Kazumi et al., 1997; Phelps et al., 1998; Caldwell nome. The protein products of ORF 137, ORF 139 and Suflita, 2000; Abu Laban et al., 2009), denitrifying and ORF 140 showed sequence identities of 37% to (Burland and Edwards, 1999; Coates et al., 2001; Ulrich phenylphosphate carboxylase PpcD of A. aromaticum and Edwards, 2003) and methanogenic (Ulrich and strain EbN1, 56% to benzoate-CoA ligase (BamY) of Edwards, 2003; Chang et al., 2005) conditions. So far, Geobacter metallireducens and 67% to 3-octaprenyl- only two anaerobic benzene-degrading strains were iso- 4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. lated to purity, which were described as denitrifying aromaticum strain EbN1 respectively. These genes members of the genera Dechloromonas and Azoarcus are proposed as constituents of a putative ben- (Coates et al., 2001; Kasai et al., 2006). Despite the zene degradation gene cluster (~17 kb) composed cultivation of benzene-degrading cultures, a profound of carboxylase-related genes. The identified gene knowledge of the biochemical mechanism and the enzymes and genes of anaerobic benzene degradation is still lacking. Received 13 October, 2009; accepted 5 April, 2010. *For Correspon- dence. E-mail: [email protected]; Tel. In the presence of molecular oxygen, benzene is (+49) 89 3187 2561; Fax (+49) 89 3187 3361. activated by oxygenases introducing one or two oxygen

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd 2784 N. Abu Laban et al. atoms from O2 yielding phenol, benzene epoxide or (Caldwell and Suflita, 2000; Phelps et al., 2001; Kunapuli catechol respectively (Bugg, 2003). Under anaerobic et al., 2008; Abu Laban et al., 2009) or (iii) a methylation conditions, the reactive oxygen is missing and degrada- to toluene (Ulrich et al., 2005) (Fig. 1). Recent studies tion requires other biochemical reactions to overcome showed an abiotic formation of phenol from benzene in the extreme chemical stability of benzene. Examples of culture media for iron- and sulfate-reducing organisms by anaerobic activation reactions of hydrocarbons include contact with air during sampling (Kunapuli et al., 2008; oxygen-independent hydroxylation of alkyl substituent via, Abu Laban et al., 2009). This indicated that phenol as a e.g. ethylbenzene dehydrogenase (Rabus and Widdel, putative intermediate of benzene degradation has to be 1995; Ball et al., 1996; Kniemeyer and Heider, 2001), or interpreted with caution. in p-cresol degradation (Hopper et al., 1991; Peters et al., Recently, the genomes of five anaerobic aromatics- 2007), fumarate addition by glycyl radical enzymes to degrading microorganisms, including the phototrophic methyl carbon atoms in toluene (Biegert et al., 1996; organism Rhodopseudomonas palustris strain CGA009 Beller and Spormann, 1998; Leuthner et al., 1998), m-, o-, (Larimer et al., 2004), the denitrifying organisms Mag- p-xylene (Krieger et al., 1999; Achong et al., 2001; netospirillum magneticum strain AMB-1 (Matsunaga et al., Morasch et al., 2004; Morasch and Meckenstock, 2005) 2005) and Aromatoleum aromaticum strain EbN1 (Rabus and p-cresol (Müller et al., 2001), and anaerobic carbo- et al., 2005), the iron reducer Geobacter metallireducens xylation of phenol (biological Kolbe-Schmitt reaction) strain GS-15 (Butler et al., 2007) and the fermenter Syn- (Schmeling et al., 2004; Schühle and Fuchs, 2004; trophus aciditrophicus strain SB (McInerney et al., 2007), Narmandakh et al., 2006). have been sequenced and several operons coding for Although the mechanism of benzene activation under enzymes of anaerobic aromatic hydrocarbon degradation anaerobic conditions is still unclear, putative reactions have been identified (Carmona et al., 2009). The overall 13 were proposed based on metabolite detection using C6- organization of anaerobic catabolic gene clusters was 18 13 benzene, H2 Oor C-labelled bicarbonate buffer. Such conserved across a wide variety of microorganisms. reactions included (ii) hydroxylation of benzene to phenol However, genus- and species-specific variations account (Vogel and Grbic-Galic, 1986; Grbic-Galic and Vogel, for differences in gene arrangements, substrate specifi- 1987; Caldwell and Suflita, 2000; Chakraborty and cities and regulatory elements. Although the genome Coates, 2005), (ii) direct carboxylation to benzoate sequence of the benzene-degrading Dechloromonas

- - COO COO COSCoA

-OOC O CH3 - COO COO- BssABCD BbsGHCD Tolue ne BbsAB a Benzylsuccinate Benzylsuccinyl-CoA Benzoyl-CoA CO 2ATP 2ADP 2 COOH COSCoA COSCoA CoA+ATP AMP+PPi 2[H] 2Pi b Acetyl-CoA + CO BadA, BclA, BzdA BcrCBAD 2 Benzene BamY BzdNOPQ Benzoate + c 2H + H2O + 2Fdred 2Fdox 2[H] BamB-I

HbaBCD HO AMP+Pi - COO- ATP OH Pi CO2 CoA+ATP AMP+PPi COSCoA HcrCAB C- PcmRST

PpsABC PpcABCD PpcABCD HbaA Phenol HcrL O O OH - OH O PO 4-hydroxybenzoate 4-hydroxybenzoyl-CoA OH Phenylphosphate

CO2

Ohb1

Fig. 1. Proposed options for anaerobic biodegradation of benzene. (a) Methylation to toluene followed by fumarate addition to form benzylsuccinate that is subsequently metabolized to benzoyl-CoA. (b) Carboxylation to benzoate and ligation of CoA forming benzoyl-CoA. (c) Hydroxylation to phenol, and subsequent degradation via 4-hydroxybenzoate and benzoyl-CoA. The names of the enzymes in the different organisms are: BssABC, benzylsuccinate synthase; BbsEF, succinyl-CoA:(R)-benzylsuccinate CoA-transferase; BbsG (R)-benzylsuccinyl-CoA dehydrogenase; BbsH, phenylitaconyl-CoA hydratase; BbsCD, 2-[hydroxy(phenyl)methyl]-succinyl-CoA dehydrogenase; BbsAB, benzoylsuccinyl-CoA thiolase; BadA, BclA, BzdA and BamY, benzoate-CoA ligase; BcrCBAD, BzdNOQP and BamB-I, benzoyl-CoA reductase; PpsABC, phenylphosphate synthase; PpcABCD, phenylphosphate carboxylase; HbaA, HcrL, 4-hydroxybenzoate-CoA ligase; and HbaBCD, HcrCAB and PcmRST, 4-hydroxybenzoyl-CoA reductase.

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 2783–2796 Enzymes involved in anaerobic benzene degradation 2785 aromatica strain RCB has been elucidated (accession the culture was transferred from benzene to a different number NC_007298), no genes coding for putative substrate, the community structure of benzene-, phenol- enzymes involved in anaerobic benzene degradation and benzoate-grown cultures was assessed by T-RFLP were indentified (Salinero et al., 2009). analysis when approximately 25 mM ferrous iron was pro- In the present study, we describe for the first time a duced (Fig. S1). With all substrates, the analysis showed combined proteomic and genomic approach to elucidate the same dominance of the Peptococcaceae-related the biochemical mechanism of anaerobic benzene degra- microorganisms forming a T-RF of 289 bp (Fig. S2). This dation. Whole proteomes of benzene-, phenol- and allowed a direct comparison of the expressed proteins of benzoate-grown cells were compared by a LC/ESI-MS/ cells grown on the respective substrates. MS-based shotgun proteomic analysis and were corre- lated to the high-throughput sequenced metagenome The metagenome of the iron-reducing culture BF information of the iron-reducing, benzene-degrading The draft metagenomic sequences (10.13 Mb) of the enrichment culture BF. Additionally, specific protein bands iron-reducing culture BF contained 14 270 open read- expressed in benzene-grown cells were subjected ing frames (ORFs) and 5832 contigs, while the DNA to N-terminal sequence analysis. A putative benzene sequence length of the contigs ranged between 0.064 and carboxylase-related protein was identified as key protein 537.470 kb. BLASTP search of the translated ORFs indi- that might be responsible for the initial activation of cated that 21.3% of the total identified ORFs could be benzene, and thus provide new evidence for carboxyla- assigned with more than 50% identity to genes in the tion of benzene under iron-reducing condition. NCBI non-redundant protein sequence database, and around 40.7% of the total ORFs did not show any relevant identity to genes with known function. Details about Results and discussion general genomic features are summarized in Table S1. Community structures of the benzene-degrading culture About 205 genes were identified to have closest BF with different growth substrates sequence similarity to genes encoding enzymes and transcriptional regulators known to be involved in anaerobic In the present study, we investigated the anaerobic aromatic hydrocarbon degradation indicating the impor- benzene degradation pathways of the iron-reducing tance of aromatic compounds as carbon source for the enrichment culture BF isolated from soil at a former growth of culture BF. Colocalized gene clusters were manufactured gas plant site. The highly enriched iron- discovered for some aromatic hydrocarbon-degrading reducing culture BF was dominated by Gram-positive proteins. However, the majority of genes were scattered Peptococcaceae-related microorganisms (Kunapuli et al., across 63 different contigs of the metagenome (Table S2). 2007). Besides benzene, the microorganisms were able Moreover, around 90 genes were discovered to have to grow with phenol (1 mM), 4-hydroxybenzoate (1 mM) sequence similarity to genes encoding proteins related to and benzoate (1 mM), but not with toluene (1 mM), ethyl- xenobiotics transporters, e.g. ATP-binding cassette (ABC) benzene (1 mM) or xylene isomers (1 mM) as sole source and major facilitator superfamily (MFS-1). of carbon (Kunapuli et al., 2007). An interesting feature of the metagenome was the pres- With the help of differential expression analysis we ence of about 112 genes similar to genes encoding aimed at identifying proteins specifically expressed phage-like proteins related to phage attachment and inte- with benzene as growth substrate but not with putative gration, such as terminase, recombinase, integrase and metabolites of benzene degradation such as phenol or resolvase. In addition, 18 ORFs similar to genes present benzoate. By comparing the different expression profiles in transposable elements (e.g. transposase) indicating we expected benzoate degradation genes to be induced a potential mobility of the genetic elements within the with all three substrates. However, culture BF is not pure metagenome were identified. Some of the corresponding and a problem for proteome analysis might arise if differ- transposable and phage-like genes were located within ent community members grew up when the culture is the contigs that contained putative aromatic-degrading transferred from benzene to phenol or benzoate. genes (Table S3). Two different full-length (1524 bp) 16S rRNA gene sequences clustering with the Peptococcaceae (T-RF Differential comparative proteome analysis of benzene-, 289) and Desulfobulbaceae (T-RF 162) were identified phenol- and benzoate-grown cells under iron-reducing from the metagenome of the benzene-grown enrichment condition by mass spectrometric analysis and culture BF. The T-RFLP analysis indicated the dominance N-terminal sequencing of Peptococcaceae (T-RF 289) during growth on benzene (Fig. S1A). To ensure that the microbial composition of the The soluble proteomes of benzene-, phenol- and iron-reducing enrichment culture BF did not change when benzoate-grown cells of the iron-reducing culture BF were

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 2783–2796 2786 N. Abu Laban et al. separated by SDS-PAGE and differentially identified by benzoyl-CoA reduction. BamY was identified as the mass spectrometric analysis. In total, 4020 proteins were benzoate-CoA ligase (Wischgoll et al., 2005; Heintz et al., identified for all tested substrates. A total of 276 proteins 2009). were exclusively expressed in benzene-grown cells, 20 of which could be assigned to aromatic hydrocarbon Genes of benzoate metabolism in culture BF. Open degradation pathways (Table 1). In addition, proteomes of reading frames homologous to putatively benzoate phenol-grown cells revealed the exclusive expression of anaerobic metabolism (bam) genes that encode proteins 90 proteins, 3 of which were similar to proteins involved in involved in reductive dearomatization of benzoyl-CoA to a phenol degradation (Table 1). Further 200 proteins were cyclic, conjugated diene during benzoate degradation in identified to be specific for benzoate-grown cells, 13 of G. metallireducens (Wischgoll et al., 2005), were found to which were similar to putative Bam and Bzd proteins be scattered over 19 different contigs of the metagenome involved in the anaerobic degradation of benzoate sequence (Table S2). A large contig with 561 ORFs (Table 1). (contig BF_11345) contained genes similar to bamCBH- FEDI (ORFs 48–49, 52–54 and 57–58; Fig. 2B) encoding proteins responsible for benzoyl-CoA reduction in G. met- Benzene metabolism allireducens (Kung et al., 2009). The predicted gene Three different pathways have been proposed for anaero- products showed a high sequence identity (> 60%) to bic benzene degradation, including a direct methylation the respective proteins described in Geobacter sp. FRC- to toluene and subsequent degradation via the benzylsuc- 32 (YP_002535701, YP_002535686, YP_002535687, cinate pathway to benzoyl-CoA (Ulrich et al., 2005), a YP_002535704, YP_0025-35705 and YP_002535691; hydroxylation of benzene to phenol and further degrada- Table S2). Furthermore, the metagenome of culture BF tion by the well-known phenol pathway to benzoyl-CoA contained one gene cluster putatively encoding proteins (Caldwell and Suflita, 2000; Chakraborty and Coates, catalysing subsequent reactions in benzoate degradation. 2005), and a direct carboxylation to benzoate and activa- The formation of a hydrolytic ring cleavage product in tion by CoA to benzoyl-CoA (Caldwell and Suflita, 2000; A. aromaticum strain EbN1 involves the enoyl-CoA Phelps et al., 2001; Kunapuli et al., 2008; Musat and hydratase (BzdW), a short-chain alcohol dehydrogenase Widdel, 2008; Abu Laban et al., 2009). If benzene degra- (BzdX) and the ring opening hydrolase (BzdY), which are dation would proceed via a direct methylation as the first probably encoded by ORFs 95–96 and 98 in culture BF activation reaction of benzene we would expect the (Table 1). benzoyl-CoA pathway enzymes to be expressed, the enzymes of the benzylsuccinate degradation pathway, Benzoate specific protein expression. In benzoate-grown plus an unknown methylase. If benzene would be acti- cells different putatively bam-related ORFs probably vated by a direct hydroxylation to phenol, one would encoding benzoate-CoA ligase (ORF 194; Table 1) and expect the same phenol degradation enzymes expressed benzoyl-CoA reductase (ORFs 53, 56, 86, 152, 155, 157, as in phenol grown cells plus some unknown hydroxylase 159, 168 and 178; Table 1) were expressed. The enzyme. Finally, for a carboxylation as the first activation expressed bam-like genes were located on different reaction of benzene, we would expect all benzoyl-CoA contigs of the metagenome sequence. Some of these degradation enzymes to be expressed plus the initial car- genes were also expressed in benzene- and phe- boxylase but no phenol degradation enzymes. nol-grown cells (ORFs 56, 86, 155, 168, 178 and 194). In addition, specific genes probably encoding enzymes involved in the lower, ring-opening pathway of anaerobic Benzoate metabolism-benzoyl-CoA pathway benzoate degradation were found to be expressed in In facultative anaerobes, benzoate degradation to the culture BF. bzdY- and bzdW-like genes (ORFs 95–97) benzoyl-CoA involved benzoate activation to benzoyl- encoding a putative ring-opening hydrolase and dienoyl- CoA catalysed by benzoate-CoA ligase (Schühle et al., CoA hydratases were expressed in benzoate-grown cells, 2003), and ring reduction of benzoate catalysed by the whereas one of the bzdW genes (ORF 96) was induced key enzyme benzoyl-CoA reductase (Bcr) (Boll and with all substrates. However, no protein products of the Fuchs, 1995; Boll, 2005). A Bcr enzyme has been isolated bzdX could be identified from phenol- and benzoate- and studied in the denitrifying Thauera aromatica (Boll grown cultures probably because expression of bzdX and Fuchs, 1995). It is an ATP-dependent oxygen- genes was below the detection limit of the mass spectro- sensitive enzyme with a abgd heterotetramere structure metric analysis. It is not clear from the obtained data (Boll et al., 2000). In the strictly anaerobic organism G. why the bzdX gene is so little expressed in phenol and metallireducens, the membrane protein complex Bam benzoate-grown cells that the protein could not be BCDEFGI (BamB-I) was proposed to be responsible for detected by mass spectrometric analysis. A possible

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 2783–2796 00SceyfrApidMcoilg n lcwl ulsigLtd, Publishing Blackwell and Microbiology Applied for Society 2010 © Table 1. Benzene-, phenol-, and benzoate-expressed ORFs of culture BF related to known genes involved in anaerobic aromatic hydrocarbon degradation and transport.

Expressed proteinsa Related gene product (BLASTP annotation)c

MS/MS peptides identiﬁcationb Gene Length Size Identity product Contig No. (aa) (kDa) Benzene Phenol Benzoate Gene Protein E-value (%) Accession No. Organismd

ORF 10 BF_5887 185 21.7 2 (7) –– Acetophenone carboxylase, gamma subunit 1.E-38 50 ZP_03494850 Aliac ORF 25 BF_5966 199 21.8 2 (5) 2 (5) – Probable UbiX-like carboxylase 2.E-72 65 YP_158781 Azose ORF 29 BF_5978 479 53.2 – 2 (6) 2 (5) Phenylphosphate carboxylase, alpha subunit 4.E-177 64 YP_158784 Azose ORF 33 BF_5983 397 44.3 – 2 (10) – bamI Formate dehydrogenase, alpha subunit 1.E-130 61 ZP_04353073 NA ORF 34 BF_6004 289 30.9 13 (50) – 2(11) pcmR Molybdopterin dehydrogenase, FAD-binding subunit 1.E-84 58 YP_002535583 Geosf ORF 35 BF_6004 114 12.2 2 (18) ––pcmS (2Fe-2S)-binding domain protein 9.E-44 71 YP_385089 Geomg ORF 53 BF_11345 143 15.9 –– 2(5) bamF Methyl-viologen-reducing hydrogenase, delta subunit 2.E-45 56 YP_002535705 Geosf ORF 56 BF_11345 76 8.0 – 2(5) 2(5) bamE 4Fe-4S ferredoxin, iron-sulfur binding protein 2.E-26 85 YP_461442 Synas ORF 70 BF_11345 326 37.6 2 (8) –– Benzoyl-CoA reductase/2-hydroxyglutaryl-CoA dehydratase 1.E-132 67 YP_001211338 Pelts ORF 77 BF_11348 95 10.1 – 2 (16) 2 (16) pcmS (2Fe-2S)-binding domian protein 6.E-33 73 YP_002535582 Geosf ORF 83 BF_11348 82 9.4 – 2 (28) – ppcD Phenylphosphate carboxylase, delta subunit 2.E-14 44 YP_158783 Azose ORF 86 BF_11348 652 74.2 2 (5) – 2(5) bamB Aldehyde ferredoxin oxidoreductase 0.E+00 73 YP_002535685 Geosf ORF 87 BF_11352 514 57.0 –– – bamY Benzoate-CoA ligase 2.E-157 54 YP_385097 Geomg ORF 90 BF_11370 587 66.8 – 2 (15) 2 (10) UbiD-like carboxylase 0.E+00 58 ZP_02849982 Dprot ORF 95 BF_11393 376 42.4 –– 2(9) bzdY 6-oxocyclohex-1-ene-1-carbonyl-CoA hydratase 3.E-148 66 AAQ08805 Azoev ORF 96 BF_11393 171 18.5 5 (43) 2 (12) 2 (44) bzdW Putative dienoyl-CoA hydratase 3.E-42 51 CAD21636 Azoev ORF 97 BF_11393 70 8.0 2 (36) – 2 (36) bzdW Dienoyl-CoA hydratase 7.E-03 35 CAD21628 Azoev ORF 98 BF_11393 183 20.1 2 (15) ––bzdX 6-hydroxycylohex-1-ene-1-carboxyl-CoA dehydrogenase 4.E-51 61 CAI78830 NA ORF 99 BF_11393 138 15.2 2 (18) ––bzdX 6-hydroxycylohex-1-ene-1-carboxyl-CoA dehydrogenase 4.E-32 45 CAI78830 NA ORF 115 BF_11406 481 53.2 – 2(5) – ppcA Phenylphosphate carboxylase, alpha subunit 4.E-160 59 YP_158784 Azose ORF 117 BF_11411 199 21.7 2 (18) –– 3-polyprenyl-4-hydroxybenzoate decarboxy-lyase 8.E-67 64 YP_160255 Azose ORF 120 BF_11415 302 33.2 2 (9) –– Probable UbiD-like carboxylase 6.E-52 41 YP_158782 Azose ORF 121 BF_11416 421 47.4 18 (38) – – ppcA Phenylphosphate carboxylase, alpha subunit 1.E-45 31 YP_158784 Azose

ORF 122 BF_11416 206 23.0 5 (30) – – 3-polyprenyl-4-hydroxybenzoate decarboxy-lyase 1.E-19 31 ZP_04376160 NA degradation benzene anaerobic in involved Enzymes ORF 123 BF_11416 286 32.5 7 (23) – – ppcA Phenylphosphate carboxylase, alpha subunit 7.E-24 30 YP_158784 Azose ORF 126 BF_11418 498 56.1 11 (17) – – Hypothetical protein 1.E-44 29 ZP_04375589 Catad niomna Microbiology Environmental ORF 127 BF_11418 309 34.2 2 (9) – – MRP protein homologue, conserved ATPase 8.E-57 44 AY_001436025 NA ORF 132 BF_11418 224 25.4 3 (13) – – Hypothetical protein 3.E-18 48 NP_143765 Pyrho ORF 133 BF_11418 131 15.2 7 (51) – – Hypothetical protein 3.E-00 28 AAP35074 Derfa ORF 136 BF_11418 317 34.2 2 (8) – – Multidrug resistance protein, MRP family 3.E-55 42 YP_002352437 Dictd ORF 137 BF_11418 126 14.7 13 (85) – 9 (48) ppcD Phenylphosphate carboxylase, delta subunit 2.E-02 37 YP_158783 Azose ORF 138 BF_11418 508 57.4 30 (85) – 15 (43) ppcA Phenylphosphate carboxylase, alpha subunit 2.E-106 43 YP_158784 Azose ORF 139 BF_11418 541 60.7 7 (12) ––bamY Benzoate-CoA ligase 1.E-172 56 YP_385097 Geomg ORF 140 BF_11418 153 16.8 5 (29) –– Probable UbiX-like carboxylase 2.E-52 68 P57767 Thaar ORF 152 BF_11447 102 11.5 –– 2(8) bamF Methyl-viologen-reducing hydrogenase, delta subunit 5.E-09 50 YP_002535689 Geosf ORF 155 BF_11452 654 74.3 17 (29) – 8(7) bamB Aldehyde ferredoxin oxidoreductase 0.E+00 73 YP_002535685 Geosf ORF 157 BF_11452 184 20.7 –– 4 (30) bamC 4Fe-4S ferredoxin, iron-sulfur binding protein 1.E-54 57 YP_384757 Geomg ORF 159 BF_11460 637 72.6 –– 2 (10) bamB Aldehyde ferredoxin oxidoreductase 0.E+00 70 YP_002535685 Geosf ORF 161 BF_11460 289 30.9 –– 5 (10) pcmR Molybdopterin dehydrogenase, FAD-binding subunit 1.E-54 57 YP_384757 Geomg ORF 163 BF_11460 405 45.2 2 (7) ––bamM Acyl-CoA dehydrogenase domain protein 1.E-118 51 ZP_04150428 Bac11 ,

12 ORF 168 BF_11492_ 365 40.5 10 (32) – 25 (31) bamI 4Fe-4S ferredoxin, iron-sulfur binding protein 8.E-38 29 YP_002430370 Geomg ORF 178 BF_11515 732 82.0 22 (20) – 25 (31) bamF Acetyl-CoA decarbonylase, beta subunit 0.E+00 74 YP_360060 Carhz 2783–2796 , ORF 184 BF_11550 339 36.6 – 2 (12) – hbaD 4-hydroxybenzoyl-CoA reductase, beta subunit 1.E-47 37 NP_946025 Rhop2 ORF 186 BF_11550 543 58.7 –– – pcmT 4-hydroxybenzoyl-CoA reductase, alpha subunit 3.E-109 40 YP_385090 Geomg ORF 194 BF_11554 521 57.7 10 (20) – 2(5) bamY Benzoate-CoA ligase family 7.E-168 55 YP_002535674 Geosf ORF 197 BF_11554 774 85.0 18 (32) – 9 (12) pcmT 4-hydroxybenzoyl-CoA reductase, alpha subunit 0.E+00 77 YP_002535581 Geosf ORF 199 BF_11555 167 17.8 2 (14) ––pcmS 4-hydroxybenzoyl-CoA reductase, gamma subunit 2.E-67 70 ZP_03023308 Geosf ORF 201 BF_11556 773 84.9 2 (5) ––pcmT 4-hydroxybenzoyl-CoA reductase, alpha subunit 0.E+00 76 YP_002535581 Geosf

a. ‘aa’ is the number amino acids; ‘kDa’ is the size of proteins in kiloDalton. b. Numbers without parenthesis indicate unique peptides. Numbers with parenthesis give the percentage of coverage (%) of the amino acid sequences of the identified proteins by the detected peptides. c. Hits were obtained from BLASTP comparison of predicted proteins with NCBI data bank (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The E-value, gives expectation value. Identity, gives the percentage of amino acids that are identical between the ORF and the next related protein in the data bank. Accession no., the accession number from NCBI database. d. Species names are abbreviated according to the list of organism identification code (SWISS-PROT): Azose, Aromatoleum aromaticum strain EbN1; Pelts, Pelotomaculum thermopropionicum strain S1; Geosf, Geobacter sp. strain FRC-32; Geomg, Geobacter metallireducens strain GS-15; Aliac, Alicyclobacillus acidocaldarius; Dprot, Delta-proteobacterium; Azoev, Azoarcus evansii; Carhz, Carboxydothermus hydrogenoformans; Rhop2, Rhodopseudomonas palustri; Synas, Syntrophus aciditrophicus; Thaar, Thauera aromatica; Bac11, Bacillus sp.; Catad, Catenulispora acidiphila; Pyrho, Pyrococcus horikoshii; Derfa, Dermatophagoides farinae; Dictd, Dictyoglomus turgidum; NA, Name of the organism is not available 2787 in SWISS-PROT. The specifically benzene-expressed protein of 60 kDa identified by the direct N-terminal sequencing from the SDS gels is depicted in bold. 2788 N. Abu Laban et al.

A ORF 11 12 1314 15 17 18 Contig BF_ 5887 bssC bssA bssB bssEbssF bssF bssD

170 171 172 173 174 175 Contig BF_ 11494 bbsA bbsB bbsD bbsE bbsG bbsF B

ORF 47 48 49 52 5354 55 56 57 58 Contig BF_11345 bamC bamBbamH bamF bamE bamE bamE bamD bamI C ORF 72 73 77 78 79 80 81 82 83 84 85 Contig BF_11348 ubiX ubiX pcmS pcmT ppsA ppcB ppcC ppcA ppcD pcmR

ORF 104 105 106 107 110 111 112114 115 116 Contig BF_ 11406 ppcD ppcA ppcC ppcB ppsA ppcD ppcA ppcC

51015 Kb Benzylsuccinate synthase β-oxidation of benzylsuccinate Benzoate-CoA ligase Benzoate degradation bamB-I 3-octaprenyl-4-hydroxybenzoate carboxy-lyase Phenylphosphate carboxylase Phenylphosphate synthase 4-hydroxybenzoyl-CoA reductase Sigma 54 specific transcriptional regulator Putative uncharacterized protein

Fig. 2. Genes identified in the metagenome of the iron-reducing culture BF that encode putative proteins of anaerobic aromatic degradation: (A) toluene, (B) benzoate and (C) phenol and 4-hydroxybenzoate. Predicted functions of ORFs were based on sequence similarities to known genes. The gene clusters were discovered on different contigs of the metagenome of culture BF (Table S2). explanation would be that for benzene degradation fast that this phylogenetic linage uses a combination of the processing of down stream metabolites in the pathway benzoyl-CoA pathways of strict anaerobes and facultative is required to enhance the initial reaction. A higher expres- anaerobes. sion of such down stream enzymes would therefore increase the kinetics of the pathway and help removing Methylation as a possible initial activation metabolites of the first reaction steps such as benzoate. reaction of benzene The identification of bam-like proteins suggests that culture BF utilizes a benzoyl-CoA pathway whose activa- Putative genes of toluene metabolism. On one contig tion and reduction steps are analogous to the one pro- (BF_5902; Table S2) of the metagenome of culture BF posed for strictly anaerobic microorganisms, such as we identified ORFs (ORFs 11–13), which are similar to G. metallireducens or Syntrophus gentiani (Wischgoll bssCAB toluene catabolic genes encoding benzylsucci- et al., 2005; McInerney et al., 2007; Heintz et al., 2009). nate synthase of A. aromaticum strain EbN1 (YP_158059 Moreover, the expression of the putative bzdXYW-likes to YP_1580561) and bssD (ORF 18) encoding the acti- genes suggests that the genes encoding the modified vating enzyme. These genes were part of a large toluene- b-oxidation of the benzoate degradation pathway using like cluster which contained also ORFs similar to bssF dienoyl-CoA as substrate and finally generating (ORFs 15 and 17) and bssE (ORF 14) encoding proteins 3-hydroxypimelyl-CoA (Laempe et al., 1998; Laempe with so far unknown functions in toluene degradation of et al., 1999) appear to be more closely related to those A. aromaticum strain EbN1 (Fig. 2A). In addition to the of denitrifying bacteria (Carmona et al., 2009). As we putative bss genes, a further contig (BF_11494; Table S2) present here for the first time a putative benzoyl-CoA containing 123 ORFs harboured putative bbsCABEFD pathway of a Gram-positive organism, this might indicate genes whose products might be involved in the

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 2783–2796 Enzymes involved in anaerobic benzene degradation 2789 b-oxidation of benzylsuccinate or some analogue (ORFs BF (BF_11348 and BF_11406; Table S2). The respective 170–175; Fig. 2A). contigs contained 72 and 60 ORFs respectively. Open reading frames similar to the putative a-subunit of the Protein expression of toluene metabolism. Although phenylphosphate synthase (PpsA) were located in both genes were identified in the metagenome that might clusters (ORFs 79 and 110; Fig. 2C) and contained the encode the toluene degradation enzymes, gene products characteristic conserved His-522 residue, which is the could be identified from protein extracts of neither specific binding site for ATP. ORFs similar to genes coding benzene-, nor phenol- or benzoate-grown cells. This for the phenylphosphate synthase b- and g-subunits strongly suggests that benzene is not degraded via (PpsB and PpsC) were absent in both gene clusters. toluene as an intermediate, which would be the first However, such sequences were identified on other product of a putative methylation reaction. Moreover, contigs of the metagenome sequence (ORFs 36-37, 89 toluene was not utilized as growth substrate by culture BF and 156; Table S2). Adjacent to ORFs 79 and 110, which (Kunapuli et al., 2008). Recent studies of benzene degra- are putatively encoding phenylphosphate synthase dation by other sulfate-reducing enrichment cultures also a-subunit (PpsA), two clusters of ORFs were found, indicated that toluene could neither be used as a sub- respectively, that are similar to genes encoding phe- strate nor as a co-substrate with benzene (Ulrich et al., nylphosphate carboxylase ppcBCAD (ORFs 80–83 and 2005; Musat et al., 2008; Oka et al., 2008; Abu Laban 104–107; Fig. 2C). The identified subunits of the putative et al., 2009). Furthermore, benzylsuccinate as the specific phenylphosphate carboxylases showed more than 45% metabolite of toluene degradation was also detected sequence identity to the respective proteins of A. aromati- neither in the iron-reducing culture BF nor in the sulfate- cum strain EbN1 (YP_158783 to YP_158786). reducing culture BPL (Kunapuli et al., 2008; Abu Laban Degradation of 4-hydroxybenzoate as an intermediate et al., 2009). Therefore, our proteomic data support the compound of anaerobic phenol degradation requires recent metabolite analyses that excluded the formation of 4-hydroxybenzoate-CoA ligase and 4-hydroxybenzoyl- toluene during benzene degradation, and we therefore CoA reductase producing benzoyl-CoA. No ORFs similar exclude methylation as an initial activation mechanism for to genes encoding 4-hydroxybenzoate-CoA ligase could the anaerobic benzene degradation in our iron-reducing be identified in the shotgun metagenome of culture BF. culture BF. Nevertheless, we propose that the putative However, several ORFs orthologous to the gene coding bss and bbs genes identified in the metagenome of BF for benzoate-CoA ligase (BamY) of G. metallireducens culture are involved in catalysing a fumarate addition (YP_385097) were found within a cluster of putative reaction of non-identified alkylated aromatic compounds phenol degradation genes mentioned above (ORF 111) other than toluene. and another gene cluster (ORFs 189–198) related to 4-hydroxybenzoate degradation (ORF 194). In general, amino acid sequences of aromatic CoA-ligases are Hydroxylation as a possible initial activation very similar to each other and it is difficult to differentiate reaction of benzene such ligases only based on the sequence (Butler The hydroxylation of benzene to phenol was previously et al., 2007; Peters et al., 2007). Open reading frames proposed as an initial activation mechanism based on the similar to genes pcmRST encoding three subunits of identification of phenol as metabolite in culture super- 4-hydroxybenzoyl-CoA reductase (ORFs 77–78 and 85; natants (Caldwell and Suflita, 2000; Chakraborty and Fig. 2C) were identified within one of the two putative Coates, 2005). Anaerobic phenol catabolism by the deni- phenol degradation gene clusters (contig BF_11348; trifying microorganisms T. aromatica and A. aromaticum Table S2), showing 73% (ORF 77: putative PcmS), 70% strain EBN1, and the iron-reducing organism G. metallire- (ORF 78: putative PcmT) and 58% (ORF 85: putative ducens, has been described in detail (Breinig et al., 2000; PcmR) amino acid sequence identities to the respective Rabus et al., 2005; Schleinitz et al., 2009), and proceeds proteins in G. metallireducens (Peters et al., 2007). via phenylphosphate synthase (PpsABC), phenylphos- Moreover, like in G. metallireducens the PcmR subunit of phate carboxylase (PpcABCD), 4-hydroxybenzoate-CoA the putative 4-hydroxybenzoyl-CoA reductase lacks the ligase (HcrL) and 4-hydroxybenzoyl-CoA reductase insertion of 40 amino acids, which carries the additional (HcrCAB/PcmRST) to benzoyl CoA. [4Fe-4S] cluster loop responsible for an inverted electron flow in facultative anaerobes. Additionally, several ORFs Genes of phenol metabolism. Open reading frames orthologous to 4-hydroxybenzoyl-CoA reductase encod- coding for enzymes similar to phenylphosphate synthase ing genes were discovered on 9 different contigs of and phenylphosphate carboxylase in A. aromaticum the BF metagenome sequence (ORFs 34, 35, 62, 63, 64, strain EbN1 were found in two different gene clusters 103, 161, 162, 183, 186, 197, 198, 199, 201, 202 and located on separate contigs of the metagenome of culture 203 respectively; Table S2).

Phenol-specific protein expression. The majority of pro- 34, 35, 197, 199 and 201 in benzene-grown cells of teins specifically expressed in phenol-grown cells were culture BF (Table 1), which are similar to PcmRST might related to phenylphosphate carboxylase (Ppc; ORFs not necessarily be active. 29, 83, and 115), 4-hydroxybenzoyl-CoA reductase Our data provide no direct indications that the (Hba/Pcm; ORFs 77 and 184) and 3-octaprenyl- expressed pcmRST-like genes are involved in phenol 4-hydroxybenzoate carboxy-lyase (UbiD/UbiX; ORF 90) degradation in culture BF. At present we can therefore enzymes. In addition, ORFs 77 and 184 encoding PcmS- not propose a clear function for the expressed pcm-like and HbaD-like proteins were expressed. Noteworthy, genes in benzene-grown cells. Together with the lack of ORFs 77, 83 and 115 were located in the putative phenol- proteomic indications that benzene might be hydroxylated degrading clusters that are described in Fig. 2C. We con- and further processed via a phenol degradation pathway, clude that these clusters are responsible for phenol we do not support a direct hydroxylation of benzene as degradation in culture BF. Unfortunately, the other phenol- the initial activation mechanism. expressed ORFs 29 and 184 are located on very short contigs, and therefore putative information concerning Carboxylation as a possible initial activation the phenylphosphate synthase (PpsABC) and carboxy- reaction of benzene lase (PpcBC) is not available. Our proteomic data revealed that the phenol degra- Benzene specific protein expression. Electrophore- dation genes described above were not expressed in tic separation of benzene-, phenol- and benzoate- benzene-grown cells. This finding supports recent studies expressed proteins revealed a very prominent protein showing that phenol detected in benzene-degrading band with a mass of about 60 kDa specifically expressed cultures was formed abiotically during sampling due to with benzene as growth substrate (Fig. 3). The contact of reduced ferrous iron in the culture medium with N-terminal sequence of the first 31 amino acids of the oxygen (Kunapuli et al., 2008; Abu Laban et al., 2009). polypeptide was determined by Edman sequencing and A theoretical possibility for the hydroxylation of ben- was identical to the amino acid sequence predicted from zene could be by xanthine-oxidase-like proteins (Enroth ORF 138 (contig BF_11418; Table 1). The gene product et al., 2000), which showed high sequence identity of ORF 138 [molecular mass (Mw) 57.4 kDa] showed to 4-hydroxybenzoyl-CoA reductase PcmRST subunits 43% sequence identity to genes coding for the a-subunit of Geobacter sp. strain FRC-32 (YP_002535581 to of phenylphosphate carboxylase (PpcA) in A. aromati- YP_002535583) (ORFs 34, 35, 197, 199 and 201). cum strain EbN1 (YP_158784). These data were also PcmRST of G. metallireducens is a special protein confirmed by mass spectrometric identification of pep- belonging to the xanthine-oxidase family of molybdenum tides of the 60 kDa band. Whereas no expression of cofactor containing enzymes that catalyse the irreversible ORF 138 could be detected in protein extracts from reductive dehydroxylation of 4-hydroxybenzoyl-CoA yield- phenol-grown cultures and no pronounced band was ing benzoyl-CoA and water (Peters et al., 2007). It is very visible in the SDS gels, the much more sensitive mass unlikely that such an enzyme could catalyse the direct spectrometric analysis showed that ORF 138 was as well hydroxylation of benzene to phenol, which would formally be a reversal of the dehydroxylation reaction. Never- ACMB kDa theless, if the gene products of pcmRST catalyse the 200 4-hydroxybenzoyl-CoA reduction, their expression in 120 benzene-grown cells would indicate an active phenol 100 degradation pathway while the initial hydroxylation 70 60 of benzene to phenol would be performed by other 50 enzymes. Furthermore, the pcmRST-like ORFs in culture 40 BF were unspecifically expressed with all three growth substrates. This finding was in accordance with the results 30 of other studies, showing that pcmRST genes were con- 25 stitutively expressed with all tested aromatic substrates utilized by G. metallireducens. On the other hand, 20 4-hydroxybenzoyl-CoA reductase activity could only 15 be measured with cells grown on p-cresol and 4-hydroxybenzoate as substrates. Therefore, the authors Fig. 3. Coomassie-stained SDS-PAGE of proteins extracted from suggested a posttranscriptional regulation of the PcmRST the iron-reducing culture BF grown on either benzene (lane A), phenol (lane B) or benzoate (lane C). The arrow indicates the enzyme activity (Peters et al., 2007). These observations specific benzene-induced protein of 60 kDa. Lane M represents might indicate that the expressed gene products of ORFs the molecular mass standard.

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 2783–2796 Enzymes involved in anaerobic benzene degradation 2791 expressed with benzoate as growth substrate. As this Putative genes of benzene metabolism. Open reading band was strongly expressed only during growth on frames coding for the putative subunits PpcD (ORF 137) benzene and not on phenol, the protein is probably not and PpcA (ORF 138) of phenylphosphate carboxylase involved in phenol metabolism. We rather propose that were discovered on a 47-ORF-containing contig (BF_ this enzyme is involved in the direct carboxylation of 11418; Table S2). The two ORFs clustered together benzene. Furthermore, the absence of the 60 kDa gene with genes encoding an UbiD/UbiX-like carboxylase product of ORF 138 in SDS gels from benzoate grown (3-octaprenyl-4-hydroxybenzoate carboxy-lyase; ORFs cells might indicate that the gene is expressed to a minor 124 and 140; Table S2) (Fig. 4A). The predicted gene extent but not upregulated. Thus, the polypeptide was products of ORF 137 (14.7 kDa) and ORF 138 (57.4 kDa) detected by the qualitative mass spectrometry but not displayed markedly reduced sequence identities 35% and visible in the less sensitive protein gel. However, a quan- 37% to putative ppcD (ORF 83 and ORF 104) and 43% titative proteomic analysis could give further insight in and 46% to putative ppcA (ORF 82 and ORF 105) genes the specific regulation of the genes involved in benzene of the other putative phenol degradation gene clusters degradation. mentioned above. As the gene cluster containing ORFs Furthermore, a product of ORF 139 (Mw 60.7 kDa; 137 and 138 did not contain ORFs similar to ppcB and Table 1), which is specifically expressed during growth ppcC needed for phenol carboxylation or ORFs similar to on benzene, encodes a protein with 56% sequence iden- ppsABC genes encoding for phenylphosphate synthase tity to benzoate-CoA ligase of G. metallireducens (PpsABC), we conclude that this gene cluster is not (BamY). This protein was also identified by mass spec- involved in anaerobic phenol metabolism but rather trometry analysis from a gel slice containing peptides contained the putative anaerobic benzene carboxylase with masses around 60 kDa. However, this polypeptide (Abc) genes (Fig. 4A). was not detected in the Edman sequencing of the The location in one gene cluster and the simultaneous 60 kDa band probably because it was present in only expression of ORFs 126–140 (Table 1), all related to minor amounts as compared with the prominent ORF either carboxylation or transport, together with the specific 138 gene product. We propose that the benzene-induced benzoate-CoA ligase (BzlA), strongly suggest that we gene product of ORF 139 constitutes a benzoate-CoA have identified the best candidates for genes encoding ligase channelling the product of benzene carboxylation, an enzyme, which is directly carboxylating benzene to benzoate, into the benzoyl-CoA reduction pathway. We benzoate. Based on the genomic and proteomic data name the enzyme BzlA for benzoate-CoA ligase of discussed above we propose a direct carboxylation of culture BF. benzene to benzoate as the initial activation mechanism Additionally, a product of ORF 137 encoding a protein in anaerobic benzene degradation (Fig. 4B). We name (Mw 14.7 kDa; Table 1) similar to the d-subunit of phe- this enzyme Abc, which most likely belongs to a carboxy- nylphosphate carboxylase (PpcD) was identified by mass lase family including phenylphosphate carboxylase and spectrometry. However, the later ORF was not visible in UbiX (3-octaprenyl-4-hydroxybenzoate carboxy-lyase). SDS-PAGE of benzene-grown cells because of the low Anaerobic benzene carboxylase probably consists of resolution for small-molecular-weight proteins in the SDS several subunits two of which are encoded by ORFs 137 gel. Moreover, other carboxylase related proteins were and 138, whereas the other potential subunits might expressed with benzene. These were encoded by ORFs be encoded by, e.g. ORFs 124, 126, 132, 133 and 140 121 and 123, which are located on another contig (Fig. 4A). The direct association of the genes to the dif- (BF_11416; Table 1). The later ORFs might be homo- ferent subunits and functions of the enzyme has to be logues to ORF138 and showed low similarity (30% solved by measuring the enzyme reaction and subse- sequence identity) to PpcA of A. aromaticum strain EbN1 quent purification of Abc. Nevertheless, the anaerobic (YP_158784). carboxylation of benzene constitutes a novel enzyme Other proteins specifically expressed on benzene reaction, which is unprecedented in biochemistry. A par- were UbiD/UbiX-like carboxylase (3-octaprenyl-4- allel in chemistry would be a Friedl–Crafts acylation of hydroxybenzoate carboxy-lyase; ORFs 122, 124 and aromatic compounds, but it is out of the scope of such a 140; Table 1) and an MRP family ATP-binding protein- study to speculate about possible reaction mechanisms. homologue (multidrug-resistant protein; ORFs 127 and 136; Table 1). Enzyme assays of putative benzene carboxylase, Moreover, proteins similar to 4-hydroxybenzoyl- 4-hydroxybenzoate-CoA ligase and CoA reductase subunits (PcmRST; ORFs 34, 35, 197, 4-hydroxybenzoyl-CoA reductase 199 and 201) were identified from benzene-grown cells (Table 1). Open reading frames 34 and 197 were also Enzymes activity tests of putative benzene carboxylase, expressed in benzoate but not in phenol-grown cells. 4-hydroxybenzoate-CoA ligase and 4-hydroxybenzoyl-

A 137* 138* ORF 124 126* 127* 132*133* 136* 139* 140* 141 142 δ α Contig BF_11418 ubiX abcD abcA bzlA ubiX

2Kb Putative benzene carboxylase Benzoate-CoA ligase Carboxylase-like Transcriptional regulator, MarR family Multidrug resistance protein MRP homologue, conserved ATPase Putative uncharacterized protein

B CO 2 COOH CoA + ATP AMP + PPi COSCoA

AbcDA BzlA Benzene Benzoate Benzoyl-CoA

Fig. 4. Putative benzene degradation gene cluster of the iron-reducing culture BF. A. Genes that encode proteins speciﬁcally expressed during anaerobic benzene degradation are indicated with stars. Putative genes involved in the initial activation of benzene are shown in a rectangle box. Predicted functions of ORFs are based on sequence similarities to known genes. B. Proposed roles of predicted genes in the initial activation of anaerobic benzene degradation to benzoate and benzoyl-CoA. The carboxylation step catalysed by anaerobic benzene carboxylase AbcDA might involve further, not speciﬁed gene products, whereas the benzoate-ligation step is probably catalysed by benzoate-CoA ligase BzlA.

CoA reductase were performed with extracts of cells tiﬁcation of phage-related and transposable element grown on benzene and phenol as control. However, we genes within contigs containing aromatic-degrading gene were not able to measure any activity so far, which might (Table S3). be related to several reasons. First, culture BF is mainly composed of Gram-positive bacterial cells, which are not Experimental procedures easily disrupted. We might have failed to open these cells, Growth conditions of anaerobic benzene-degrading e.g. due to the presence of ferric iron minerals. Second, enrichment cultures the yield of cells biomass and proteins (25–40 mgl-1)of the culture BF was extremely low and might not have The iron-reducing enrichment culture BF was cultivated as been sufficient for the enzyme assay. Third, the presence previously described (Kunapuli et al., 2007). For differential of insoluble ferrihydrite in the cell extract might have inter- protein expression analyses, cultivation was performed in 4 l growth medium. Inocula (10%, v/v) from benzene-grown pre- fered with the enzyme activity. cultures of culture BF were transferred into separate bottles containing either benzene (1 mM), phenol (1 mM) or ben- Possible horizontal gene transfer of the putative zoate (1 mM) as growth substrates in the presence of XAD7 as substrate reservoir (Morasch et al., 2001) and 50 mM aromatic-degrading genes of culture BF ferrihydrite as electron acceptor. All culture bottles were incu- The metagenome analysis indicated that most of the gene bated at 30°C in the dark. products likely to be involved in phenol carboxylation Microbial community structure analysis of the (e.g. PpsA, PpcBCAD) and a modiﬁed b-oxidation of the iron-reducing enrichment culture BF benzoyl-CoA pathway (e.g. BzdYWX) are similar to those of denitrifying Betaproteobacteria. However, gene prod- The total genomic DNA of benzene-, phenol- and benzoate- ucts likely to be involved 4-hydroxybenzoyl-CoA reduction grown cells of the iron-reducing enrichment culture BF was (e.g. PcmSTR), benzoate activation (BamY) and dearo- extracted during the exponential growth phase with the matization (e.g. Bam-like) appear to be more similar to FastDNA Spin Kit for Soil (MP Biomedicals, Illkirch, France) those of strict anaerobic Deltaproteobacteria (Table 1). according to the manufacturer’s protocol. The microbial community structure was analysed by terminal restriction frag- These data may suggest that the aromatic-degrading ment length polymorphism (T-RFLP) analysis as previously gene clusters of culture BF might be acquired from two described by Winderl and colleagues (2008). The analysis phylogenetically different microorganisms through hori- was carried out in three biological replicates for each aro- zontal gene transfer. This might be explained by the iden- matic substrate.

Shotgun genomic DNA sequencing of the lane was cut into 10 slices, washed with H2O for 30 min, iron-reducing culture BF reduced with 5 mM DTT (15 min, 20°C), and acetylated with 25 mM iodacetamide (15 min, 20°C). Then, the gel slices The whole metagenome of the iron-reducing culture BF were washed twice with 40% acetonitrile and once for 5 min grown on benzene was extracted with the FastDNA Spin Kit with 100% acetonitrile. Subsequently, proteins were digested for Soil (MP Biomedicals) according to the manufacturer’s with trypsin (0.03 mg ml-1 of 50 mM ammonium bicarbonate) protocol. Genomic information was obtained by shotgun overnight at 37°C. The digested peptides were separated sequencing using a GS FLX sequencer (performed by Euro- by reversed phase chromatography [PepMap, 75 mm (inside fins MWG Operon, Ebersberg, Germany). The sequence diameter) ¥ 250 mm, LC Packings] operated on a nano- reads have been automatically assembled into contigs. HPLC (Ultimate 3000, Dionex) with a non-linear 170 min Automated annotation of the assembled sequences was per- gradient using 2% acetonitrile and 0.1% formic acid in water formed using the PEDANT (PMID 18940859) software (A) and 0.1% formic acid in 98% acetonitrile (B) as eluents system. The prediction of coding sequences was performed with a flow rate of 250 nl min-1. The gradient settings were with a combination of Genemark 2.6r (PMID 11410670) and subsequently: 0–140 min: 2–30% B, 140–150 min: 30–60% Glimmer 3.02 (PMID 17237039). Homology to already pub- B, 150–160 min: 60–99% B, 160–170 min: stay at 99% B. lished proteins in Uniref100 was used to decide about the The nano-LC was connected to a linear quadrupole ion trap- best gene models and in order to decide about the best gene Orbitrap (LTQ Orbitrap) mass spectrometer (ThermoElectron, starts. Coding sequences were automatically assigned by Bremen, Germany) equipped with a nano-ESI source. The PEDANT to functional categories according to the functional mass spectrometer was operated in the data-dependent role catalogues FunCat (PMID 15486203) and Gene Ontol- mode to automatically switch between Orbitrap-MS and LTQ- ogy (PMID 14681407). All sequence data from this study MS/MS acquisition. Survey full scan MS spectra (from m/z were deposited at GenBank under the accession numbers 300 to 1500) were acquired in the Orbitrap with resolution GU357855 to GU358059. R = 60 000 at m/z 400 (after accumulation to a target of 1 000 000 charges in the LTQ). The method used allowed Protein extraction and SDS-PAGE sequential isolation of the most intense ions, up to five, depending on signal intensity, for fragmentation on the linear Cells were harvested from 4 l culture bottles by centrifugation ion trap using collisionally induced dissociation at a target at 9000 g for 15 min. The cell pellet was washed three times value of 100 000 charges. Target ions already selected with 50 mM Tris-HCl buffer (pH 7.5) and incubated overnight for MS/MS were dynamically excluded for 30 s. General at -20°C. One gram of cell pellet was resuspended in 1 ml mass spectrometry conditions were: electrospray voltage, lysis buffer (9 M urea, 2% CHAPS, 1% DTT; GE Healthcare 1.25–1.4 kV; no sheath and auxiliary gas flow. The ion selec- Europe GmbH, Freiburg, Germany). The cell extracts were tion threshold was 500 counts for MS/MS. Furthermore, -1 treated with a 7¥ stock solution (167 mlml lysis buffer) of an activation Q-value of 0.25 and activation time of 30 ms Complete Mini EDTA-free Protease Inhibitor Cocktail Tablet were applied for MS/MS. The resulted peptide MS/MS (Roche Diagnostics GmbH, Penzberg, Germany) and incu- spectra were identified by Mascot search (http://www. bated at 15°C for 30 min. Extracts were transferred into matrixscience.com). Mascot was searched with a fragment matrix lysis tubes B (MP Biomedicals) and were treated in a ion mass tolerance of 1.00 Da and a parent ion tolerance of bead beating FastPrep Instrument 120 (MP Biomedicals) at 10.0 p.p.m. Iodacetamide derivatives of cysteine were speci- -1 6.0ms for 35 s. After centrifugation for 1 min at 20 000 g fied in Mascot as a fixed modification. Protein identification the supernatant was incubated with nuclease mix (1 ml per was carried out by blast of peptides fragments against NCBI- 100 ml of supernatant; GE Healthcare) at 20°C for 30 min and BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or against again purified by centrifugation (20 000 g, 1 h). the translated metagenome sequence of the iron-reducing Quantification of protein concentrations was performed culture BF in a pedant database (Munich information centre using 2D Quant Kit according to the manufacturer’s instruc- for protein sequences) by using the Scaffold 2.02.03 software tions (GE Healthcare). Proteins were precipitated with (Proteome Software, Portland, OR, USA). Protein identi- acetone for 10 min at -20°C, and collected by centrifugation fication was accepted if the probability was greater than at 15 000 g for 30 min. Some 30 mg of proteins was resus- 95% and when at least two peptides were identified. Protein pended in 20 ml lysis buffer and subsequently separated by probabilities were assigned by the Protein Prophet algorithm sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Nesvizhskii et al., 2003). (SDS-PAGE; 4% stacking gel, 12 % separating gel) with a running buffer containing glycine 14.4 g l-1, Tris-base 3 g l-1 (pH 8.3) and SDS 1 g l-1. After electrophoresis, the gels were N-terminal sequence analysis stained with colloidal Coomassie brilliant blue (Zehr et al., 1989). Protein extracts were prepared from three biological A 60 kDa protein band specifically expressed in benzene- replicates for each substrate. Three parallel gels for each grown cells was transferred from SDS gels onto a PVDF protein extract were analysed to account for technical varia- membrane using a semi-dry blotting apparatus (Bio-RAD tions in proteomic analysis. Trans-Blot SD) at 1 mA cm-2 for 2 h. The PVDF membrane was stained with Coomassie brilliant blue and washed with Protein identification by ESI/LC-MS/MS 50% methanol. Coomassie-stained proteins were excised from the PVDF membrane and collected fractions were sub- For identification of proteins from the SDS-PAGE of jected to N-terminal sequence analysis using a 492-protein benzene-, phenol- and benzoate-grown cells, the complete sequencer (Applied Biosystems, Darmstadt, Germany)

© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 2783–2796 2794 N. Abu Laban et al. according to the manufacturer’s instructions. The obtained Butler, J., He, Q., Nevin, K., He, Z., Zhou, J., and Lovley, D. N-terminal sequence was used for protein identification by (2007) Genomic and microarray analysis of aromatics search against the translated metagenome sequence of the degradation in Geobacter metallireducens and comparison iron-reducing culture BF. The analysis was carried out with to a Geobacter isolate from a contaminated field site. BMC two separate biological replicates. Genomics 8: 180. Caldwell, M., and Suflita, J.M. (2000) Detection of phenol and Acknowledgements benzoate as intermediates of anaerobic benzene biodegradation under different terminal electron-accepting condi- The authors would like to thank Hakan Sarioglou for the tions. Environ Sci Technol 34: 1216–1220. assistance in proteomic analysis, and Reinhard Mentele for Carmona, M., Zamarro, M., Blazquez, B., Durante- the Edman sequencing. This project was supported by the Rodriguez, G., Juarez, J., Valderrama, J., et al. (2009) DAAD-Helmholtz PhD exchange program through an operat- Anaerobic catabolism of aromatic compounds: a genetic ing grant awarded to Nidal Abu Laban and by priority program and genomic view. Microbiol Mol Biol Rev 73: 71–133. 1319 funded by the German Research Foundation (DFG). Chakraborty, R., and Coates, J. (2005) Hydroxylation and carboxylation – two crucial steps of anaerobic benzene References degradation by Dechloromonas strain RCB. Appl Environ Microbiol 71: 5427–5432. Abu Laban, N., Selesi, D., Jobelius, C., and Meckenstock, R. Chang, W., Um, Y., and Pulliam Holoman, T. (2005) Molecular (2009) Anaerobic benzene degradation by Gram-positive characterization of anaerobic microbial communities from sulfate-reducing bacteria. 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