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

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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
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