Combined Genomic and Proteomic Approaches Identify Gene ...Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed

JOURNAL OF BACTERIOLOGY, Jan. 2010, p. 295–306 Vol. 192, No. 10021-9193/10/$12.00 doi:10.1128/JB.00874-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Combined Genomic and Proteomic Approaches Identify Gene ClustersInvolved in Anaerobic 2-Methylnaphthalene Degradation in the

Sulfate-Reducing Enrichment Culture N47�

Drazenka Selesi,1 Nico Jehmlich,2 Martin von Bergen,2,3 Frank Schmidt,4 Thomas Rattei,5Patrick Tischler,5 Tillmann Lueders,1 and Rainer U. Meckenstock1*

Institute of Groundwater Ecology, Helmholtz Zentrum Munchen—German Research Center for Environmental Health,Ingolstadter Landstrasse 1, D-85764 Neuherberg, Germany1; UFZ-Helmholtz Center for Environmental Research,

Department of Proteomics, Permoserstrasse 15, D-04318 Leipzig, Germany2; UFZ-Helmholtz Center forEnvironmental Research, Department of Metabolomics, Permoserstrasse 15, D-04318 Leipzig, Germany3;Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Friedrich-Ludwig-Jahn-Strasse 15a,

D-17487 Greifswald, Germany4; and Chair for Genome-Oriented Bioinformatics, Technische Universitat Munchen,Life and Food Science Center Weihenstephan, Am Forum 1, D-85354 Freising-Weihenstephan, Germany5

Received 3 July 2009/Accepted 14 October 2009

The highly enriched deltaproteobacterial culture N47 anaerobically oxidizes the polycyclic aromatic hydro-carbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genomesequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses wereperformed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaer-obic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductivering cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO2. The glycyl radical-catalyzedfumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinatesynthase (Nms), composed of �-, �-, and �-subunits that are encoded by the genes nmsABC. Located upstreamof nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe4S4] clustersequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involvedin beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four inter-vening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) cor-responding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradationwithin the denitrifiers “Aromatoleum aromaticum” EbN1, Azoarcus sp. strain T, and Thauera aromatica. Anothercontiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes thatwere found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that weresupposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetyl-CoA and CO2. Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the geneproduct to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work providesthe first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implicationsfor understanding contaminant degradation.

Polycyclic aromatic hydrocarbons (PAHs) are constantly re-leased into the environment by anthropogenic activities such asindustrial use or by accidental contamination. Due to the lowchemical reactivity caused by the resonance energy of the ar-omatic ring structure and the low bioavailability of PAHs, theyare persistent in the environment (15). The understanding ofmicrobial metabolic capabilities in terms of anaerobic PAHdegradation is in its infancy. However, natural amelioration ofcontaminated sites relies on the degradation capacities of mi-croorganisms, and therefore, it is an essential prerequisite tobroaden knowledge about the microorganisms involved andtheir potentials concerning PAH breakdown.

Numerous microorganisms that can degrade PAHs underaerobic conditions have already been identified, but only asmall number of anaerobic cultures that degrade PAHs likenaphthalene, 2-methylnaphthalene, and phenanthrene havebeen isolated so far (17, 20, 24, 31, 46–48, 50, 52, 66). It hasbeen shown that these anaerobic degraders activate aromatichydrocarbons by very unusual biochemical reactions which dif-fer completely from those of aerobic degradation. The periph-eral pathway of 2-methylnaphthalene degradation occurs inanalogy to anaerobic toluene degradation by the addition offumarate to the methyl group, catalyzed by the glycyl radicalenzyme naphthyl-2-methyl-succinate synthase (Nms) (Fig. 1)(3). In subsequent reactions, naphthyl-2-methyl-succinate isactivated to yield the coenzyme A (CoA) ester and oxidized toform naphthyl-2-methylene-succinyl-CoA. The following beta-oxidation of the side chain results in the formation of 2-naph-thoyl-CoA and succinate (3, 53). The first three enzyme reac-tions of this pathway have been measured in vitro (3, 53).Recently, Musat et al. (48) identified the gene coding for the

* Corresponding author. Mailing address: Institute of GroundwaterEcology, Helmholtz Zentrum Munchen—German Research Centerfor Environmental Health, Ingolstadter Landstrasse 1, D-85764 Neu-herberg, Germany. Phone: 49 (0) 89 3187 2561. Fax: 49 (0) 89 31873361. E-mail: [email protected].

� Published ahead of print on 23 October 2009.

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�-subunit of a putative naphthyl-2-methyl-succinate synthase(nmsA) in 2-methylnaphthalene-grown bacterial cultures. Themolecular composition of the nmsA gene is analogous to thatof the benzylsuccinate synthase �-subunit gene (bssA). The Bssenzyme is a well-investigated close homolog of Nms, catalyzingfumarate addition in the initial reaction of anaerobic toluenedegradation (34, 40). Based on findings from comparative se-quence studies, glycine radical-catalyzed fumarate addition hasbeen shown to be a widely distributed initial reaction mecha-nism for anaerobic hydrocarbon degradation involving tolueneand 2-methylnaphthalene, n-alkanes (12, 13, 25, 51), m-xylene(33), m- and p-cresols (9), and ethylbenzene (32).

In a process analogous to the anaerobic benzoyl-CoA degra-dation pathway (7), 2-naphthoyl-CoA is subjected to aromaticring reduction by a putative naphthoyl-CoA reductase, probablygenerating 5,6,7,8-tetrahydro-naphthoyl-CoA and further octahy-dro-2-naphthoic acid (4, 46). In the subsequent reactions, the ringsystem should be thiolytically cleaved and subjected to beta-oxi-dation, leading to the formation of acetyl-CoA and CO2.

In contrast to the first enzymatic reaction in the degradationof methylated aromatics, the first enzymatic reaction in anaer-obic degradation of unsubstituted aromatic compounds such asnaphthalene is still unresolved. In order to determine the ini-tial activation reaction of anaerobic naphthalene degradation,studies based on the analysis of metabolites have been per-formed. Zhang and Young (66) observed the incorporation of13C-labeled bicarbonate from the buffer into the carboxylgroup of 2-naphthoic acid, hypothesizing that carboxylation isthe initial activation reaction of anaerobic naphthalene degra-dation in the culture studied. Recently, Safinowski and Meck-enstock (54) identified the deuterated metabolites naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate, whichare exclusive intermediates of anaerobic 2-methylnaphthalenedegradation, in the enrichment culture N47 when the culturewas cultivated on fully deuterated naphthalene. Moreover,specific enzyme activities of the anaerobic 2-methylnaphtahl-ene degradation pathway have been detected in naphthalene-grown cells (54). Therefore, methylation of naphthalene toyield 2-methylnaphthalene as the initial activation reaction andsubsequent degradation via the 2-methylnaphthalene pathwaywere proposed for this bacterial culture. The elucidation of2-methylnaphthalene degradation may therefore reveal an im-portant part of the naphthalene degradation pathway. How-ever, Musat et al. (48) questioned methylation as the firstreaction in naphthalene degradation for their marine naphtha-lene-degrading deltaproteobacterial NaphS strains.

Whereas molecular components involved in anaerobic deg-radation of monoaromatic hydrocarbons are well known,knowledge about genes and enzymes involved in anaerobicPAH degradation is still missing (14). Here, we provide thefirst results of a whole-proteome- and whole-genome-basedinvestigation of the sulfate-reducing enrichment culture N47

FIG. 1. Proposed pathway for anaerobic 2-methylnaphthalene deg-radation and reductive dearomatization of 2-naphthoyl-CoA (3, 4, 53).Genes found in the N47 genome encode the following enzymes(shown in gray boxes): NmsABC, naphthyl-2-methyl-succinate syn-thase; BnsEF, naphthyl-2-methyl-succinate CoA transferase; BnsG,naphthyl-2-methyl-succinyl-CoA dehydrogenase; BnsH, naphthyl-2-

methylene-succinyl-CoA hydratase; BnsCD, naphthyl-2-hydroxymethyl-succinyl-CoA dehydrogenase; BnsAB, naphthyl-2-oxomethyl-succinyl-CoA thiolase; and NcrABCD, 2-naphthoyl-CoA reductase. The posi-tion of the double bond is not known for octahydro-2-naphthoyl-CoA.COSCoA, thioester of CoA and the respective carboxyl group.

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degrading naphthalene and 2-methylnaphtalene. We haveidentified some gene clusters encoding enzymes involved in2-methylnaphthalene degradation, 2-naphthoyl-CoA dearoma-tization, and subsequent ring cleavage reactions in 2-methyl-naphthalene-grown N47 cells.

MATERIALS AND METHODS

Cultivation of the enrichment culture N47. The sulfate-reducing culture N47was enriched with naphthalene, obtained from contaminated sediment collectedat a former coal gasification site, as a carbon source and cultivated as reportedpreviously (53). The culture is able to grow with the aromatic hydrocarbonsnaphthalene, 2-methylnaphthalene, 2-naphthoic acid, para-cresol, phenol, benz-aldehyde, and 3-hydroxybenzaldehyde as sole sources of carbon. Neither ben-zene nor toluene can be utilized. Additionally, growth of the microorganisms inthe culture occurred with the nonaromatic organic compounds glucose, pyruvate,and acetate. Besides SO4

2�, the culture is able to use S0 as an electron acceptor.The substrates naphthalene and 2-methylnaphthalene were added as 1.5%

solutions in 2,2,4,4,6,8,8-heptamethylnonane (1 ml/50 ml medium; Sigma-Al-drich, Steinheim, Germany) to the cultivation bottles after autoclaving. Culturesin 1:10 dilutions were inoculated into the bottles. Substrate utilization was mon-itored by colorimetric measurement of sulfide (16).

16S rRNA gene sequence analysis and phylogenetic affiliations of microor-ganisms in the enrichment culture N47. Cells were harvested by centrifugationfor 20 min at 3,300 � g and washed with 0.5� phosphate-buffered saline.Genomic DNA from naphthalene- and 2-methylnaphthalene-grown cells wasextracted with the FastDNA spin kit for soil according to the protocol of themanufacturer (MP Biomedicals, Illkirch, France). For terminal restriction frag-ment length polymorphism (T-RFLP) analysis, 16S rRNA gene sequences fromtwo separate incubations were obtained using the primer set Ba27f-FAM/907r(38). PCR products were digested with the restriction enzyme MspI (Fermentas,St. Leon-Rot, Germany) and analyzed as described previously (62). Amplifica-tion, cloning, and sequencing of almost-full-length bacterial 16S rRNA genesequences were performed with DNA extracted from naphthalene-grown cellswith the primer set Ba27f-Ba1492r (61) as described previously (55). Archaeal16S RNA gene sequences were amplified from extracted DNA with the primerset Ar109f and Ar912r (45). The 16S rRNA gene sequences were manuallyassembled, checked for quality by using the SeqMan II software module (Laser-gene 6 suite; DNASTAR, Madison, WI), and tested for chimerical structures byusing the Chimera Check analysis function of Ribosomal Database Project II(http://rdp.cme.msu.edu/). Phylogenetic analysis of the 16S rRNA sequences wasperformed with the ARB software package (http://www.arb-home.de) (44).Alignments were checked visually. Phylogenetic analyses based on nucleotidesequences were performed, and results were verified by applying maximumlikelihood, maximum parsimony, and neighbor-joining methods using the respec-tive tools in the ARB software package.

Proteome analysis. After several transfers of culture N47 grown on 2-methyl-naphthalene, an 800-ml culture sample that accumulated 2.5 mM sulfide as agrowth measure was harvested by centrifugation (20 min at 3,300 � g and 4°C).The cell pellet was washed three times with 50 mM Tris-HCl, pH 7.5, andresuspended in a mixture of 400 �l lysis buffer {9 M urea, 2% 3-[(3-cholamido-propyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol;GE Healthcare Europe GmbH, Freiburg, Germany} and 67 �l of a 7� stocksolution containing a Complete EDTA-free mini-protease inhibitor cocktail tab-let (Roche Diagnostics GmbH, Penzberg, Germany). After 30 min of incubationat room temperature, the cell-buffer mixture was transferred into a lysing matrixB tube (MP Biomedicals) and processed for 35 s at speed 6.0 in a FastPrepinstrument (MP Biomedicals). The homogenized solution was centrifuged for 2min at 20,000 � g and 4°C. The supernatant was treated for 30 min at theambient temperature with 3 �l nuclease mix (GE Healthcare) and centrifugedfor 1 h at 15,000 � g and 4°C. Estimation of the protein level in the supernatantwas performed with the two-dimensional Quant kit according to the protocol ofthe manufacturer (GE Healthcare).

For mass spectrometric analysis of peptides, a 30-�g sample of proteins ex-tracted from 2-methylnaphthalene-grown N47 cells was separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (37) and visualized by Coo-massie brilliant blue staining (65). The complete one-dimensional gel lanescontaining the separated proteins were cut into 24 equal pieces, which werewashed two times with 50% methanol–5% acetic acid (vol/vol) and digested withtrypsin (Sigma). After digestion, peptides were concentrated and desalted usingZipTip C18 columns (Millipore, Bedford, MA). The separation of the complexpeptide solutions was achieved by reversed-phase chromatography with a Pep-

Map column (internal diameter, 75 �m; length, 250 mm [LC Packings]) operatedon a nano-high-performance liquid chromatography system (Agilent Technolo-gies, Waldbronn, Germany) with a nonlinear 90-min gradient using 2% aceto-nitrile in 0.05% acetic acid in water (solution A) and 0.05% acetic acid in 90%acetonitrile (solution B) as eluents at a flow rate of 300 nl/min. The gradientsettings were as follows: 0 to 2 min, 1 to 5% solution B; 2 to 65 min, 5 to 25%solution B; 65 to 80 min, 25 to 60% solution B; and 80 to 91 min, 60 to 99%solution B. The nano-liquid chromatograph was connected to a linear trapquadrupole Orbitrap (LTQ Orbitrap) mass spectrometer (ThermoElectron, Bre-men, Germany) equipped with a nano-electrospray ionization source. The massspectrometer was operated in the data-dependent mode to automatically switchbetween Orbitrap mass spectrometry (MS) and LTQ tandem MS (MS/MS)acquisition. Survey full-scan MS spectra (from m/z 300 to 2,000) were acquiredby using the Orbitrap with a resolution power of 60,000 at m/z 400 (afteraccumulation to a target of 1,000,000 charges in the LTQ). The method em-ployed allowed sequential isolation of the most intense ions—up to six, depend-ing on the signal intensity—for fragmentation on the linear ion trap usingcollision-induced dissociation at a target value of 100,000 charges. Target ionsalready selected for MS/MS were dynamically excluded for 60 s. General MSconditions were an electrospray voltage of 1.5 kV, no sheath, and auxiliary gasflow. The ion selection threshold for MS/MS was 500 counts, and an activationQ-value (effective collision energy) of 0.25 and an activation time of 30 ms werealso applied for MS/MS.

For data analysis, tandem mass spectra were extracted by Sorcerer version 3.5(Sage-N Research, Inc.). All MS/MS samples were analyzed using Sequest (ver-sion 27, revision 11; ThermoFinnigan, San Jose, CA). Sequest was set up tosearch the N47 genome database (4,755 entries), assuming digestion by trypsin.Sequest was searched with a precursor ion tolerance of 20 ppm and a fragmention mass tolerance of 1.00 Da. The oxidation of methionine and the carb-amidomethylation of cysteine residues were specified in Sequest as variable mod-ifications. Peptide identifications were accepted if the data exceeded specific data-base search engine thresholds. Sequest identifications required deltaCn scores ofgreater than 0.10 and cross correlation scores (XCorr) of greater than 1.9, 2.2, 3.8,and 3.8 for singly, doubly, triply, and quadruply charged peptides. Protein identifi-cations were accepted if the proteins contained at least two identified peptides.Proteins that contained similar peptides and could not be differentiated based onMS/MS analysis alone were grouped to satisfy the principles of parsimony.

Sequencing and annotation of the genomic information from the enrichmentculture N47. Isolation of genomic DNA from a 400-ml naphthalene-grown cul-ture was performed with the Wizard genomic DNA purification kit by followingthe protocol of the manufacturer (Promega, Madison, WI). Whole-genome in-formation for the enrichment culture N47 was obtained by combining 454-pyrosequencing and Sanger sequencing of plasmid libraries by Roche GmbH(Penzberg, Germany) and AGOWA GmbH (Berlin, Germany). The automatedassembly of the sequences was checked manually. Automated annotation of theassembled sequences was performed using the PEDANT (PubMed identificationnumber [PMID] 18940859) software system, and predicted coding sequenceswere identified with GeneMark (5) (PMID 18428700). Coding sequences wereautomatically assigned by PEDANT to functional categories according to thefunctional role catalogues FunCat (PMID 15486203) and Gene Ontology (PMID14681407). The two contigs were searched for taxonomic markers correspondingto the 50 most universal bacterial clusters of orthologous groups (COGs) ofproteins in the eggNOG database (30) by using BLAST (2) (E value � 1E�10).This search resulted in the identification of 27 marker genes. To confirm that thetwo contigs carrying the 2-methylnaphthalene degradation genes belong to N47,we calculated neighbor-joining phylogenies for the COG sequences that arepresent on each contig (using MUSCLE [22] and NEIGHBOR [23] with defaultparameters).

Phylogenetic analysis of the �-subunit sequence of naphthyl-2-methyl-succi-nate synthase. A phylogenetic tree was reconstructed from all publicly availabledata for pure-culture benzylsuccinate synthases and homologous gene operonsby using amino acid alignment in ARB (44). We used the quartet puzzlingalgorithm (59) as implemented in ARB with 10,000 puzzling steps, the Jones-Taylor-Thornton model of substitution, and a uniform model of rate heteroge-neities. A positional filter was generated from the data set to include only aminoacid positions covered for all sequences (256 positions) for tree inference. Treetopology and branching orders were also verified using Fitch distance matrix andneighbor-joining algorithms as described previously (63).

Nucleotide sequence accession numbers. All 16S rRNA gene sequences weredeposited in GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbersGU080088 and GU080089. All other sequence data from this study were depos-ited in GenBank under accession numbers GU080116 to GU080137 andGU080090 to GU080115.

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RESULTS

16S rRNA gene sequence analysis of the enrichment cultureN47. The main bacterial members of the enrichment culturegrown with naphthalene or 2-methylnaphthalene as the carbon

source were assessed by T-RFLP analyses and sequencing of16S rRNA genes. The fingerprints of the enrichment culturewere clearly dominated by a 16S rRNA gene sequence forminga 513-bp terminal restriction fragment (T-RF). One further,inferior 207-bp T-RF was also consistently detected with bothgrowth substrates (Fig. 2). Based on cloning and sequencing of16S rRNA gene sequences, the 513-bp T-RF was clearly as-signed to an unidentified member of the Deltaproteobacteria(Fig. 3). The corresponding 16S rRNA gene is most closelyrelated (99.5% sequence similarity) to an environmental se-quence detected in a sample from a subsurface acid minedrainage system (GenBank accession number AY082457) butis also more distantly related to the sequence from the nextcultivated relative, Desulfobacterium cetonicum (GenBank ac-cession number AJ237603), with 92.9% sequence similarity,which is usually considered to be beyond the genus level. Thedominant phylotype in the enrichment culture N47 is relatedonly distantly to the naphthalene-degrading strains NaphS2and NaphS3 (with 87% sequence similarity to NaphS2) (24,48). Of the sequences in the clone library, 93% (27 of 29)

FIG. 2. T-RFLP analysis of bacterial 16S rRNA gene sequencesamplified from the enrichment culture N47 grown on naphthalene(A) and 2-methylnaphthalene (B). The lengths of major T-RFs areindicated.

FIG. 3. Phylogenetic tree reflecting the relationships of 16S rRNA gene sequences identified in the naphthalene-grown enrichment culture N47to selected sequences of Deltaproteobacteria and Spirochaetes. Sequences obtained from culture N47 are listed in bold. n indicates the number ofidentified sequences in the clone library. A selection of 16S rRNA genes representing selected lineages of Bacteria and Archaea was used as theout-group. The bar indicates 10% estimated sequence divergence. TCE, trichloroethene; BTEX, benzene, toluene, ethylbenzene, and xylene; TCB,trichlorobenzene.


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belonged to this deltaproteobacterial lineage. The remaining7% of the clone sequences (2 of 29) represented the 207-bpT-RF, belonging to members of the Spirochaetes. This se-quence type showed 100% sequence identity to the 16S rRNAgene of the spirochaetal isolate SA-8 (GenBank accessionnumber AY695839) (Fig. 3). Archaeal 16S rRNA gene se-quences were not detectable in the naphthalene-grown culturevia standard PCR assays (data not shown). The enrichmentculture N47 is capable of growing with nonaromatic hydrocar-bons like acetate, glucose, and pyruvate. Nevertheless, growthwith nonaromatic hydrocarbons results in a shift of the domi-nant microbial community, as detected by 16S rRNA-basedT-RFLP analyses (data not shown). Consequently, the enrich-ment culture N47 contains additional strains which were lessrepresented when the culture was grown with naphthalene.

Genes coding for the naphthyl-2-methyl-succinate synthase.In order to correlate the identified peptides to sequence infor-mation for the culture N47, the whole genome of the culturewas sequenced. Because of repeated sequence elements, gapsin the genome of the dominant deltaproteobacterium couldnot be completely closed. Any efforts to close the gaps byapplying different methods remained unsuccessful. While onecopy of the deltaproteobacterial 16S rRNA gene sequence wasdetected in the genome, no 16S rRNA gene sequences relatedto spirochaete sequences were identified. In the present study,two contigs harboring genes involved in 2-methylnaphthalenedegradation are described. The phylogenetic comparison ofuniversal COGs located on these contigs confirmed that thecontigs belong to the Deltaproteobacteria and not to the Spiro-chaetes.

The MS analysis of the whole proteome of 2-methylnaph-thalene-grown N47 cells led to the identification of 629 pro-teins that could all be mapped to the genome. The draft ge-nome of culture N47 contains, at this writing, 4,755 putativeprotein coding genes. Thus, the identified proteins cover about13% of the total putative proteome. Since the analysis wasperformed with a draft genome and not all genes are expressedunder any given growth conditions, the coverage of the pre-dicted proteome is within common ranges observed for otherorganisms. The number of peptides matching an identifiedprotein and the total coverage of the protein are listed in Table1. Among the corresponding genes, we identified several se-quences that are highly similar to those of the bss and bbsoperons involved in anaerobic toluene degradation and to therecently identified nmsA gene, encoding the �-subunit of thenaphthyl-2-methyl-succinate synthase (Nms) (48). We assignedthese genes the function of coding for enzymes catalyzing theconversion of 2-methylnaphthalene to 2-naphthoyl-CoA (Ta-ble 1). Moreover, further genes that are similar to bss/bbsgenes were not present in the genome. As the first enzyme ofthe degradation pathway, we identified the �-, �-, and �-sub-units of Nms, which catalyzes the addition of fumarate to themethyl group of 2-methylnaphthalene. The Nms �-subunit(NmsA) of culture N47 displays 92% amino acid sequencesimilarity to the putative NmsA of the deltaproteobacteriumNaphS6 (GenBank accession number CAO72222) (48) (Fig.4). NmsA sequences are phylogenetically related to the �-sub-unit of the benzylsuccinate synthase (BssA; �50% sequenceidentity), which catalyzes the first step of anaerobic toluenedegradation, and to the 1-methylalkyl-succinate synthase

(MasG/AssA), which activates n-alkanes by fumarate addition.The nmsA gene product of the enrichment culture N47 harborsa glycyl residue in the characteristic amino acid RVXG motifof glycine radical enzymes at amino acid position 803 and theconserved cysteine residue at position 467 (Fig. 5). NmsAsequences are clearly distinguished from those of BssA andMasG/AssA by containing an isoleucine (I) instead of a valine(V) residue at the active site (Fig. 5).

Another protein has been detected in protein extracts from2-methylnaphthalene-grown N47 cells which was distantly re-lated to the �-subunit of Bss identified in a toluene-degradingconsortium (33% sequence similarity; GenBank accessionnumber ABO30981) (60). We therefore designated the polypep-tide NmsB. Within the nms operon, the coding gene nmsB islocated upstream of nmsA (Fig. 6). Downstream of nmsA is asequence which we assigned as the gene for the �-subunit ofNms (NmsC). Moreover, a putative Nms-activating enzyme,NmsD, was identified. Based on sequence similarities, the se-quence most related to NmsD is that of the putative 1-methyl-alkyl-succinate synthase activase (MasG) of Azoarcus sp. strainHxN1 (37% sequence similarity; GenBank accession numberCAO03077) (25), which is involved in anaerobic n-alkane deg-radation. The gene encoding NmsD (nmsD) is located up-stream of the nmsB gene. Further upstream, beyond a largeintergenic region of 884 bp, is a gene (nmsF) that is similar tobssF of “Aromatoleum aromaticum” EbN1 (49). The geneproduct could not be reproducibly detected in protein extractsfrom 2-methylnaphthalene-grown N47 cells. The derivedamino acid sequence showed no similarity to known proteins,rendering it impossible to predict a potential function. Furtherupstream of nmsF, seven additional genes that are most similarto orthologs in A. aromaticum EbN1 (Table 1) are organized.Products of three of these genes (open reading frame 1[ORF1], ORF6, and ORF7) are most similar to enzymes in-volved in butanoate metabolism.

Genes encoding enzymes for the beta-oxidation of naphthyl-2-methyl-succinate. In the 2-methylnaphthalene degradationpathway, naphthyl-2-methyl-succinate is converted via beta-oxidation reactions to 2-naphthoyl-CoA (46). So far, somebiochemical reactions of the pathway are well described butthe enzymes and the respective genes are still enigmatic. Inprotein extracts from 2-methylnaphthalene-grown cells, weidentified peptides related to enzymes that catalyze similarreactions in anaerobic toluene degradation. We therefore as-signed these to enzymes necessary to perform the reactionsconverting naphthyl-2-methyl-succinate to 2-naphthoyl-CoAand succinyl-CoA, and we successfully identified the corre-sponding genes from the genome. In analogy to genes foranaerobic toluene degradation, we named the genes coding forthe beta-oxidation of naphthyl-2-methyl-succinate to 2-naph-thoyl-CoA bns (beta-oxidation of naphthyl-2-methyl-succi-nate). The bns operon consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb (Fig. 6). Orthologs of the geneproducts have been identified in the denitrifying organisms A.aromaticum EbN1, Thauera aromatica, and Azoarcus sp. strainT, with amino acid similarities ranging from 44 to 78%, makingthe prediction of functions for the gene products possible (Ta-ble 1). The bnsH gene most likely codes for a putative naph-thyl-2-methylene-succinyl-CoA hydratase, bnsG for naphthyl-2-methyl-succinyl-CoA dehydrogenase, bnsEF for the subunits


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of naphthyl-2-methyl-succinate CoA transferase, bnsCD forthe subunits of naphthyl-2-hydroxymethyl-succinyl-CoA dehy-drogenase, and bnsAB for the subunits of naphthyl-2-oxom-ethyl-succinyl-CoA thiolase. Four further genes are positionedbetween the nms and bns genes. The products of ORF15 andORF16 display high similarities to the �- and �-subunits ofelectron-transferring flavoproteins (ETFs).

Genes coding for enzymes of dearomatization and ringcleavage of 2-naphthoyl-CoA. Subsequent reactions of the2-naphthoyl-CoA degradation pathway include reductivedearomatization, hydrolytic ring cleavage, and beta-oxidationto yield acetyl-CoA and CO2. The combined proteome andgenome analyses of the enrichment culture N47 have givenaccess to the coding genes for a putative 2-naphthoyl-CoAreductase that catalyzes the dearomatization of the ring struc-ture distal to the carboxyl group (4). During growth of theculture N47 with 2-methylnaphthalene, four subunits of a pu-tative 2-naphthoyl-CoA reductase were expressed that weremost similar to the benzoyl-CoA reductase subunits of Azoar-cus sp. strain CIB (43) (Table 1). In analogy to genes foranaerobic benzoyl-CoA degradation, the genes coding for2-naphthoyl-CoA reductase were designated ncr (2-naphthoyl-CoA reductase). The ncr gene cluster contains the genesncrCBAD, which code for the � (NcrC)-, � (NcrB)-, � (NcrA)-,and � (NcrD)-subunits of the 2-naphthoyl-CoA reductase(Ncr) and which correspond to a sequence length of 4.2 kb(Fig. 7). In the subsequent reactions, the product of 2-naph-thoyl-CoA reductase undergoes beta-oxidation and hydrolyticcleavage of the ring system, leading to the formation of acetyl-CoA and CO2. Downstream of the ncr gene cluster are 16additional genes (ORF33 to ORF48) that were identified byusing protein extracts from 2-methylnaphthalene-grown N47cells and that may code for candidate enzymes that catalyzethese reactions (Table 1; Fig. 7). The subsequent metabolicpathway is hypothetical, therefore, and the roles of these ORFscannot be specified.

The metagenome of culture N47 was screened for additionalputative genes involved in anaerobic degradation of aromatichydrocarbons. Genes coding for the �-, �-, and �-subunits of4-hydroxybenzoyl-CoA reductase involved in anaerobic phenoldegradation were organized adjacent to one another on onecontig. In addition, genes coding for two �-subunits and forone �-subunit of 4-hydroxybenzoyl-CoA reductase were foundscattered throughout the genome. Three genes encoding pu-tative subunits of the 3-octaprenyl-4-hydroxybenzoate carbox-ylase were also present in the N47 genome. Besides genes foranaerobic phenol degradation, the genome contained threegenes for additional �-, �-, and �-subunits of benzoyl-CoAreductase that were distributed throughout the genome. Ex-cept for the three 3-octaprenyl-4-hydroxybenzoate carboxylasegenes, none of the additional genes were found to be expressedin 2-methylnaphthalene-grown cells.

DISCUSSION

Phylogenetic characterization of the enrichment cultureN47. So far, a small number of sulfate-reducing naphthalene-oxidizing bacteria have been brought into culture. The delta-proteobacterial strains NaphS2 and NaphS3 were isolatedfrom marine sediments and phylogenetically belong to theO

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Desulfobacteraceae (24, 48). 16S rRNA gene sequences closelyrelated to the phylotype of NaphS2 were also identified ashighly abundant in PAH-contaminated marine sediments (26),demonstrating that similar sulfate-reducing and maybe naph-thalene-degrading bacteria are present in contaminated ma-rine harbor sediments of geographically distinct sites such asthe North Sea and the Pacific Ocean.

Culture N47 was enriched almost 10 years ago, and all effortsto obtain a pure culture were not successful until now. Sur-prisingly, members of the Spirochaetes are stable members ofthe consortium. This fact may hint toward a so far unknownnecessity for a spirochaetal partner organism in the enrichmentculture to sustain biodegradation. However, the exact effect ofthese Spirochaetes on the growth of the sulfate-reducing keydegrader remains to be elucidated. Potentially, the spiro-

chaetal bacterium releases a compound or vitamin needed bythe sulfate-reducing Desulfobacterium.

In addition, the enrichment culture N47 is able to grow with2-methylnaphthalene as the sole source of carbon. Based onT-RFLP analysis, we could show that the bacterial communitystructure does not change when the enrichment culture isgrowing on either 2-methylnaphthalene or naphthalene. Thecapability to metabolize 2-methylnaphthalene is a generalproperty of all anaerobic naphthalene-degrading bacteria in-vestigated to date (24, 48, 54).

Genes coding for the naphthyl-2-methyl-succinate synthase.In the present study, a combined genomic and proteomic studywas applied to analyze the expression of genes involved inanaerobic 2-methylnaphthalene degradation. In 2-methylnaph-thalene-grown cultures, proteins that were similar to the four

FIG. 4. Phylogenetic tree showing the relationships of the N47 NmsA enzyme to available pure-culture BssA enzymes and homologousfumarate-adding enzymes based on amino acid sequences. The tree was constructed by quartet puzzling. Numbers at nodes show branchingconfidence values deduced from 10,000 intermediate trees. GenBank accession numbers of available reference sequences are indicated. The scalebar represents 10% sequence divergence.

��HWALVLCMAPGVGK.....EPEKWQSLIVRIAGYSARF�� NmsA N47��HWALVLCMAPGVGK.....EPEKWQSMIVRIAGYSARF�� HWALVLCMAPGVGK.....EPEKWQSMIVRIAGYSARF�� HWALVLCMAPGVSK.....EPEKWESLIVRIAGYSARF�� TWVHMACMSPNPTT.....DPEGYQEVIVRVAGYSAHF�� Azoarcus��TWVHQACMSPCPTT.....DPEKYSEVIVRVAGYSAHF�� TWVHQACMSPCPTT.....DPEKYSEVIVRVAGYSAHF �� YWVNVLCMAPGVAG.....EPEKHQDLIVRVSGFSARF�� A. aromaticum�� YWVNVLCMAPGLAG.....EPEKHHDLIVRVSGYSARF�� T. aromatica�!��NWVNVLCMSPGIHG.....EPEKHSDLIVRVSGYSARF 833 ��A.��! 480�NWANVLCMSPGLCG.....EPEKHQDLIVRVSGFSSRF�� BssA D. toluolica TOL��NWVNVLCMSPGLAG.....EPEKHHDLIVRVSGYSARF�� Magnetosp.��! ��YWGLVLCMSPGVCG.....EPEKHQDLIVRVSGFSARF�� G. metallireducens

FIG. 5. Partial alignment of the amino acid sequence of NmsA from the enrichment culture N47 with those of other NmsA, MasG/AssA, andBssA enzymes. The depicted alignment site contains the catalytic active cysteine and glycine residues with the characteristic sequence motif forglycine radical enzymes (shown in bold). Numbers refer to amino acid positions in the respective proteins. A. sp. T, Azoarcus sp. strain T;Magnetosp., Magnetospirillum.


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subunits and the activating enzyme of the benzylsuccinate syn-thase (Bss) were expressed. As the initial activation reactionsof toluene and 2-methylnaphthalene degradation are similar(53), the predicted functions of the corresponding genes can becorrelated to enzymes of anaerobic 2-methylnaphthalene deg-radation. Analogous to the genes for Bss, the nms genes codefor a putative large subunit (�; 96 kDa) and two putative smallsubunits (� and �; 7.9 and 7.8 kDa) of Nms. The large subunitsof Nms enzymes from N47 and NaphS2 had a high degree ofidentity and were more distantly related to those of Bss (Fig.4). In addition, the phylogenetic affiliation supports the clus-tering of �-subunit sequences based on specificities for sub-strates (2-methylnaphthalene, toluene, or n-alkanes) describedearlier (48). Therefore, we conclude that Nms constitutes anovel subgroup of glycine radical enzymes. This finding is alsosupported by an apparently NmsA-specific RIXG amino acidsequence motif instead of the motif RVXG, which had so farbeen assumed to be conserved in all glycine radical enzymes.As the exchange of a valine for an isoleucine residue is con-sistent in all known NmsA enzymes, it may play a role insubstrate specificity. In addition to this sequence motif, NmsAharbors the characteristic cysteinyl residue, which is involved inradical formation (40), at position 467. The electron of theglycyl radical is transferred to the cysteinyl residue, and a thioyl

radical is formed, which then probably abstracts a hydrogenatom from the methyl group of 2-methylnaphthalene (27).

Besides nmsA, the gene cluster contains two further ORFswhich code for putative �- and �-subunits of Nms. Homologysearching in public databases revealed weak identity (33%)between the putative NmsB enzyme and a BssB enzyme re-trieved from a toluene-degrading methanogenic enrichmentculture (60). As the identity value is very low and no similar-ities to other known BssB enzymes were present, it is ques-tionable whether the ORF really codes for NmsB. The nmsCgene, located downstream of nmsA, encodes a putative �-sub-unit of Nms. Like BssC from T. aromatica (40), NmsC fromculture N47 is rich in cysteines and charged amino acids, whichtogether account for 47% of the total amino acids. Althoughthe roles of the two small subunits cannot be concluded be-cause the functions of the corresponding Bss subunits are notyet clarified, it has been demonstrated previously by gene in-activation experiments that the small subunits of Bss are es-sential for Bss activity (1, 18, 42). Thus, we can assume thatNmsB and NmsC may be necessary for the proper activity ofNms in culture N47.

As part of the nms gene cluster, the gene nmsD encodes theputative naphthyl-2-methyl-succinate-activating enzyme, an S-adenosylmethionine (SAM) radical enzyme, which catalyzes

FIG. 6. Organization of the nms and bns gene clusters of anaerobic 2-methylnaphthalene catabolism in the sulfate-reducing culture N47 incomparison with those of the bss and bbs genes for anaerobic toluene degradation in Magnetospirillum sp. strain TS-6 (57), Azoarcus sp. strain T(1), T. aromatica strain K172 (28, 39), T. aromatica strain T1 (19), A. aromaticum strain EbN1 (35), and G. metallireducens strain GS15 (11). Genesare depicted as arrowheads. Annotation data for nms and bns genes are provided in Table 1. Genes for anaerobic toluene degradation are asfollows: bssABCD, encoding benzylsuccinate synthase and the activating enzyme; bssE, encoding a putative chaperone; bssH, encoding a putativetransporter; bbsEF, encoding succinyl-CoA:(R)-benzylsuccinate CoA-transferase; bbsG, encoding (R)-benzylsuccinyl-CoA dehydrogenase; bbsH,encoding phenylitaconyl-CoA hydratase; bbsCD, encoding 2-[hydoxy(phenyl)methyl]-succinyl-CoA dehydrogenase; bbsAB, encoding benzoylsuc-cinyl-CoA thiolase; and bssF, bssG, bbsI, and bbsJ, encoding hypothetical proteins.

FIG. 7. Scale model of the organization of the ncr genes coding for the 2-naphthoyl-CoA reductase and additional genes coding for putativeenzymes catalyzing ring cleavage and beta-oxidation, leading to the formation of acetyl-CoA and CO2. Enzymes were detected in protein extractsfrom 2-methylnaphthalene-grown N47 cells. Symbols for the genes encoding the four subunits of 2-naphthoyl-CoA reductase are highlighted ingray. The scale indicates the nucleotide position on the DNA contig.


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glycyl radical formation by abstracting a hydrogen atom fromthe reactive glycine. The orthologous proteins BssD and MasGcontain three conserved, cysteine-rich sequence motifs in theN-terminal region (36, 58). Reflected also in culture N47,NmsD contains the sequence C32LLNCAWC, correspondingto the specific CX3CX2C motif characteristic of SAM radicalenzymes which coordinates a [Fe4S4] cluster. In addition, theconserved sequence motif CX2CX2CX3C, a typical [Fe4S4]ferredoxin motif, is present as C58VRCGTCVAAC and C100

TLCMKCVDVC in NmsD from culture N47. For the bssoperon, it could be shown that the activase coding gene isalways located upstream of the genes coding for the threesubunits of Bss (56). In the nms gene cluster of N47, analogousgene organization is present.

Four additional genes (ORF13 to ORF16) are located be-tween the nms and bns gene clusters of culture N47. Two ofthem, ORF15 and ORF16, code for the �- and �-subunits of aputative ETF. The ETF may possibly serve as an electronacceptor for the reaction with naphthyl-2-methyl-succinyl-CoAdehydrogenase (41), which can be replaced by phenazinemethosulfate in in vitro enzyme tests (53). A third gene,ORF14, codes for a protein that contains iron-sulfur clustersand thus may also be involved in the electron transfer reaction.Interestingly, this gene organization is similar to the arrange-ment of the bss gene cluster in the deltaproteobacterial ironreducer Geobacter metallireducens, whereas the genetic orga-nization within the betaproteobacterial denitrifiers T. aro-matica and A. aromaticum EbN1 is distinct (35). The similarityof the gene organization in N47 to that in G. metallireducens isalso obvious from the arrangement of the bbs operon in G.metallireducens, in which no sequences coding for BssI andBssJ are present. Genes coding for the corresponding prod-ucts, with so far unknown functions, are also absent from thebns operon in culture N47 (Fig. 6).

A putative transcriptional regulator (ORF25) is locateddownstream of the bns gene cluster and is transcribed in theopposite direction. The N-terminal region of the regulatorprotein contains an XylR domain that is significant for �54-dependent transcriptional activators, including those for an-aerobic phenol degradation (10). However, we could notobserve expression of ORF25 during growth on 2-methyl-naphthalene.

The nms/bns gene cluster is flanked by genes coding for areverse transcriptase and a transposase. Upstream, a gene en-codes a retron-type reverse transcriptase that exhibits 67%amino acid sequence similarity to a reverse transcriptase fromDesulfotomaculum acetoxidans (GenBank accession numberZP_04352877). The transposase encoded by the gene down-stream of the nms/bns gene cluster is characterized by an in-tegrase core domain closely related to the transposase ofDethiosulfovibrio peptidovorans (GenBank accession numberZP_04341313). The presence of these genes indicates geneticmobility of the nms/bns operons and may hint at a putativehorizontal gene transfer event in the evolution of aromatichydrocarbon degraders. The pattern in which transposase orintegrase coding genes are present with gene clusters for aro-matic degradation is widely distributed and occurs, e.g., in A.aromaticum EbN1 (14, 49). Respective events of lateral genetransfer also have been interpreted from previous phylogeneticanalyses of bssA gene relationships (57, 63).

Genes coding for enzymes involved in dearomatization andring cleavage of 2-naphthoyl-CoA. In 2-methylnaphthalene-grown N47 cells, we could identify peptides similar to enzymesof the anaerobic benzoyl-CoA degradation pathway, which ini-tiates ring cleavage by reducing the aromatic ring of benzoyl-CoA. Genes similar to bamB to bamI, encoding reductivelydearomatizing enzymes of the G. metallireducens type, werenot present in the genome of N47 (64). Recently, it was dem-onstrated that 5,6,7,8-tetrahydro-2-naphthoyl-CoA is formedas a major metabolite during anaerobic 2-methylnaphthalenedegradation (4). Therefore, it was concluded that further deg-radation of 2-naphthoyl-CoA proceeds via reduction of thebicyclic ring system to cyclohexanoic compounds and not viathe monoaromatic benzoyl-CoA. Indeed, the reduced metab-olite octahydro-2-naphthoyl-CoA was detected in naphtha-lene-grown N47 cultures (46). As the genome of culture N47does not contain further gene clusters similar to that for ben-zoyl-CoA reductase, we correlate the identified proteins withthe enzyme naphthoyl-CoA reductase of the 2-methylnaphtha-lene pathway.

Based on amino acid sequence similarities, the 2-naphthoyl-CoA reductase of culture N47 is related to the Azoarcus type ofbenzoyl-CoA reductases (7). The reduction of benzoyl-CoA toa nonaromatic cyclic compound via an Azoarcus-like benzoyl-CoA reductase is catalyzed by an ATP-dependent two-electrontransfer to the aromatic ring via ferredoxin (43). The transferof electrons to the benzene ring requires electrons at a very lowredox potential (6). In Azoarcus evansii, ferredoxin serves asthe electron donor for the benzoyl-CoA reductase (21) and theoxidized ferredoxin is reduced by a 2-oxoglutarate:acceptoroxidoreductase in combination with a NADPH:acceptor oxi-doreductase. In Azoarcus sp. strain CIB, the genes encodingthe 2-oxoglutarate:acceptor oxidoreductase are not present inthe bzd gene cluster (43). Interestingly, like the correspondingoperon from Azoarcus sp. strain CIB, the bns operon fromculture N47 contains no genes that could code for a putative2-oxoglutarate:acceptor oxidoreductase. Genes for a putativeNADPH:acceptor oxidoreductase (ORF34) and ferredoxin(ORF33) are directly associated downstream of the ORFs cod-ing for the four subunits of the putative 2-naphthoyl-CoAreductase (ncrABCD). At the N-terminal part of the enzyme,the putative NADPH:acceptor oxidoreductase from cultureN47 harbors a conserved domain that is characteristic ofNADPH-ubiquinone oxidoreductase iron-sulfur-binding re-gions.

In benzoyl-CoA reductase, the �- and �-subunits contain theactive sites where electrons become ATP-dependently acti-vated. The aromatic compound becomes dearomatized at the�- and �-subunits (8). For 2-naphthoyl-CoA reductase, foursubunits were identified, whereas the �- and �-subunits arealso characterized by the presence of an Hsp70 class ATPasedomain. It was demonstrated earlier that similar �- and �-sub-units contain the ATP-binding sites of the acetate kinase/sugarkinase/Hsp70 actin family (29). As these subunits of naphthoyl-CoA reductase are also characterized by an ATP-binding site,ATP-dependent reduction of the aromatic ring can be pro-posed. However, ATP-dependent ring reduction has beenshown to occur only in facultative anaerobes but not in strictanaerobes like sulfate reducers (8). Due to the low energy gainfor sulfate reducers, it is unlikely that culture N47 has ATP-


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driven ring reduction. ORF27 is located upstream of the ncrgene cluster and codes for a putative TetR family transcrip-tional regulator.

Comparative sequence analyses showed that the genes cod-ing for 2-methylnaphthalene and 2-naphthoyl-CoA degrada-tion are related to those involved in anaerobic toluene andbenzoate metabolism. Nevertheless, they are apparently differ-ent enough to probably enable specific tracking of 2-methyl-naphthalene degradation genes in other bacterial cultures oreven environmental samples. Genes like nmsA and nmsD cod-ing for key enzymes of the 2-methylnaphthalene degradationpathway may be useful as functional marker genes in anthro-pogenically impacted environments in order to predict thepotential for natural attenuation processes for aromatic con-taminants. This approach would provide essential knowledgeof the abundance and diversity of PAH-degrading microorgan-isms, which have so far been insufficiently investigated becauseof the limited availability of appropriate molecular markers. Asan example, specific toluene degrader communities could becorrelated to distinct biogeochemical conditions within thedepth profile of a tar-oil-contaminated aquifer based on thedetection of bssA, the gene coding for the key enzyme ofanaerobic toluene degradation (63).

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