Combined Genomic and Proteomic Approaches Identify Gene ...

JOURNAL OF BACTERIOLOGY, Jan. 2010, p. 295–306 0021-9193/10/$12.00 doi:10.1128/JB.00874-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 192, No. 1

Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47䌤 Drazˇenka Selesi,1 Nico Jehmlich,2 Martin von Bergen,2,3 Frank Schmidt,4 Thomas Rattei,5 Patrick Tischler,5 Tillmann Lueders,1 and Rainer U. Meckenstock1* Institute of Groundwater Ecology, Helmholtz Zentrum Mu ¨ nchen—German Research Center for Environmental Health, Ingolsta ¨dter Landstrasse 1, D-85764 Neuherberg, Germany1; UFZ-Helmholtz Center for Environmental Research, Department of Proteomics, Permoserstrasse 15, D-04318 Leipzig, Germany2; UFZ-Helmholtz Center for Environmental 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 Universita ¨t Mu ¨nchen, 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 hydrocarbons naphthalene and 2-methylnaphthalene, with sulfate as the electron acceptor. Combined genome sequencing and liquid chromatography-tandem mass spectrometry-based shotgun proteome analyses were performed to identify genes and proteins involved in anaerobic aromatic catabolism. Proteome analysis of 2-methylnaphthalene-grown N47 cells resulted in the identification of putative enzymes catalyzing the anaerobic conversion of 2-methylnaphthalene to 2-naphthoyl coenzyme A (2-naphthoyl-CoA), as well as the reductive ring cleavage of 2-naphthoyl-CoA, leading to the formation of acetyl-CoA and CO2. The glycyl radical-catalyzed fumarate addition to the methyl group of 2-methylnaphthalene is catalyzed by naphthyl-2-methyl-succinate synthase (Nms), composed of ␣-, ␤-, and ␥-subunits that are encoded by the genes nmsABC. Located upstream of nmsABC is nmsD, encoding the Nms-activating enzyme, which harbors the characteristic [Fe4S4] cluster sequence motifs of S-adenosylmethionine radical enzymes. The bns gene cluster, coding for enzymes involved in beta-oxidation reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA, was found four intervening open reading frames further downstream. This cluster consists of eight genes (bnsABCDEFGH) corresponding to 8.1 kb, which are closely related to genes for enzymes involved in anaerobic toluene degradation within the denitrifiers “Aromatoleum aromaticum” EbN1, Azoarcus sp. strain T, and Thauera aromatica. Another contiguous DNA sequence harbors the gene for 2-naphthoyl-CoA reductase (ncr) and 16 additional genes that were found to be expressed in 2-methylnaphthalene-grown cells. These genes code for enzymes that were supposed to catalyze the dearomatization and ring cleavage reactions converting 2-naphthoyl-CoA to acetylCoA and CO2. Comparative sequence analysis of the four encoding subunits (ncrABCD) showed the gene product to have the closest similarity to the Azoarcus type of benzoyl-CoA reductase. The present work provides the first insight into the genetic basis of anaerobic 2-methylnaphthalene metabolism and delivers implications for understanding contaminant degradation. Numerous microorganisms that can degrade PAHs under aerobic conditions have already been identified, but only a small number of anaerobic cultures that degrade PAHs like naphthalene, 2-methylnaphthalene, and phenanthrene have been isolated so far (17, 20, 24, 31, 46–48, 50, 52, 66). It has been shown that these anaerobic degraders activate aromatic hydrocarbons by very unusual biochemical reactions which differ completely from those of aerobic degradation. The peripheral pathway of 2-methylnaphthalene degradation occurs in analogy to anaerobic toluene degradation by the addition of fumarate to the methyl group, catalyzed by the glycyl radical enzyme naphthyl-2-methyl-succinate synthase (Nms) (Fig. 1) (3). In subsequent reactions, naphthyl-2-methyl-succinate is activated to yield the coenzyme A (CoA) ester and oxidized to form naphthyl-2-methylene-succinyl-CoA. The following betaoxidation of the side chain results in the formation of 2-naphthoyl-CoA and succinate (3, 53). The first three enzyme reactions of this pathway have been measured in vitro (3, 53). Recently, Musat et al. (48) identified the gene coding for the

Polycyclic aromatic hydrocarbons (PAHs) are constantly released into the environment by anthropogenic activities such as industrial use or by accidental contamination. Due to the low chemical reactivity caused by the resonance energy of the aromatic ring structure and the low bioavailability of PAHs, they are persistent in the environment (15). The understanding of microbial metabolic capabilities in terms of anaerobic PAH degradation is in its infancy. However, natural amelioration of contaminated sites relies on the degradation capacities of microorganisms, and therefore, it is an essential prerequisite to broaden knowledge about the microorganisms involved and their potentials concerning PAH breakdown.

* Corresponding author. Mailing address: Institute of Groundwater Ecology, Helmholtz Zentrum Mu ¨nchen—German Research Center for Environmental Health, Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany. Phone: 49 (0) 89 3187 2561. Fax: 49 (0) 89 3187 3361. E-mail: [email protected]. 䌤 Published ahead of print on 23 October 2009. 295

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␣-subunit of a putative naphthyl-2-methyl-succinate synthase (nmsA) in 2-methylnaphthalene-grown bacterial cultures. The molecular composition of the nmsA gene is analogous to that of the benzylsuccinate synthase ␣-subunit gene (bssA). The Bss enzyme is a well-investigated close homolog of Nms, catalyzing fumarate addition in the initial reaction of anaerobic toluene degradation (34, 40). Based on findings from comparative sequence studies, glycine radical-catalyzed fumarate addition has been shown to be a widely distributed initial reaction mechanism for anaerobic hydrocarbon degradation involving toluene and 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 degradation pathway (7), 2-naphthoyl-CoA is subjected to aromatic ring reduction by a putative naphthoyl-CoA reductase, probably generating 5,6,7,8-tetrahydro-naphthoyl-CoA and further octahydro-2-naphthoic acid (4, 46). In the subsequent reactions, the ring system should be thiolytically cleaved and subjected to beta-oxidation, leading to the formation of acetyl-CoA and CO2. In contrast to the first enzymatic reaction in the degradation of methylated aromatics, the first enzymatic reaction in anaerobic degradation of unsubstituted aromatic compounds such as naphthalene is still unresolved. In order to determine the initial activation reaction of anaerobic naphthalene degradation, studies based on the analysis of metabolites have been performed. Zhang and Young (66) observed the incorporation of 13 C-labeled bicarbonate from the buffer into the carboxyl group of 2-naphthoic acid, hypothesizing that carboxylation is the initial activation reaction of anaerobic naphthalene degradation in the culture studied. Recently, Safinowski and Meckenstock (54) identified the deuterated metabolites naphthyl-2methyl-succinate and naphthyl-2-methylene-succinate, which are exclusive intermediates of anaerobic 2-methylnaphthalene degradation, in the enrichment culture N47 when the culture was cultivated on fully deuterated naphthalene. Moreover, specific enzyme activities of the anaerobic 2-methylnaphtahlene degradation pathway have been detected in naphthalenegrown cells (54). Therefore, methylation of naphthalene to yield 2-methylnaphthalene as the initial activation reaction and subsequent degradation via the 2-methylnaphthalene pathway were proposed for this bacterial culture. The elucidation of 2-methylnaphthalene degradation may therefore reveal an important part of the naphthalene degradation pathway. However, Musat et al. (48) questioned methylation as the first reaction in naphthalene degradation for their marine naphthalene-degrading deltaproteobacterial NaphS strains. Whereas molecular components involved in anaerobic degradation of monoaromatic hydrocarbons are well known, knowledge about genes and enzymes involved in anaerobic PAH degradation is still missing (14). Here, we provide the first results of a whole-proteome- and whole-genome-based investigation of the sulfate-reducing enrichment culture N47

FIG. 1. Proposed pathway for anaerobic 2-methylnaphthalene degradation 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 synthase; BnsEF, naphthyl-2-methyl-succinate CoA transferase; BnsG, naphthyl-2-methyl-succinyl-CoA dehydrogenase; BnsH, naphthyl-2-

methylene-succinyl-CoA hydratase; BnsCD, naphthyl-2-hydroxymethylsuccinyl-CoA dehydrogenase; BnsAB, naphthyl-2-oxomethyl-succinylCoA thiolase; and NcrABCD, 2-naphthoyl-CoA reductase. The position 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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GENES FOR ANAEROBIC 2-METHYLNAPHTHALENE DEGRADATION

degrading naphthalene and 2-methylnaphtalene. We have identified some gene clusters encoding enzymes involved in 2-methylnaphthalene degradation, 2-naphthoyl-CoA dearomatization, and subsequent ring cleavage reactions in 2-methylnaphthalene-grown N47 cells. MATERIALS AND METHODS Cultivation of the enrichment culture N47. The sulfate-reducing culture N47 was enriched with naphthalene, obtained from contaminated sediment collected at a former coal gasification site, as a carbon source and cultivated as reported previously (53). The culture is able to grow with the aromatic hydrocarbons naphthalene, 2-methylnaphthalene, 2-naphthoic acid, para-cresol, phenol, benzaldehyde, and 3-hydroxybenzaldehyde as sole sources of carbon. Neither benzene nor toluene can be utilized. Additionally, growth of the microorganisms in the culture occurred with the nonaromatic organic compounds glucose, pyruvate, and acetate. Besides SO42⫺, 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-Aldrich, Steinheim, Germany) to the cultivation bottles after autoclaving. Cultures in 1:10 dilutions were inoculated into the bottles. Substrate utilization was monitored by colorimetric measurement of sulfide (16). 16S rRNA gene sequence analysis and phylogenetic affiliations of microorganisms in the enrichment culture N47. Cells were harvested by centrifugation for 20 min at 3,300 ⫻ g and washed with 0.5⫻ phosphate-buffered saline. Genomic DNA from naphthalene- and 2-methylnaphthalene-grown cells was extracted with the FastDNA spin kit for soil according to the protocol of the manufacturer (MP Biomedicals, Illkirch, France). For terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene sequences from two 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). Amplification, cloning, and sequencing of almost-full-length bacterial 16S rRNA gene sequences were performed with DNA extracted from naphthalene-grown cells with the primer set Ba27f-Ba1492r (61) as described previously (55). Archaeal 16S RNA gene sequences were amplified from extracted DNA with the primer set Ar109f and Ar912r (45). The 16S rRNA gene sequences were manually assembled, checked for quality by using the SeqMan II software module (Lasergene 6 suite; DNASTAR, Madison, WI), and tested for chimerical structures by using the Chimera Check analysis function of Ribosomal Database Project II (http://rdp.cme.msu.edu/). Phylogenetic analysis of the 16S rRNA sequences was performed with the ARB software package (http://www.arb-home.de) (44). Alignments were checked visually. Phylogenetic analyses based on nucleotide sequences were performed, and results were verified by applying maximum likelihood, maximum parsimony, and neighbor-joining methods using the respective tools in the ARB software package. Proteome analysis. After several transfers of culture N47 grown on 2-methylnaphthalene, an 800-ml culture sample that accumulated 2.5 mM sulfide as a growth 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, and resuspended in a mixture of 400 ␮l lysis buffer {9 M urea, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol; GE Healthcare Europe GmbH, Freiburg, Germany} and 67 ␮l of a 7⫻ stock solution containing a Complete EDTA-free mini-protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Penzberg, Germany). After 30 min of incubation at room temperature, the cell-buffer mixture was transferred into a lysing matrix B tube (MP Biomedicals) and processed for 35 s at speed 6.0 in a FastPrep instrument (MP Biomedicals). The homogenized solution was centrifuged for 2 min at 20,000 ⫻ g and 4°C. The supernatant was treated for 30 min at the ambient temperature with 3 ␮l nuclease mix (GE Healthcare) and centrifuged for 1 h at 15,000 ⫻ g and 4°C. Estimation of the protein level in the supernatant was performed with the two-dimensional Quant kit according to the protocol of the manufacturer (GE Healthcare). For mass spectrometric analysis of peptides, a 30-␮g sample of proteins extracted from 2-methylnaphthalene-grown N47 cells was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (37) and visualized by Coomassie brilliant blue staining (65). The complete one-dimensional gel lanes containing the separated proteins were cut into 24 equal pieces, which were washed two times with 50% methanol–5% acetic acid (vol/vol) and digested with trypsin (Sigma). After digestion, peptides were concentrated and desalted using ZipTip C18 columns (Millipore, Bedford, MA). The separation of the complex peptide solutions was achieved by reversed-phase chromatography with a Pep-

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Map column (internal diameter, 75 ␮m; length, 250 mm [LC Packings]) operated on a nano-high-performance liquid chromatography system (Agilent Technologies, Waldbronn, Germany) with a nonlinear 90-min gradient using 2% acetonitrile 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 gradient settings 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 trap quadrupole Orbitrap (LTQ Orbitrap) mass spectrometer (ThermoElectron, Bremen, Germany) equipped with a nano-electrospray ionization source. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap mass spectrometry (MS) and LTQ tandem MS (MS/MS) acquisition. Survey full-scan MS spectra (from m/z 300 to 2,000) were acquired by using the Orbitrap with a resolution power of 60,000 at m/z 400 (after accumulation to a target of 1,000,000 charges in the LTQ). The method employed allowed sequential isolation of the most intense ions—up to six, depending on the signal intensity—for fragmentation on the linear ion trap using collision-induced dissociation at a target value of 100,000 charges. Target ions already selected for MS/MS were dynamically excluded for 60 s. General MS conditions were an electrospray voltage of 1.5 kV, no sheath, and auxiliary gas flow. The ion selection threshold for MS/MS was 500 counts, and an activation Q-value (effective collision energy) of 0.25 and an activation time of 30 ms were also 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 (version 27, revision 11; ThermoFinnigan, San Jose, CA). Sequest was set up to search the N47 genome database (4,755 entries), assuming digestion by trypsin. Sequest was searched with a precursor ion tolerance of 20 ppm and a fragment ion mass tolerance of 1.00 Da. The oxidation of methionine and the carbamidomethylation of cysteine residues were specified in Sequest as variable modifications. Peptide identifications were accepted if the data exceeded specific database search engine thresholds. Sequest identifications required deltaCn scores of greater 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 identifications were accepted if the proteins contained at least two identified peptides. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Sequencing and annotation of the genomic information from the enrichment culture N47. Isolation of genomic DNA from a 400-ml naphthalene-grown culture was performed with the Wizard genomic DNA purification kit by following the protocol of the manufacturer (Promega, Madison, WI). Whole-genome information for the enrichment culture N47 was obtained by combining 454pyrosequencing and Sanger sequencing of plasmid libraries by Roche GmbH (Penzberg, Germany) and AGOWA GmbH (Berlin, Germany). The automated assembly of the sequences was checked manually. Automated annotation of the assembled sequences was performed using the PEDANT (PubMed identification number [PMID] 18940859) software system, and predicted coding sequences were identified with GeneMark (5) (PMID 18428700). Coding sequences were automatically assigned by PEDANT to functional categories according to the functional role catalogues FunCat (PMID 15486203) and Gene Ontology (PMID 14681407). The two contigs were searched for taxonomic markers corresponding to the 50 most universal bacterial clusters of orthologous groups (COGs) of proteins in the eggNOG database (30) by using BLAST (2) (E value ⱕ 1E⫺10). This search resulted in the identification of 27 marker genes. To confirm that the two contigs carrying the 2-methylnaphthalene degradation genes belong to N47, we calculated neighbor-joining phylogenies for the COG sequences that are present on each contig (using MUSCLE [22] and NEIGHBOR [23] with default parameters). Phylogenetic analysis of the ␣-subunit sequence of naphthyl-2-methyl-succinate synthase. A phylogenetic tree was reconstructed from all publicly available data for pure-culture benzylsuccinate synthases and homologous gene operons by using amino acid alignment in ARB (44). We used the quartet puzzling algorithm (59) as implemented in ARB with 10,000 puzzling steps, the JonesTaylor-Thornton model of substitution, and a uniform model of rate heterogeneities. A positional filter was generated from the data set to include only amino acid positions covered for all sequences (256 positions) for tree inference. Tree topology and branching orders were also verified using Fitch distance matrix and neighbor-joining algorithms as described previously (63). Nucleotide sequence accession numbers. All 16S rRNA gene sequences were deposited in GenBank (http://www.ncbi.nlm.nih.gov/) under accession numbers GU080088 and GU080089. All other sequence data from this study were deposited in GenBank under accession numbers GU080116 to GU080137 and GU080090 to GU080115.

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

RESULTS 16S rRNA gene sequence analysis of the enrichment culture N47. The main bacterial members of the enrichment culture grown with naphthalene or 2-methylnaphthalene as the carbon

source were assessed by T-RFLP analyses and sequencing of 16S rRNA genes. The fingerprints of the enrichment culture were clearly dominated by a 16S rRNA gene sequence forming a 513-bp terminal restriction fragment (T-RF). One further, inferior 207-bp T-RF was also consistently detected with both growth substrates (Fig. 2). Based on cloning and sequencing of 16S rRNA gene sequences, the 513-bp T-RF was clearly assigned to an unidentified member of the Deltaproteobacteria (Fig. 3). The corresponding 16S rRNA gene is most closely related (99.5% sequence similarity) to an environmental sequence detected in a sample from a subsurface acid mine drainage system (GenBank accession number AY082457) but is also more distantly related to the sequence from the next cultivated relative, Desulfobacterium cetonicum (GenBank accession number AJ237603), with 92.9% sequence similarity, which is usually considered to be beyond the genus level. The dominant phylotype in the enrichment culture N47 is related only distantly to the naphthalene-degrading strains NaphS2 and NaphS3 (with 87% sequence similarity to NaphS2) (24, 48). Of the sequences in the clone library, 93% (27 of 29)

FIG. 3. Phylogenetic tree reflecting the relationships of 16S rRNA gene sequences identified in the naphthalene-grown enrichment culture N47 to selected sequences of Deltaproteobacteria and Spirochaetes. Sequences obtained from culture N47 are listed in bold. n indicates the number of identified sequences in the clone library. A selection of 16S rRNA genes representing selected lineages of Bacteria and Archaea was used as the out-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 remaining 7% of the clone sequences (2 of 29) represented the 207-bp T-RF, belonging to members of the Spirochaetes. This sequence type showed 100% sequence identity to the 16S rRNA gene of the spirochaetal isolate SA-8 (GenBank accession number AY695839) (Fig. 3). Archaeal 16S rRNA gene sequences were not detectable in the naphthalene-grown culture via standard PCR assays (data not shown). The enrichment culture N47 is capable of growing with nonaromatic hydrocarbons like acetate, glucose, and pyruvate. Nevertheless, growth with nonaromatic hydrocarbons results in a shift of the dominant microbial community, as detected by 16S rRNA-based T-RFLP analyses (data not shown). Consequently, the enrichment culture N47 contains additional strains which were less represented 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 information for the culture N47, the whole genome of the culture was sequenced. Because of repeated sequence elements, gaps in the genome of the dominant deltaproteobacterium could not be completely closed. Any efforts to close the gaps by applying different methods remained unsuccessful. While one copy of the deltaproteobacterial 16S rRNA gene sequence was detected in the genome, no 16S rRNA gene sequences related to spirochaete sequences were identified. In the present study, two contigs harboring genes involved in 2-methylnaphthalene degradation are described. The phylogenetic comparison of universal COGs located on these contigs confirmed that the contigs belong to the Deltaproteobacteria and not to the Spirochaetes. The MS analysis of the whole proteome of 2-methylnaphthalene-grown N47 cells led to the identification of 629 proteins that could all be mapped to the genome. The draft genome of culture N47 contains, at this writing, 4,755 putative protein coding genes. Thus, the identified proteins cover about 13% of the total putative proteome. Since the analysis was performed with a draft genome and not all genes are expressed under any given growth conditions, the coverage of the predicted proteome is within common ranges observed for other organisms. The number of peptides matching an identified protein and the total coverage of the protein are listed in Table 1. Among the corresponding genes, we identified several sequences that are highly similar to those of the bss and bbs operons involved in anaerobic toluene degradation and to the recently identified nmsA gene, encoding the ␣-subunit of the naphthyl-2-methyl-succinate synthase (Nms) (48). We assigned these genes the function of coding for enzymes catalyzing the conversion of 2-methylnaphthalene to 2-naphthoyl-CoA (Table 1). Moreover, further genes that are similar to bss/bbs genes were not present in the genome. As the first enzyme of the degradation pathway, we identified the ␣-, ␤-, and ␥-subunits of Nms, which catalyzes the addition of fumarate to the methyl group of 2-methylnaphthalene. The Nms ␣-subunit (NmsA) of culture N47 displays 92% amino acid sequence similarity to the putative NmsA of the deltaproteobacterium NaphS6 (GenBank accession number CAO72222) (48) (Fig. 4). NmsA sequences are phylogenetically related to the ␣-subunit of the benzylsuccinate synthase (BssA; ⬍50% sequence identity), which catalyzes the first step of anaerobic toluene degradation, and to the 1-methylalkyl-succinate synthase

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(MasG/AssA), which activates n-alkanes by fumarate addition. The nmsA gene product of the enrichment culture N47 harbors a glycyl residue in the characteristic amino acid RVXG motif of glycine radical enzymes at amino acid position 803 and the conserved cysteine residue at position 467 (Fig. 5). NmsA sequences are clearly distinguished from those of BssA and MasG/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 from 2-methylnaphthalene-grown N47 cells which was distantly related to the ␤-subunit of Bss identified in a toluene-degrading consortium (33% sequence similarity; GenBank accession number ABO30981) (60). We therefore designated the polypeptide NmsB. Within the nms operon, the coding gene nmsB is located upstream of nmsA (Fig. 6). Downstream of nmsA is a sequence which we assigned as the gene for the ␥-subunit of Nms (NmsC). Moreover, a putative Nms-activating enzyme, NmsD, was identified. Based on sequence similarities, the sequence most related to NmsD is that of the putative 1-methylalkyl-succinate synthase activase (MasG) of Azoarcus sp. strain HxN1 (37% sequence similarity; GenBank accession number CAO03077) (25), which is involved in anaerobic n-alkane degradation. The gene encoding NmsD (nmsD) is located upstream of the nmsB gene. Further upstream, beyond a large intergenic region of 884 bp, is a gene (nmsF) that is similar to bssF of “Aromatoleum aromaticum” EbN1 (49). The gene product could not be reproducibly detected in protein extracts from 2-methylnaphthalene-grown N47 cells. The derived amino acid sequence showed no similarity to known proteins, rendering it impossible to predict a potential function. Further upstream of nmsF, seven additional genes that are most similar to 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 involved in butanoate metabolism. Genes encoding enzymes for the beta-oxidation of naphthyl2-methyl-succinate. In the 2-methylnaphthalene degradation pathway, naphthyl-2-methyl-succinate is converted via betaoxidation reactions to 2-naphthoyl-CoA (46). So far, some biochemical reactions of the pathway are well described but the enzymes and the respective genes are still enigmatic. In protein extracts from 2-methylnaphthalene-grown cells, we identified peptides related to enzymes that catalyze similar reactions in anaerobic toluene degradation. We therefore assigned these to enzymes necessary to perform the reactions converting naphthyl-2-methyl-succinate to 2-naphthoyl-CoA and succinyl-CoA, and we successfully identified the corresponding genes from the genome. In analogy to genes for anaerobic toluene degradation, we named the genes coding for the beta-oxidation of naphthyl-2-methyl-succinate to 2-naphthoyl-CoA bns (beta-oxidation of naphthyl-2-methyl-succinate). The bns operon consists of eight genes (bnsABCD EFGH) corresponding to 8.1 kb (Fig. 6). Orthologs of the gene products have been identified in the denitrifying organisms A. aromaticum EbN1, Thauera aromatica, and Azoarcus sp. strain T, with amino acid similarities ranging from 44 to 78%, making the prediction of functions for the gene products possible (Table 1). The bnsH gene most likely codes for a putative naphthyl-2-methylene-succinyl-CoA hydratase, bnsG for naphthyl2-methyl-succinyl-CoA dehydrogenase, bnsEF for the subunits

255

249

381

152

ORF21/bnsD

ORF22/bnsC

ORF23/bnsB

ORF24/bnsA

108 840

427

ORF20/bnsE

ORF33 ORF34

409

ORF19/bnsF

302

407

ORF18/bnsG

ORF32/ncrD

251

ORF17/bnsH

250

251

ORF16

ORF31/ncrA

296

ORF15

436

296 664

ORF13 ORF14

ORF30/ncrB

66

ORF12/nmsC

209

847

ORF11/nmsA

271 376

73

ORF10/nmsB

ORF28 ORF29/ncrC

316 302 202 188 294 358 299

ORF2 ORF3 ORF4 ORF5/nmsG ORF6 ORF7 ORF9/nmsD

ORF27

538

Product length (aa)a

ORF1

ORF/gene

11,866 92,151

32,886

Unknown 2-Naphthoyl-CoA ␥-subunit 2-Naphthoyl-CoA ␤-subunit 2-Naphthoyl-CoA ␣-subunit 2-Naphthoyl-CoA ␦-subunit Ferredoxin Unknown reductase,

reductase,

reductase,

reductase,

Naphthyl-2-methylenesuccinyl-CoA hydratase Naphthyl-2-methyl-succinylCoA dehydrogenase Naphthyl-2-methyl-succinate CoA transferase subunit Naphthyl-2-methyl-succinate CoA transferase subunit Naphthyl-2-hydroxymethylsuccinyl-CoA dehydrogenase subunit Naphthyl-2-hydroxymethylsuccinyl-CoA dehydrogenase subunit Naphthyl-2-oxomethylsuccinyl-CoA thiolase subunit Naphthyl-2-oxomethylsuccinyl-CoA thiolase subunit Unknown

Unknown

Unknown

Unknown Unknown Unknown Hypothetical protein NmsG Unknown Unknown Naphthyl-2-methyl-succinate synthase-activating enzyme Naphthyl-2-methyl-succinate synthase, ␤-subunit Naphthyl-2-methyl-succinate synthase, ␣-subunit Naphthyl-2-methyl-succinate synthase, ␥-subunit Unknown Unknown

Unknown

Description of putative gene product in culture N47

3E⫺106

Benzoyl-CoA reductase, ␦-subunit

5E⫺18 1E⫺175

9E⫺67

Benzoyl-CoA reductase, ␣-subunit

Ferredoxin (BzdM) Putative NADPH:acceptor oxidoreductase (BzdV)

3E⫺136

Benzoyl-CoA reductase, ␤-subunit

3E⫺16 3E⫺64 1E⫺121

7E⫺48

1E⫺168

3E⫺51

2E⫺98

2E⫺154

4E⫺177

6E⫺166

6E⫺115

4E⫺95

9E⫺98

2E⫺68 2E⫺121

3E⫺14

0

3E⫹00

3E⫺82 3E⫺88 2E⫺34 5E⫺48 6E⫺56 2E⫺104 8E⫺57

7E⫺166

E value

TetR family transcriptional regulator Enoyl-CoA hydratase Benzoyl-CoA reductase, ␥-subunit

Benzoylsuccinyl-CoA thiolase subunit (BbsA)

Benzylsuccinate synthase, ␤-subunit (BssB) Benzylsuccinate synthase, ␣-subunit (BssA) Benzylsuccinate synthase, ␥-subunit (BssC) Isoprenoid biosynthesis protein Putative iron-sulfur-binding reductase Electron transfer flavoprotein, ␣subunit Electron transfer flavoprotein, ␤subunit Putative E-phenylitaconyl-CoA hydratase (BbsH) Benzylsuccinyl-CoA dehydrogenase (BbsG) Succinyl-CoA:(R)-benzylsuccinateCoA transferase (BbsF) Succinyl-CoA:(R)-benzylsuccinateCoA transferase (BbsE) 2-关Hydroxy(phenyl)methyl兴-succinylCoA dehydrogenase subunit (BbsD) 2-关Hydroxy(phenyl)methyl兴-succinylCoA dehydrogenase subunit (BbsC) Benzoylsuccinyl-CoA thiolase subunit (BbsB)

AMP-dependent synthetase and ligase Sugar dehydratase Hypothetical protein c2A200 Hypothetical protein c2B001 Hypothetical protein c2A197 (BssG) Phosphate butyryltransferase Butyrate kinase 1-Methylalkyl-succinate synthase activase (MasD)

Description

49 41

65

56

52

47 54

25

66

72

44

68

63

70

68

78

66

62

61 37

59

92

33

48 52 42 47 43 52 37

55

% Identity Organism

Azoarcus evansii Azoarcus sp. strain CIB

Aromatoleum aromaticum EbN1

Azoarcus evansii


Acidobacterium Ellin345 Azoarcus sp. strain CIB

Nocardia farcinica

Thauera aromatica



Thauera aromatica

Azoarcus sp. strain T

Thauera aromatica



Geobacter metallireducens GS-15

Geobacter sp. strain FRC-32

Syntrophus aciditrophicus SB Carboxydothermus hydrogenoformans


Deltaproteobacterium NaphS6

Bacterium bssA-1

Aromatoleum aromaticum EbN1 Aromatoleum aromaticum EbN1 Aromatoleum aromaticum EbN1 Aromatoleum aromaticum EbN1 Clostridium perfringens D strain Syntrophus aciditrophicus SB Azoarcus sp. strain HxN1

Desulfatibacillum alkenivorans AK-01

Related gene product

CAD21632 AAQ08814

YP_157401

CAD21630

YP_157403

YP_590289 AAQ08806

YP_118409

YP_158078

YP_158077

AAF89841

AAF89839

AAU45405

AF173961

YP_158072

YP_158071

YP_384484

YP_002537879

YP_461301 YP_36042

YP_158059

CAO72222

ABO30981

YP_158068 YP_158067 YP_158066 YP_158064 ZP_02953579 YP_462111 CAO03077

YP_002430902

Accession no.

2 31

11

11

14

14 17

4

9

15

15

19

24

12

20

15

11

17

9 9

2

49

3

8 2 12 14 12 12 14

8

No. of matching peptidesb

21 46

48

62

40

76 36

29

77

51

68

80

59

33

50

65

59

58

67 9

53

55

47

35 14 65 67 52 36 54

18

% Coveragec

SELESI ET AL.

26,339

50,331

28,874 44,082

23,962

17,441

40,641

26,550

27,330

47,448

45,280

45,811

27,999

27,466

31,092

22,437 73,808

7,842

95,851

7,919

36,512 34,865 22,660 22,006 31,906 39,198 33,606

60,304

Product mass (Da)

TABLE 1. Genes expressed during anaerobic 2-methylnaphthalene metabolism in the sulfate-reducing culture N47 300 J. BACTERIOL.

44 47 35 50 45 47 46 48 45 70

Thermoanaerobacter tengcongensis MB4 Desulfatibacillum alkenivorans AK-01 Desulfatibacillum alkenivorans AK-01 Carboxydothermus hydrogenoformans Clostridium tetani Bordetella petrii DSM 12804 Bordetella petrii DSM 12804 Carboxydothermus hydrogenoformans Archaeoglobus fulgidus DSM 4304 Desulfatibacillum alkenivorans AK-01

NP_622216 YP_002432948 YP_002430691 YP_360557 NP_781174 YP_001629755 YP_001629756 YP_360183 NP_069271 YP_002429235

7 6 10 20 11 22 6 23 12 12

30 21 36 61 38 48 68 64 39 41


b

a

aa, amino acids. Number of peptides with masses matching those of regions in the identified ORF product. c Percentage of the total protein covered by the matching peptides.

2E⫺50 3E⫺60 3E⫺29 9E⫺96 4E⫺109 2E⫺118 1E⫺18 1E⫺91 4E⫺54 6E⫺171 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown 28,194 29,465 31,768 41,710 46,464 50,652 13,924 42,928 30,160 43,007 259 272 277 381 430 457 127 385 272 401 ORF39 ORF40 ORF41 ORF42 ORF43 ORF44 ORF45 ORF46 ORF47 ORF48

16,585 16,319 48,945 31,586 ORF35 ORF36 ORF37 ORF38

145 144 449 292

Unknown Unknown Unknown Unknown

Hypothetical protein RPC_3995 MaoC-like dehydratase Acetyl-CoA acetyltransferase 3-Hydroxybutyryl-CoA dehydrogenase Enoyl-CoA hydratase/isomerase 3-Hydroxybutyryl-CoA dehydratase Amidohydrolase 2 Acyl-CoA dehydrogenase 4-Hydroxybutyrate CoA transferase Hypothetical protein Bpet1151 Hypothetical protein Bpet1152 Butyryl-CoA dehydrogenase Enoyl-CoA hydratase Acetyl-CoA acetyltransferase

4E⫺15 2E⫺18 5E⫺84 3E⫺72

32 39 43 49

Rhodopseudomonas palustris BisB18 Anaeromyxobacter sp. strain Fw109-5 Burkholderia sp. strain H160 Thermoanaerobacter tengcongensis MB4

YP_533840 YP_001377590 ZP_03266598 NP_622220

7 5 22 9

64 45 51 36

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301

of naphthyl-2-methyl-succinate CoA transferase, bnsCD for the subunits of naphthyl-2-hydroxymethyl-succinyl-CoA dehydrogenase, and bnsAB for the subunits of naphthyl-2-oxomethyl-succinyl-CoA thiolase. Four further genes are positioned between the nms and bns genes. The products of ORF15 and ORF16 display high similarities to the ␣- and ␤-subunits of electron-transferring flavoproteins (ETFs). Genes coding for enzymes of dearomatization and ring cleavage of 2-naphthoyl-CoA. Subsequent reactions of the 2-naphthoyl-CoA degradation pathway include reductive dearomatization, hydrolytic ring cleavage, and beta-oxidation to yield acetyl-CoA and CO2. The combined proteome and genome analyses of the enrichment culture N47 have given access to the coding genes for a putative 2-naphthoyl-CoA reductase that catalyzes the dearomatization of the ring structure distal to the carboxyl group (4). During growth of the culture N47 with 2-methylnaphthalene, four subunits of a putative 2-naphthoyl-CoA reductase were expressed that were most similar to the benzoyl-CoA reductase subunits of Azoarcus sp. strain CIB (43) (Table 1). In analogy to genes for anaerobic benzoyl-CoA degradation, the genes coding for 2-naphthoyl-CoA reductase were designated ncr (2-naphthoylCoA reductase). The ncr gene cluster contains the genes ncrCBAD, 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-naphthoyl-CoA reductase undergoes beta-oxidation and hydrolytic cleavage of the ring system, leading to the formation of acetylCoA and CO2. Downstream of the ncr gene cluster are 16 additional genes (ORF33 to ORF48) that were identified by using protein extracts from 2-methylnaphthalene-grown N47 cells and that may code for candidate enzymes that catalyze these reactions (Table 1; Fig. 7). The subsequent metabolic pathway is hypothetical, therefore, and the roles of these ORFs cannot be specified. The metagenome of culture N47 was screened for additional putative genes involved in anaerobic degradation of aromatic hydrocarbons. Genes coding for the ␣-, ␤-, and ␥-subunits of 4-hydroxybenzoyl-CoA reductase involved in anaerobic phenol degradation were organized adjacent to one another on one contig. In addition, genes coding for two ␣-subunits and for one ␤-subunit of 4-hydroxybenzoyl-CoA reductase were found scattered throughout the genome. Three genes encoding putative subunits of the 3-octaprenyl-4-hydroxybenzoate carboxylase were also present in the N47 genome. Besides genes for anaerobic phenol degradation, the genome contained three genes for additional ␦-, ␥-, and ␤-subunits of benzoyl-CoA reductase that were distributed throughout the genome. Except for the three 3-octaprenyl-4-hydroxybenzoate carboxylase genes, none of the additional genes were found to be expressed in 2-methylnaphthalene-grown cells. DISCUSSION Phylogenetic characterization of the enrichment culture N47. So far, a small number of sulfate-reducing naphthaleneoxidizing bacteria have been brought into culture. The deltaproteobacterial strains NaphS2 and NaphS3 were isolated from marine sediments and phylogenetically belong to the

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J. BACTERIOL.

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

Desulfobacteraceae (24, 48). 16S rRNA gene sequences closely related to the phylotype of NaphS2 were also identified as highly abundant in PAH-contaminated marine sediments (26), demonstrating that similar sulfate-reducing and maybe naphthalene-degrading bacteria are present in contaminated marine harbor sediments of geographically distinct sites such as the North Sea and the Pacific Ocean. Culture N47 was enriched almost 10 years ago, and all efforts to obtain a pure culture were not successful until now. Surprisingly, members of the Spirochaetes are stable members of the consortium. This fact may hint toward a so far unknown necessity for a spirochaetal partner organism in the enrichment culture to sustain biodegradation. However, the exact effect of these Spirochaetes on the growth of the sulfate-reducing key degrader remains to be elucidated. Potentially, the spiro-

461 461 461 461 470 467 467 483 482 482

chaetal bacterium releases a compound or vitamin needed by the sulfate-reducing Desulfobacterium. In addition, the enrichment culture N47 is able to grow with 2-methylnaphthalene as the sole source of carbon. Based on T-RFLP analysis, we could show that the bacterial community structure does not change when the enrichment culture is growing on either 2-methylnaphthalene or naphthalene. The capability to metabolize 2-methylnaphthalene is a general property of all anaerobic naphthalene-degrading bacteria investigated to date (24, 48, 54). Genes coding for the naphthyl-2-methyl-succinate synthase. In the present study, a combined genomic and proteomic study was applied to analyze the expression of genes involved in anaerobic 2-methylnaphthalene degradation. In 2-methylnaphthalene-grown cultures, proteins that were similar to the four

HWALVLCMAPGVGK.....EPEKWQSLIVRIAGYSARF HWALVLCMAPGVGK.....EPEKWQSMIVRIAGYSARF HWALVLCMAPGVGK.....EPEKWQSMIVRIAGYSARF HWALVLCMAPGVSK.....EPEKWESLIVRIAGYSARF TWVHMACMSPNPTT.....DPEGYQEVIVRVAGYSAHF TWVHQACMSPCPTT.....DPEKYSEVIVRVAGYSAHF TWVHQACMSPCPTT.....DPEKYSEVIVRVAGYSAHF YWVNVLCMAPGVAG.....EPEKHQDLIVRVSGFSARF YWVNVLCMAPGLAG.....EPEKHHDLIVRVSGYSARF NWVNVLCMSPGIHG.....EPEKHSDLIVRVSGYSARF 480 NWANVLCMSPGLCG.....EPEKHQDLIVRVSGFSSRF 481 NWVNVLCMSPGLAG.....EPEKHHDLIVRVSGYSARF 480 YWGLVLCMSPGVCG.....EPEKHQDLIVRVSGFSARF

808 808 808 808 819 815 815 830 833

833

831 829 831

NmsA N47

NmsA NaphS2 NmsA NaphS6 NmsA Naphs3 MasD Azoarcus sp. HxN1 AssA2 Strain AK-01 AssA1 Strain AK-01 BssA A. aromaticum EbN1 BssA T. aromatica T1 BssA A. sp T BssA D. toluolica TOL

BssA Magnetosp. sp. TS BssA 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, and BssA enzymes. The depicted alignment site contains the catalytic active cysteine and glycine residues with the characteristic sequence motif for glycine 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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FIG. 6. Organization of the nms and bns gene clusters of anaerobic 2-methylnaphthalene catabolism in the sulfate-reducing culture N47 in comparison 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). Genes are depicted as arrowheads. Annotation data for nms and bns genes are provided in Table 1. Genes for anaerobic toluene degradation are as follows: bssABCD, encoding benzylsuccinate synthase and the activating enzyme; bssE, encoding a putative chaperone; bssH, encoding a putative transporter; 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 benzoylsuccinyl-CoA thiolase; and bssF, bssG, bbsI, and bbsJ, encoding hypothetical proteins.

subunits and the activating enzyme of the benzylsuccinate synthase (Bss) were expressed. As the initial activation reactions of toluene and 2-methylnaphthalene degradation are similar (53), the predicted functions of the corresponding genes can be correlated to enzymes of anaerobic 2-methylnaphthalene degradation. Analogous to the genes for Bss, the nms genes code for a putative large subunit (␣; 96 kDa) and two putative small subunits (␤ and ␥; 7.9 and 7.8 kDa) of Nms. The large subunits of Nms enzymes from N47 and NaphS2 had a high degree of identity and were more distantly related to those of Bss (Fig. 4). In addition, the phylogenetic affiliation supports the clustering of ␣-subunit sequences based on specificities for substrates (2-methylnaphthalene, toluene, or n-alkanes) described earlier (48). Therefore, we conclude that Nms constitutes a novel subgroup of glycine radical enzymes. This finding is also supported by an apparently NmsA-specific RIXG amino acid sequence motif instead of the motif RVXG, which had so far been assumed to be conserved in all glycine radical enzymes. As the exchange of a valine for an isoleucine residue is consistent in all known NmsA enzymes, it may play a role in substrate specificity. In addition to this sequence motif, NmsA harbors the characteristic cysteinyl residue, which is involved in radical formation (40), at position 467. The electron of the glycyl radical is transferred to the cysteinyl residue, and a thioyl

radical is formed, which then probably abstracts a hydrogen atom from the methyl group of 2-methylnaphthalene (27). Besides nmsA, the gene cluster contains two further ORFs which code for putative ␤- and ␥-subunits of Nms. Homology searching in public databases revealed weak identity (33%) between the putative NmsB enzyme and a BssB enzyme retrieved from a toluene-degrading methanogenic enrichment culture (60). As the identity value is very low and no similarities to other known BssB enzymes were present, it is questionable whether the ORF really codes for NmsB. The nmsC gene, located downstream of nmsA, encodes a putative ␥-subunit of Nms. Like BssC from T. aromatica (40), NmsC from culture N47 is rich in cysteines and charged amino acids, which together account for 47% of the total amino acids. Although the roles of the two small subunits cannot be concluded because the functions of the corresponding Bss subunits are not yet clarified, it has been demonstrated previously by gene inactivation experiments that the small subunits of Bss are essential for Bss activity (1, 18, 42). Thus, we can assume that NmsB and NmsC may be necessary for the proper activity of Nms in culture N47. As part of the nms gene cluster, the gene nmsD encodes the putative naphthyl-2-methyl-succinate-activating enzyme, an Sadenosylmethionine (SAM) radical enzyme, which catalyzes

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

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glycyl radical formation by abstracting a hydrogen atom from the reactive glycine. The orthologous proteins BssD and MasG contain three conserved, cysteine-rich sequence motifs in the N-terminal region (36, 58). Reflected also in culture N47, NmsD contains the sequence C32LLNCAWC, corresponding to the specific CX3CX2C motif characteristic of SAM radical enzymes which coordinates a [Fe4S4] cluster. In addition, the conserved sequence motif CX2CX2CX3C, a typical [Fe4S4] ferredoxin motif, is present as C58VRCGTCVAAC and C100 TLCMKCVDVC in NmsD from culture N47. For the bss operon, it could be shown that the activase coding gene is always located upstream of the genes coding for the three subunits of Bss (56). In the nms gene cluster of N47, analogous gene organization is present. Four additional genes (ORF13 to ORF16) are located between the nms and bns gene clusters of culture N47. Two of them, ORF15 and ORF16, code for the ␣- and ␤-subunits of a putative ETF. The ETF may possibly serve as an electron acceptor for the reaction with naphthyl-2-methyl-succinyl-CoA dehydrogenase (41), which can be replaced by phenazine methosulfate in in vitro enzyme tests (53). A third gene, ORF14, codes for a protein that contains iron-sulfur clusters and thus may also be involved in the electron transfer reaction. Interestingly, this gene organization is similar to the arrangement of the bss gene cluster in the deltaproteobacterial iron reducer Geobacter metallireducens, whereas the genetic organization within the betaproteobacterial denitrifiers T. aromatica and A. aromaticum EbN1 is distinct (35). The similarity of the gene organization in N47 to that in G. metallireducens is also obvious from the arrangement of the bbs operon in G. metallireducens, in which no sequences coding for BssI and BssJ are present. Genes coding for the corresponding products, with so far unknown functions, are also absent from the bns operon in culture N47 (Fig. 6). A putative transcriptional regulator (ORF25) is located downstream of the bns gene cluster and is transcribed in the opposite direction. The N-terminal region of the regulator protein contains an XylR domain that is significant for ␴54dependent transcriptional activators, including those for anaerobic phenol degradation (10). However, we could not observe expression of ORF25 during growth on 2-methylnaphthalene. The nms/bns gene cluster is flanked by genes coding for a reverse transcriptase and a transposase. Upstream, a gene encodes a retron-type reverse transcriptase that exhibits 67% amino acid sequence similarity to a reverse transcriptase from Desulfotomaculum acetoxidans (GenBank accession number ZP_04352877). The transposase encoded by the gene downstream of the nms/bns gene cluster is characterized by an integrase core domain closely related to the transposase of Dethiosulfovibrio peptidovorans (GenBank accession number ZP_04341313). The presence of these genes indicates genetic mobility of the nms/bns operons and may hint at a putative horizontal gene transfer event in the evolution of aromatic hydrocarbon degraders. The pattern in which transposase or integrase coding genes are present with gene clusters for aromatic degradation is widely distributed and occurs, e.g., in A. aromaticum EbN1 (14, 49). Respective events of lateral gene transfer also have been interpreted from previous phylogenetic analyses of bssA gene relationships (57, 63).

J. BACTERIOL.

Genes coding for enzymes involved in dearomatization and ring cleavage of 2-naphthoyl-CoA. In 2-methylnaphthalenegrown N47 cells, we could identify peptides similar to enzymes of the anaerobic benzoyl-CoA degradation pathway, which initiates ring cleavage by reducing the aromatic ring of benzoylCoA. Genes similar to bamB to bamI, encoding reductively dearomatizing enzymes of the G. metallireducens type, were not present in the genome of N47 (64). Recently, it was demonstrated that 5,6,7,8-tetrahydro-2-naphthoyl-CoA is formed as a major metabolite during anaerobic 2-methylnaphthalene degradation (4). Therefore, it was concluded that further degradation of 2-naphthoyl-CoA proceeds via reduction of the bicyclic ring system to cyclohexanoic compounds and not via the monoaromatic benzoyl-CoA. Indeed, the reduced metabolite octahydro-2-naphthoyl-CoA was detected in naphthalene-grown N47 cultures (46). As the genome of culture N47 does not contain further gene clusters similar to that for benzoyl-CoA reductase, we correlate the identified proteins with the enzyme naphthoyl-CoA reductase of the 2-methylnaphthalene pathway. Based on amino acid sequence similarities, the 2-naphthoylCoA reductase of culture N47 is related to the Azoarcus type of benzoyl-CoA reductases (7). The reduction of benzoyl-CoA to a nonaromatic cyclic compound via an Azoarcus-like benzoylCoA reductase is catalyzed by an ATP-dependent two-electron transfer to the aromatic ring via ferredoxin (43). The transfer of electrons to the benzene ring requires electrons at a very low redox potential (6). In Azoarcus evansii, ferredoxin serves as the electron donor for the benzoyl-CoA reductase (21) and the oxidized ferredoxin is reduced by a 2-oxoglutarate:acceptor oxidoreductase in combination with a NADPH:acceptor oxidoreductase. In Azoarcus sp. strain CIB, the genes encoding the 2-oxoglutarate:acceptor oxidoreductase are not present in the bzd gene cluster (43). Interestingly, like the corresponding operon from Azoarcus sp. strain CIB, the bns operon from culture N47 contains no genes that could code for a putative 2-oxoglutarate:acceptor oxidoreductase. Genes for a putative NADPH:acceptor oxidoreductase (ORF34) and ferredoxin (ORF33) are directly associated downstream of the ORFs coding for the four subunits of the putative 2-naphthoyl-CoA reductase (ncrABCD). At the N-terminal part of the enzyme, the putative NADPH:acceptor oxidoreductase from culture N47 harbors a conserved domain that is characteristic of NADPH-ubiquinone oxidoreductase iron-sulfur-binding regions. In benzoyl-CoA reductase, the ␣- and ␦-subunits contain the active sites where electrons become ATP-dependently activated. The aromatic compound becomes dearomatized at the ␤- and ␥-subunits (8). For 2-naphthoyl-CoA reductase, four subunits were identified, whereas the ␣- and ␦-subunits are also characterized by the presence of an Hsp70 class ATPase domain. It was demonstrated earlier that similar ␣- and ␦-subunits contain the ATP-binding sites of the acetate kinase/sugar kinase/Hsp70 actin family (29). As these subunits of naphthoylCoA reductase are also characterized by an ATP-binding site, ATP-dependent reduction of the aromatic ring can be proposed. However, ATP-dependent ring reduction has been shown to occur only in facultative anaerobes but not in strict anaerobes like sulfate reducers (8). Due to the low energy gain for sulfate reducers, it is unlikely that culture N47 has ATP-

VOL. 192, 2010


driven ring reduction. ORF27 is located upstream of the ncr gene cluster and codes for a putative TetR family transcriptional regulator. Comparative sequence analyses showed that the genes coding for 2-methylnaphthalene and 2-naphthoyl-CoA degradation are related to those involved in anaerobic toluene and benzoate metabolism. Nevertheless, they are apparently different enough to probably enable specific tracking of 2-methylnaphthalene degradation genes in other bacterial cultures or even environmental samples. Genes like nmsA and nmsD coding for key enzymes of the 2-methylnaphthalene degradation pathway may be useful as functional marker genes in anthropogenically impacted environments in order to predict the potential for natural attenuation processes for aromatic contaminants. This approach would provide essential knowledge of the abundance and diversity of PAH-degrading microorganisms, which have so far been insufficiently investigated because of the limited availability of appropriate molecular markers. As an example, specific toluene degrader communities could be correlated to distinct biogeochemical conditions within the depth profile of a tar-oil-contaminated aquifer based on the detection of bssA, the gene coding for the key enzyme of anaerobic toluene degradation (63). REFERENCES 1. Achong, G. R., A. M. Rodriguez, and A. M. Spormann. 2001. Benzylsuccinate synthase of Azoarcus sp. strain T: cloning, sequencing, transcriptional organization, and its role in anaerobic toluene and m-xylene mineralization. J. Bacteriol. 183:6763–6770. 2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410. 3. Annweiler, E., A. Materna, M. Safinowski, A. Kappler, H. H. Richnow, W. Michaelis, and R. U. Meckenstock. 2000. Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture. Appl. Environ. Microbiol. 66:5329–5333. 4. Annweiler, E., W. Michaelis, and R. U. Meckenstock. 2002. Identical ring cleavage products during anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin indicate a new metabolic pathway. Appl. Environ. Microbiol. 68:852–858. 5. Besemer, J., A. Lomsadze, and M. Borodovsky. 2001. GeneMarkS: a selftraining method for prediction of gene starts in microbial genomes. Nucleic Acids Res. 29:2607–2618. 6. Birch, A. J., A. K. Linde, and L. Radom. 1980. A theoretical approach to the Birch reduction. Structures and stabilities of the radical anions of substituted benzenes. J. Am. Chem. Soc. 102:3370–3376. 7. Boll, M. 2005. Dearomatizing benzene ring reductases. J. Mol. Microbiol. Biotechnol. 10:132–142. 8. Boll, M. 2005. Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions. Biochim. Biophys. Acta 1707:34–50. 9. Boll, M., G. Fuchs, and J. Heider. 2002. Anaerobic oxidation of aromatic compounds and hydrocarbons. Curr. Opin. Chem. Biol. 6:604–611. 10. Breinig, S., E. Schiltz, and G. Fuchs. 2000. Genes involved in anaerobic metabolism of phenol in the bacterium Thauera aromatica. J. Bacteriol. 182:5849–5863. 11. Butler, J., Q. He, K. Nevin, Z. He, J. Zhou, and D. Lovley. 2007. Genomic and microarray analysis of aromatics degradation in Geobacter metallireducens and comparison to a Geobacter isolate from a contaminated field site. BMC Genomics 8:180. 12. Callaghan, A. V., L. M. Gieg, K. G. Kropp, J. M. Suflita, and L. Y. Young. 2006. Comparison of mechanisms of alkane metabolism under sulfate-reducing conditions among two bacterial isolates and a bacterial consortium. Appl. Environ. Microbiol. 72:4274–4282. 13. Callaghan, A. V., B. Wawrik, S. M. N. Chadhain, L. Y. Young, and G. J. Zylstra. 2008. Anaerobic alkane-degrading strain AK-01 contains two alkylsuccinate synthase genes. Biochem. Biophys. Res. Commun. 366:142–148. 14. Carmona, M., M. T. Zamarro, B. Blazquez, G. Durante-Rodriguez, J. F. Juarez, J. A. Valderrama, M. J. L. Barragan, J. L. Garcia, and E. Diaz. 2009. Anaerobic catabolism of aromatic compounds: a genetic and genomic view. Microbiol. Mol. Biol. Rev. 73:71–133. 15. Cerniglia, C. E. 1992. Biodegradation of polycyclic hydrocarbons. Biodegradation 3:351–368. 16. Cline, J. D. 1969. Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol. Oceanogr. 14:454–458.

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