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J Bacteriol. 2010 January; 192(1): 295–306.
Published online 2009 October 23. doi:  10.1128/JB.00874-09
PMCID: PMC2798259

Combined Genomic and Proteomic Approaches Identify Gene Clusters Involved in Anaerobic 2-Methylnaphthalene Degradation in the Sulfate-Reducing Enrichment Culture N47[down-pointing small open triangle]

Abstract

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 α-, β-, and γ-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 acetyl-CoA 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.

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.

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. (Fig.1)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 beta-oxidation 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 α-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 α-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).

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; ...

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 13C-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-2-methyl-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 naphthalene-grown 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 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 μl lysis buffer {9 M urea, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol; GE Healthcare Europe GmbH, Freiburg, Germany} and 67 μl 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 μl 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-μg 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 PepMap column (internal diameter, 75 μm; 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 454-pyrosequencing 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 ≤ 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 α-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 Jones-Taylor-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.

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. (Fig.2).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. (Fig.3).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) 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. (Fig.3).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.

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.
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. ...

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 Table1.1. 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 α-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 (Table1).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 α-, β-, and γ-subunits of Nms, which catalyzes the addition of fumarate to the methyl group of 2-methylnaphthalene. The Nms α-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. (Fig.4).4). NmsA sequences are phylogenetically related to the α-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 (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. (Fig.5).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. (Fig.55).

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

Another protein has been detected in protein extracts from 2-methylnaphthalene-grown N47 cells which was distantly related to the β-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. (Fig.6).6). Downstream of nmsA is a sequence which we assigned as the gene for the γ-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 (Table1)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.

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), ...

Genes encoding enzymes for the beta-oxidation of naphthyl-2-methyl-succinate.

In the 2-methylnaphthalene degradation pathway, naphthyl-2-methyl-succinate is converted via beta-oxidation 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 (bnsABCDEFGH) corresponding to 8.1 kb (Fig. (Fig.6).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 (Table1).1). The bnsH gene most likely codes for a putative naphthyl-2-methylene-succinyl-CoA hydratase, bnsG for naphthyl-2-methyl-succinyl-CoA dehydrogenase, bnsEF for the subunits 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 α- and β-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 (Table1).1). In analogy to genes for anaerobic benzoyl-CoA degradation, the genes coding for 2-naphthoyl-CoA reductase were designated ncr (2-naphthoyl-CoA reductase). The ncr gene cluster contains the genes ncrCBAD, which code for the γ (NcrC)-, β (NcrB)-, α (NcrA)-, and δ (NcrD)-subunits of the 2-naphthoyl-CoA reductase (Ncr) and which correspond to a sequence length of 4.2 kb (Fig. (Fig.7).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 acetyl-CoA 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 (Table1;1; Fig. Fig.7).7). The subsequent metabolic pathway is hypothetical, therefore, and the roles of these ORFs cannot be specified.

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 ...

The metagenome of culture N47 was screened for additional putative genes involved in anaerobic degradation of aromatic hydrocarbons. Genes coding for the α-, β-, and γ-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 α-subunits and for one β-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 δ-, γ-, and β-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 naphthalene-oxidizing bacteria have been brought into culture. The deltaproteobacterial strains NaphS2 and NaphS3 were isolated from marine sediments and phylogenetically belong to the 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 spirochaetal 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 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 (α; 96 kDa) and two putative small subunits (β and γ; 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. (Fig.4).4). In addition, the phylogenetic affiliation supports the clustering of α-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 β- and γ-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 γ-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 S-adenosylmethionine (SAM) radical enzyme, which catalyzes 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 C100TLCMKCVDVC 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 α- and β-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. (Fig.66).

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 σ54-dependent 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).

Genes coding for enzymes involved in dearomatization and ring cleavage of 2-naphthoyl-CoA.

In 2-methylnaphthalene-grown 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 benzoyl-CoA. 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-naphthoyl-CoA 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 benzoyl-CoA 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 α- and δ-subunits contain the active sites where electrons become ATP-dependently activated. The aromatic compound becomes dearomatized at the β- and γ-subunits (8). For 2-naphthoyl-CoA reductase, four subunits were identified, whereas the α- and δ-subunits are also characterized by the presence of an Hsp70 class ATPase domain. It was demonstrated earlier that similar α- and δ-subunits contain the ATP-binding sites of the acetate kinase/sugar kinase/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 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-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).

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 October 2009.

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. [PMC free article] [PubMed]
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. [PubMed]
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. [PMC free article] [PubMed]
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. [PMC free article] [PubMed]
5. Besemer, J., A. Lomsadze, and M. Borodovsky. 2001. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Nucleic Acids Res. 29:2607-2618. [PMC free article] [PubMed]
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. [PubMed]
8. Boll, M. 2005. Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions. Biochim. Biophys. Acta 1707:34-50. [PubMed]
9. Boll, M., G. Fuchs, and J. Heider. 2002. Anaerobic oxidation of aromatic compounds and hydrocarbons. Curr. Opin. Chem. Biol. 6:604-611. [PubMed]
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. [PMC free article] [PubMed]
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. [PMC free article] [PubMed]
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. [PMC free article] [PubMed]
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. [PubMed]
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. [PMC free article] [PubMed]
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.
17. Coates, J. D., R. Chakraborty, J. G. Lack, S. M. O'Connor, K. A. Cole, K. S. Bender, and L. A. Achenbach. 2001. Anoxic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas. Nature 411:1039-1043. [PubMed]
18. Coschigano, P. W. 2000. Transcriptional analysis of the tutE tutFDGH gene cluster from Thauera aromatica strain T1. Appl. Environ. Microbiol. 66:1147-1151. [PMC free article] [PubMed]
19. Coschigano, P. W., T. S. Wehrman, and L. Y. Young. 1998. Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free radical. Appl. Environ. Microbiol. 64:1650-1656. [PMC free article] [PubMed]
20. Davidova, I. A., L. M. Gieg, K. E. Duncan, and J. M. Suflita. 2007. Anaerobic phenanthrene mineralization by a carboxylating sulfate-reducing bacterial enrichment. ISME J. 1:436-442. [PubMed]
21. Ebenau-Jehle, C., M. Boll, and G. Fuchs. 2003. 2-Oxoglutarate:NADP+ oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction. J. Bacteriol. 185:6119-6129. [PMC free article] [PubMed]
22. Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792-1797. [PMC free article] [PubMed]
23. Felsenstein, J. 1989. PHYLIP—phylogeny inference package (version 3.2). Cladistics 5:164-166.
24. Galushko, A., D. Minz, B. Schink, and F. Widdel. 1999. Anaerobic degradation of naphthalene by a pure culture of a novel type of marine sulphate-reducing bacterium. Environ. Microbiol. 1:415-420. [PubMed]
25. Grundmann, O., A. Behrends, R. Rabus, J. Amann, T. Halder, J. Heider, and F. Widdel. 2008. Genes encoding the candidate enzyme for anaerobic activation of n-alkanes in the denitrifying bacterium, strain HxN1. Environ. Microbiol. 10:376-385. [PubMed]
26. Hayes, L. A., and D. R. Lovley. 2002. Specific 16S rDNA sequences associated with naphthalene degradation under sulfate-reducing conditions in harbor sediments. Microb. Ecol. 43:134-145. [PubMed]
27. Heider, J. 2007. Adding handles to unhandy substrates: anaerobic hydrocarbon activation mechanisms. Curr. Opin. Chem. Biol. 11:188-194. [PubMed]
28. Hermuth, K., B. Leuthner, and J. Heider. 2002. Operon structure and expression of the genes for benzylsuccinate synthase in Thauera aromatica strain K172. Arch. Microbiol. 177:132-138. [PubMed]
29. Hurley, J. H. 1996. The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism. Annu. Rev. Biophys. Biomol. Struct. 25:137-162. [PubMed]
30. Jensen, L. J., P. Julien, M. Kuhn, C. von Mering, J. Muller, T. Doerks, and P. Bork. 2008. eggNOG: automated construction and annotation of orthologous groups of genes. Nucleic Acids Res. 36:D250-254. [PMC free article] [PubMed]
31. Kasai, Y., Y. Kodama, Y. Katahata, T. Hoaki, and K. Watanabe. 2007. Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11. Environ. Sci. Technol. 41:6222-6227. [PubMed]
32. Kniemeyer, O., T. Fischer, H. Wilkes, F. O. Glockner, and F. Widdel. 2003. Anaerobic degradation of ethylbenzene by a new type of marine sulfate-reducing bacterium. Appl. Environ. Microbiol. 69:760-768. [PMC free article] [PubMed]
33. Krieger, C. J., H. R. Beller, M. Reinhard, and A. M. Spormann. 1999. Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp. strain T. J. Bacteriol. 181:6403-6410. [PMC free article] [PubMed]
34. Krieger, C. J., W. Roseboom, S. P. J. Albracht, and A. M. Spormann. 2001. A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T. J. Biol. Chem. 276:12924-12927. [PubMed]
35. Kube, M., J. Heider, J. Amann, P. Hufnagel, S. Kuhner, A. Beck, R. Reinhardt, and R. Rabus. 2004. Genes involved in the anaerobic degradation of toluene in a denitrifying bacterium, strain EbN1. Arch. Microbiol. 181:182-194. [PubMed]
36. Kulzer, R., T. Pils, R. Kappl, J. Huttermann, and J. Knappe. 1998. Reconstitution and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme: molecular properties of the holoenzyme form. J. Biol. Chem. 273:4897-4903. [PubMed]
37. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. [PubMed]
38. Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogint, and N. R. Pace. 1985. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82:6955-6959. [PubMed]
39. Leuthner, B., and J. Heider. 2000. Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate. J. Bacteriol. 182:272-277. [PMC free article] [PubMed]
40. Leuthner, B., C. Leutwein, H. Schulz, P. Horth, W. Haehnel, E. Schiltz, H. Schagger, and J. Heider. 1998. Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol. Microbiol. 28:615-628. [PubMed]
41. Leutwein, C., and J. Heider. 2002. (R)-benzylsuccinyl-CoA dehydrogenase of Thauera aromatica, an enzyme of the anaerobic toluene catabolic pathway. Arch. Microbiol. 178:517-524. [PubMed]
42. Li, L., D. P. Patterson, C. C. Fox, B. Lin, P. W. Coschigano, and E. N. Marsh. 2009. Subunit structure of benzylsuccinate synthase. Biochemistry 48:1284-1292. [PMC free article] [PubMed]
43. Lopez Barragan, M. J., M. Carmona, M. T. Zamarro, B. Thiele, M. Boll, G. Fuchs, J. L. Garcia, and E. Diaz. 2004. The bzd gene cluster, coding for anaerobic benzoate catabolism in Azoarcus sp. strain CIB. J. Bacteriol. 186:5762-5774. [PMC free article] [PubMed]
44. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, A. Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Förster, I. Brettske, S. Gerber, A. W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. König, T. Liss, R. Lüssmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and K. H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32:1-9. [PMC free article] [PubMed]
45. Lueders, T., and M. Friedrich. 2000. Archaeal population dynamics during sequential reduction processes in rice field soil. Appl. Environ. Microbiol. 66:2732-2742. [PMC free article] [PubMed]
46. Meckenstock, R. U., E. Annweiler, W. Michaelis, H. H. Richnow, and B. Schink. 2000. Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture. Appl. Environ. Microbiol. 66:2743-2747. [PMC free article] [PubMed]
47. Meckenstock, R. U., M. Safinowski, and C. Griebler. 2004. Anaerobic degradation of polycyclic aromatic hydrocarbons. FEMS Microbiol. Ecol. 49:27-36. [PubMed]
48. Musat, F., A. S. Galushko, J. Jacob, F. Widdel, M. Kube, R. Reinhardt, H. Wilkes, B. Schink, and R. Rabus. 2008. Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria. Environ. Microbiol. 11:209-219. [PubMed]
49. Rabus, R., M. Kube, J. Heider, A. Beck, K. Heitmann, F. Widdel, and R. Reinhardt. 2005. The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch. Microbiol. 183:27-36. [PubMed]
50. Rabus, R., and F. Widdel. 1995. Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria. Arch. Microbiol. 163:96-103. [PubMed]
51. Rabus, R., H. Wilkes, A. Behrends, A. Armstroff, T. Fischer, A. J. Pierik, and F. Widdel. 2001. Anaerobic initial reaction of n-alkanes in a denitrifying bacterium: evidence for (1-methylpentyl)succinate as initial product and for involvement of an organic radical in n-hexane metabolism. J. Bacteriol. 183:1707-1715. [PMC free article] [PubMed]
52. Rockne, K. J., J. C. Chee-Sanford, R. A. Sanford, B. P. Hedlund, J. T. Staley, and S. E. Strand. 2000. Anaerobic naphthalene degradation by microbial pure cultures under nitrate-reducing conditions. Appl. Environ. Microbiol. 66:1595-1601. [PMC free article] [PubMed]
53. Safinowski, M., and R. U. Meckenstock. 2004. Enzymatic reactions in anaerobic 2-methylnaphthalene degradation by the sulphate-reducing enrichment culture N47. FEMS Microbiol. Lett. 240:99-104. [PubMed]
54. Safinowski, M., and R. U. Meckenstock. 2006. Methylation is the initial reaction in anaerobic naphthalene degradation by a sulfate-reducing enrichment culture. Environ. Microbiol. 8:347-352. [PubMed]
55. Selesi, D., and R. U. Meckenstock. 2009. Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture. FEMS Microbiol. Ecol. 68:86-93. [PubMed]
56. Selmer, T., A. J. Pierik, and J. Heider. 2005. New glycyl radical enzymes catalysing key metabolic steps in anaerobic bacteria. Biol. Chem. 386:981-988. [PubMed]
57. Shinoda, Y., J. Akagi, Y. Uchihashi, A. Hiraishi, H. Yukawa, H. Yurimoto, Y. Sakai, and N. Kato. 2005. Anaerobic degradation of aromatic compounds by Magnetospirillum strains: isolation and degradation genes. Biosci. Biotechnol. Biochem. 69:1483-1491. [PubMed]
58. Sofia, H. J., G. Chen, B. G. Hetzler, J. F. Reyes-Spindola, and N. E. Miller. 2001. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 29:1097-1106. [PMC free article] [PubMed]
59. Strimmer, K., and A. von Haeseler. 1997. Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc. Natl. Acad. Sci. USA 94:6815-6819. [PubMed]
60. Washer, C. E., and E. A. Edwards. 2007. Identification and expression of benzylsuccinate synthase genes in a toluene-degrading methanogenic consortium. Appl. Environ. Microbiol. 73:1367-1369. [PMC free article] [PubMed]
61. Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703. [PMC free article] [PubMed]
62. Winderl, C., B. Anneser, C. Griebler, R. U. Meckenstock, and T. Lueders. 2008. Depth-resolved quantification of anaerobic toluene degraders and aquifer microbial community patterns in distinct redox zones of a tar oil contaminant plume. Appl. Environ. Microbiol. 74:792-801. [PMC free article] [PubMed]
63. Winderl, C., S. Schaefer, and T. Lueders. 2007. Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker. Environ. Microbiol. 9:1035-1046. [PubMed]
64. Wischgoll, S., D. Heintz, F. Peters, A. Erxleben, E. Sarnighausen, R. Reski, A. Van Dorsselaer, and M. Boll. 2005. Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens. Mol. Microbiol. 58:1238-1252. [PubMed]
65. Zehr, B. D., T. J. Savin, and R. E. Hall. 1989. A one-step, low background Coomassie staining procedure for polyacrylamide gels. Anal. Biochem. 182:157-159. [PubMed]
66. Zhang, X. M., and L. Y. Young. 1997. Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia. Appl. Environ. Microbiol. 63:4759-4764. [PMC free article] [PubMed]

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