Diversity of Benzyl- and Alkylsuccinate Synthase Genes in

May 26, 2010 - Neighbor-joining dendrogram of assA and bssA genes from reference strains, enrichment cultures, and environmental samples. Sequences ...
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Environ. Sci. Technol. 2010, 44, 7287–7294

Diversity of Benzyl- and Alkylsuccinate Synthase Genes in Hydrocarbon-Impacted Environments and Enrichment Cultures A M Y V . C A L L A G H A N , * ,†,‡ I R E N E A . D A V I D O V A , †,‡ K R I S T E N S A V A G E - A S H L O C K , †,| V I C T O R I A A . P A R I S I , †,‡ L I S A M . G I E G , ⊥,† J O S E P H M . S U F L I T A , †,‡ JEROME J. KUKOR,§ AND BORIS WAWRIK† Department of Botany and Microbiology and Institute for Energy and the Environment, University of Oklahoma, Norman, Oklahoma, 73019, and Biotechnology Center for Agriculture and the Environment, Rutgers University, New Brunswick, New Jersey 08901-8521

Received January 19, 2010. Revised manuscript received May 3, 2010. Accepted May 7, 2010.

Hydrocarbon-degrading microorganisms play an important role in the natural attenuation of spilled petroleum in a variety of anoxic environments. The role of benzylsuccinate synthase (BSS) in aromatic hydrocarbon degradation and its use as a biomarker for field investigations are well documented. The recent discovery of alkylsuccinate synthase (ASS) allows the opportunity to test whether its encoding gene, assA, can serve as a comparable biomarker of anaerobic alkane degradation. Degenerate assAand bssA-targeted PCR primers were designed in order to survey the diversity of genes associated with aromatic and aliphatic hydrocarbon biodegradation in petroleum-impacted environments and enrichment cultures. DNA was extracted from an anaerobic alkane-degrading isolate (Desulfoglaeba alkenexedens ALDC), hydrocarbon-contaminated river and aquifer sediments, a paraffin-degrading enrichment, and a propane-utilizing mixed culture. Partial assA and bssA genes were PCR amplified, cloned, and sequenced, yielding several novel clades of assA genes. These data expand the range of alkane-degrading conditions for which relevant gene sequences are available and indicate that considerable diversity of assA genes can be found in hydrocarbon-impacted environments. The detection of genes associated with anaerobic alkane degradation in conjunction with the in situ detection of alkylsuccinate metabolites was also demonstrated. Comparable molecular signals of assA/bssA were not found when environmental metagenome databases of uncontaminated sites were searched. These data confirm that the assA gene is a useful biomarker for anaerobic alkane metabolism. * Corresponding author phone: 405-325-1872; fax: 405-325-7619; e-mail: [email protected]. † Department of Botany and Microbiology, University of Oklahoma. ‡ Institute for Energy and the Environment, University of Oklahoma. § Biotechnology Center for Agriculture and the Environment, Rutgers University. | Current affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma, 74074. ⊥ Current affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4. 10.1021/es1002023

 2010 American Chemical Society

Published on Web 05/26/2010

Introduction Petroleum consumption in the United States exceeds 297 billion gallons annually (1), and has widespread environmental consequences, most often evident by the contamination of many ecosystems. Given the cost of remediating petroleum-impacted environments, research has been directed toward documenting both natural and enhanced attenuation processes. However, evidence for such processes is often indirect. Contamination of the environment with high organic loads results in the development of anoxic conditions, under which novel biodegradation mechanisms are involved in hydrocarbon attenuation processes. Documenting such processes can provide direct evidence of contaminant remediation. Among these mechanisms, addition of the parent substrate across the double bond of fumarate (referred to as fumarate addition) plays a particularly dominant role in the degradation of both saturated and aromatic hydrocarbons (for review, see ref 2). Fumarate addition is catalyzed by glycyl radical enzymes and has been best characterized with respect to aromatic hydrocarbons. For example, toluene, ethylbenzene, 2-methylnaphthalene, and o- and m-xylenes are anaerobically activated by the enzyme, benzylsuccinate synthase (BSS) to form benzyl-, 1-phenylethyl-, 2-(naphthylmethyl), and 2- and 3-methylbenzylsuccinate metabolites, respectively (for reviews, see refs 3 and 4). These signature metabolites, as well as the detection, quantification, and expression of bssA genes, have facilitated several field investigations that aim to document in situ biodegradation of aromatic hydrocarbons (5-12). In this context, Beller et al. (5) demonstrated the need for multiple approaches in field investigations and suggested combining the detection of signature metabolites with other techniques such as PCR amplification of catabolic genes. Although BTEX compounds (benzene, toluene, ethylbenzene, and xylenes) have garnered more attention because of their classification as priority pollutants, the anaerobic biodegradation of alkanes is equally relevant. Alkanes are quantitatively the most important hydrocarbon components in petroleum, and some are acutely toxic (13), as well as difficult to remediate. Kropp et al. (14) demonstrated that n-alkanes are activated via fumarate addition to form alkylsuccinates, and subsequent studies showed that this mode of activation applies to a range of alkane substrates (15). Alkylsuccinates have been targeted as signature metabolites in field studies of hydrocarbonimpacted aquifers (8, 9, 16). However, because of the phylogenetic diversity of anaerobic alkane degraders, it has not been possible to characterize the relevant organisms based on ribosomal gene targets, and for almost a decade, alkylsuccinates were the only “biomarkers” of anaerobic alkane metabolism. Recently, two genes (assA1 and assA2) with high similarity to the catalytic subunit gene of BSS (bssA) were identified in the alkane degrader Desulfatibacillum alkenivorans AK-01 (17). D. alkenivorans is a sulfate-reducer that utilizes C13 to C18 alkanes via fumarate addition (18). ASS-R1 was implicated in alkane degradation via proteomic experiments (17). Similar observations were made in the denitrifying, alkanedegrading strain HxN1, which utilizes C6 to C8 alkanes and contains the assA-like gene, masD (19). At the beginning of the present study, assA1 and assA2 were the only alkylsuccinate synthase sequences in GenBank. The sequence of masD became available shortly thereafter. VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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The availability of gene sequences associated with anaerobic alkane metabolism enables the application of gene detection tools during environmental assessments. Using the assA genes as metabolic biomarkers, however, requires a greater understanding of the genetic diversity of the assA genes in the context of substrate range, environmental systems, and geochemical processes. The goals of this study were therefore 2-fold. First, enrichment cultures growing on short- and long-chain alkane substrates (C3 and C28) were investigated for the presence of assA genotypes to expand the range of alkane-degrading conditions for which gene sequences are available. Second, hydrocarbon-impacted environments were assayed for the presence of assA and bssA genotypes, to document the presence of assA and bssA genes in conjunction with the detection of the requisite metabolites in situ. The results indicate that the assA gene is a useful biomarker for anaerobic alkane metabolism, and they provide a platform for further investigation of hydrocarbon-contaminated environments.

Materials and Methods assA/bssA Primer Design. A series of oligonucleotide primer pairs were designed for the amplification of assA and bssA genes by alignment of known sequences and identification of conserved sequence motifs (SI Table SI-1). At the beginning of this study, only assA1 and assA2 genes in D. alkenivorans AK-01 were known (GenBank DQ826035 and DQ826036). These sequences and several bssA sequences (GenBank CR555306, AJ001848, AB066263, AY032676, AF113168, and NC_007517) were used to design primer set 1, which was used to amplify assA from Desulfoglaeba alkanedexens ALDC and sediment samples from hydrocarbon-impacted rivers and estuaries in NY and NJ. The ALDC assA sequence (GU453656), in addition to the HxN1 masD sequence (AM748709; deposited in GenBank during the time of this study), were included in the primer design for primer sets 3 through 9 (SI Table SI-1). Primer set 2 (17) was used to amplify bssA genes. All primer combinations were tested on D. alkenivorans AK-01, D. alkanedexens ALDC, two sediment samples from hydrocarbon-impacted aquifer systems, and two enrichment cultures utilizing a range of alkanes. Environmental Samples and Enrichment Culture Information. Contaminated sites in New York and New Jersey were chosen as “proof-of-principle” examples to demonstrate the presence of assA genes in hydrocarbon-impacted environments. The following sites were chosen based on their histories of petroleum contamination: Gowanus Canal (NY), Newtown Creek (NY), the Arthur Kill waterway (NY/NJ), and the Passaic River (NJ) (SI Table SI-2). Sediments were collected in 20-L buckets, topped off with site water, and stored in the dark at 4 °C until DNA extraction. Two anaerobic enrichment cultures were interrogated with respect to the diversity of assA and bssA genes (SI Table SI-2). One of these enrichments was established with sediment from the source of Zodletone spring, which is located north of Zodletone Mountain in the Anadarko Basin, OK. Zodletone spring water is anoxic, high in sulfide (8 to10 mM), and degasses methane, ethane, and propane (20). The enrichment culture is maintained under sulfate-reducing conditions (15 mM sulfate) with propane (0.160 mM) as sole source of carbon and energy (20). Both n-propylsuccinate and iso-propylsuccinate were detected in the culture, indicative of terminal and subterminal addition of propane to fumarate (20). The culture designated as SDB is a methanogenic, octacosane (C28H58)-degrading enrichment culture that was established with oil-contaminated marine sediments from the Paletta Creek site in San Diego Bay, CA. The culture was incubated in 160-ml serum bottles containing 50 mL of basal, sulfate-free, reduced mineral medium (pH 7.2-7.3) (21) amended with 20 mg of octacosane (C28H58) (dissolved 7288

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in 1 mL of 2,2,4,4,6,8,8-heptamethylnonane (HMN)) as sole source of carbon and energy. Culture bottles were sealed with Teflon-lined stoppers, secured with aluminum crimp seals, and incubated under N2/CO2 (80:20) at 31 °C without shaking. Archived sediments from two hydrocarbon-impacted aquifer systems were selected based on the identification of metabolites consistent with the anaerobic degradation of aromatic and aliphatic hydrocarbons (SI Table SI-2). The first field site is located near Fort Lupton, CO, and is part of the South Platte alluvial aquifer, located in Weld County. The sediments and groundwater are contaminated with gas condensate (96% w/w C5-C15 compounds, including 18% w/w BTEX) (7). Sediment was collected from a depth of 5 feet, within the contaminant plume between wells 41 and 45 (see ref 7), by hand auger and placed immediately into a 1-L sterile glass jar with a rubber seal. Sediments were topped with anaerobic groundwater (from well 45), sealed without a headspace, and stored at 4 °C. Previous groundwater investigations at this site resulted in the detection of alkylsuccinic acids with and without unsaturation (C6, C7, C8, C9) and methylbenzylsuccinic acids in the groundwater (6-8). The second sampling location was the site of a former oil refinery in Casper, WY, where groundwater and aquifer sediment are contaminated from historical releases of fuel gas, liquid propane gas, motor/aviation gasoline, fluid cracking unit coke, heavy fuel oil, kerosene and distillates, asphalt, and components of nonaqueous phase liquids (9). Historical geochemical data suggest that sulfate reduction is the dominant terminal electron-accepting process. Using hollow stem augering techniques, a borehole was advanced to the water table in the vicinity of MW-439 (see ref 9). Quaternary alluvial sediment was collected at a depth of 6 m (within the hydrocarbon-laden zone) using a split spoon sampler. Sediment was transferred immediately from the split spoon to 1-L sterile glass jars with rubber seals, which were topped with groundwater from the same well, sealed without headspace and stored at 4 °C until DNA extraction. Investigations at this groundwater location resulted in the identification of benzylsuccinic acid, methylbenzylsuccinic acids, dimethylbenzylsuccinic acids, and alkylsuccinic acids with and without saturation (C5, C6, C7) (SI Table SI-2) (9). DNA Extraction and PCR Amplification. DNA was extracted by a method adapted from Rainey et al. (22). With the exception of the NJ/NY samples, which were only interrogated with primer set 1, all samples were interrogated with each of the nine primer sets. PCR SuperMix (Invitrogen, Carlsbad, CA) was used to set up 50-µL reactions containing 5-50 ng of DNA template, 1 µM of each primer, and 5 units of GoTaq (Promega). PCR conditions were as follows: 95 °C for 3 min followed by 40 cycles of 95 °C for 45 s, 55 °C for one min, 72 °C for two min, and a final extension step at 72 °C for 10 min. When unspecific amplification was observed, the band of the correct size was excised from 1% agarose gels and purified. In some cases, PCR products were reamplified for 15 cycles before cloning using the PCR conditions described above. 16S rRNA genes were amplified using the bacterial primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1525R (5′AAGGAGGTGWTCCARCC-3′) (23) and the 16S rRNA archaeal primers A8F (5′-TCCGGTTGATCCTGCC-3′) (24) and A1538R (5′-CGGTTGGATCACCTC-3′) (25). The 27F and A8F primers included the following 5′-sequencing tag: 5′-GCCTTGCCAGCCCGCTC-3′. PCR SuperMix (Invitrogen, Carlsbad, CA) was used to set up 50-µL reactions containing 5 to 50 ng DNA template, 200 nM of each primer, and 5 units of GoTaq (Promega). PCR conditions were as follows: 95 °C for 2 min, followed by 35 cycles of 96 °C for 30 s, 55 °C for 45 s, 72 °C for 1.2 min, and a final extension step at 72 °C for 10 min.

Construction of 16S rRNA, assA and bssA Gene Clone Libraries. PCR products were gel-purified (Qiagen, Valencia, CA) and cloned into either pCRII or pCR4-TOPO vector (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Colonies were picked into individual wells of 96-well microtiter plates and grown overnight. Inserts were sequenced using the M13R priming site. For the New York/ New Jersey libraries, 24 clones were sequenced for each transformation. For the Fort Lupton assA clone library, 192 colonies were picked. For the remaining assA and bssA clone libraries, 96 clones were sequenced for each. One hundred and ninety-two clones were picked and sequenced for bacterial and archaeal 16S rRNA genes, respectively (i.e., total of 384 partial 16S rRNA gene clones for each sample). After sequencing, reads were trimmed to remove vector and primer sequences before further analysis. Phylogenetic Analysis. Sequences from each respective library were assembled into operational taxonomic units (OTUs) of g97% sequence identity using Lasergene 7.2 (DNASTAR Inc., Madison, WI). For 16S rRNA gene libraries, phylogenetic classification was assigned via the Greengenes Database (26) using the Hugenholtz classification system. The assA/bssA OTUs were aligned with all known assA genes, as well as several known bssA genes. The glycyl radical enzyme, pyruvate formate lyase (PFL) served as the outgroup. A neighbor-joining tree was constructed in MEGA4 (27) using the Tajima Nei distance method, with pairwise deletion and performing 10000 bootstrap replicates. The DNA sequences of bacterial, archaeal and glycyl radical enzyme OTUs are found in GenBank under the accession numbers GU453429 through GU453677.

Results and Discussion To gain a better understanding of the genetic diversity of assA genes, both in the context of defined culture conditions and environmental systems, enrichment cultures growing on short- and long-chain alkane substrates (C3 and C28) and sediments from several hydrocarbon-impacted sites were probed for the presence of assA/bssA genotypes. New York/New Jersey Samples. NY and NJ contain an abundance of sites that have been negatively impacted by petroleum spills. Four contaminated sites were chosen, and it was hypothesized that assA and/or bssA would be present in the anaerobic sediments. Primer set 1 was designed to target both assA and bssA genes and allowed detection of assA in all of the NY and NJ samples (SI Table SI-3, Figure 1). Analysis of 96 sequenced clones resulted in 21 OTUs (>97% identity), all of which were most similar to known assA genes. These data indicated that assA genes are frequently associated with sediments with chronic exposure to petroleum contamination. Although primer set 1 amplifies bssA from isolates known to contain this gene (e.g., Thauera aromatica T1, data not shown), bssA was not detected in any of the NJ/NY samples nor in any of the other samples (see below) using this primer set. The inability to detect bssA in NJ/NY sediments using primer set 1 does not mean that bssA is not present, but it appears as though primer set 1 is a better tool for the detection of assA genes in these systems. Pure Culture. An important goal of this study was to expand the number of reference assA sequences to facilitate improved degenerate primer design. Desulfoglaeba alkanexedens ALDC activates n-alkanes (C6-C12) via fumarate addition (28, 29), and it was hypothesized that this organism would contain an assA-like gene. Primer set 1 was used to amplify a partial assA sequence, and the complete sequence was later obtained from the draft genome of D. alkanexedens. The sequence of ASS-R in D. alkanexedens is 75%, 77%, and 70% identical at the amino acid level to ASS-R2 (strain AK01), ASS-R1 (strain AK-01), and MAS-δ (strain HxN1), respectively. The remaining primer pairs (3 through 9) were

subsequently designed by considering all full-length assA gene sequences (AK-01 assA1, AK-01 assA2, ALDC assA, and HxN1 masD) (SI Table SI-1) and were shown to successfully amplify assA from D. alkanexedens (SI Table SI-3). The assA gene in D. alkanexedens could not be amplified with primer set 2 because of eight mismatches between the assA sequence and the forward bssA primer. Primer set 2 is based on bssA sequences (17), and although it facilitated the detection of assA in strain AK-01 (17), it does not appear to amplify assA easily in other samples. All primer pairs resulted in positive amplification of the assA gene(s) in D. alkenivorans AK-01 (SI Table SI-3). Propane-Utilizing Mixed Culture. Fumarate addition was only recently demonstrated for short-chain n-alkanes. Kniemeyer et al. (30) described the biodegradation of shortchain alkanes (ethane, propane and butane) under sulfatereducing conditions in enrichment cultures derived from marine hydrocarbon seeps. One of the enrichment cultures, Propane60-GuB, utilizes propane under thermophilic conditions and produces iso- and n-propylsuccinates, indicating both subterminal and terminal alkane activation. On the basis of phylogenetic analysis and fluorescent whole-cell hybridization, the culture was shown to be dominated by Desulfotomaculum (92% of cells). The propane-utilizing culture described in the present study appears to be phylogenetically quite similar to Propane60-GuB. Of 143 bacterial clone sequences, 90% are most similar to Desulfotomaculum (identities ranging from 79 to 93%) (Figure 2a). Desulfotomaculum has been detected in several other studies focusing on anaerobic hydrocarbon degradation (16, 31-33). Out of 167 archaeal clones sequenced, 47% are most closely related to Methanococcus, and 35% are most closely related to Halobacteriaceae (Figure 3a). The detection of halophilic Archaea is consistent with previous studies of Zodletone Spring (34). Similar to Propane60-GuB, the propane-utilizing culture described herein also produces iso- and n-propylsuccinates (20). All primer sets were tested to determine whether the culture contains an assA-genotype consistent with the detected fumarate addition metabolites. Primer set 9 resulted in positive identification of one assA genotype (Figure 1, SI Table SI-3). The detection of assA and metabolites derived from fumarate addition provide strong evidence that alkylsuccinate synthase is involved in the anaerobic degradation of propane in this culture. SDB Paraffin-Degrading Enrichment Culture. Paraffins are components of crude oil and consist of straight and branched long-chain alkanes with >16 carbons. During oil recovery, reduced temperatures and pressures result in the precipitation of paraffins and separation of the lighter, more volatile hydrocarbons (35, 36). This results in an annual loss of millions of dollars because of removal costs and lost production (37). Additionally, the conversion of long-chain alkanes to methane in oil reservoirs is of significant interest (38-41) and has important implications for optimizing energy recovery. Therefore, a greater understanding of microbial transformation of these compounds is desired. Very little is known about anaerobic biotransformation of paraffins. Caldwell et al. (42) reported the biodegradation of C15-C34 n-alkanes under sulfate-reducing conditions in an enrichment culture amended with artificially weathered crude oil, and Townsend et al. (43) also reported degradation of C13-C34 n-alkanes in artificially weathered crude oil under both sulfate-reducing and methanogenic conditions. It has been hypothesized that paraffins may be activated in a similar manner as shorter alkanes, but metabolites have not yet been reported. A C28-alkylsuccinate metabolite has, for example, not been detected in the SDB consortium, but the discovery of assA (17, 19) provides an opportunity to interrogate VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Neighbor-joining dendrogram of assA and bssA genes from reference strains, enrichment cultures, and environmental samples. Sequences were aligned using the ClustalW algorithm within Megalign (DNASTAR Inc., Madison, WI). The alignment was exported to MEGA4 (27), and the tree was constructed using the Tajima-Nei distance method (scale bar), with pairwise deletion and performing 10000 bootstrap replicates. Bootstrap values below 65 are not shown. Both synonymous and nonsynonymous substitutions were considered. The dendrogram was rooted with pyruvate formate lyase (PFL) paralogues as an outgroup (not shown). Numbers in parentheses represent GenBank accession numbers. Sequences in bold represent the available sequences for reference strains at the beginning of this study. Abbreviations: ass (alkylsuccinate synthase), mas (methylalkylsuccinate synthase) bss (benzylsuccinate synthase), tut (“toluene-utilizing”; benzylsuccinate synthase), and nms (naphthylmethylsuccinate synthase).

anaerobic paraffin-degrading cultures, such as SDB, for genes that may be associated with alkane degradation. 7290

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Phylogenetic analysis of 130 bacterial 16S rRNA sequences from SDB resulted in 44 OTUs, 35 of which belong to the

FIGURE 2. Class-level phylogenetic composition of bacterial 16S rRNA gene clone libraries from the consortia and environmental samples. Assignments to class-level divisions were generated using the Greengenes alignment tool and Hugenholtz classification system. The numbers following classifications indicate the number of OTUs found in the respective classification category. Curved lines surrounding the pie charts indicate which OTUs are associated with individual class-level assignments. (A) Propane-degrading mixed culture. (B) SDB paraffin-degrading enrichment culture. (C) Fort Lupton site sediment. (D) Casper site sediment. N denotes the number of high quality sequence reads that were included in the analysis.

FIGURE 3. Class-level phylogenetic composition of archaeal 16S rRNA gene clone libraries from the consortia and environmental samples. Assignments to class-level divisions were generated using the Greengenes alignment tool and Hugenholtz classification system. The numbers following classifications indicate the number of OTUs found in the respective classification category. Curved lines surrounding the pie charts indicate which OTUs are associated with individual class-level assignments. (A) Propane-degrading mixed culture. (B) SDB paraffin-degrading enrichment culture. (C) Fort Lupton site sediment. (D) Casper site sediment. N denotes the number of high quality sequence reads that were included in the analysis. class deltaproteobacteria (Figure 2b). Interestingly, seven of the deltaproteobacterial OTUs (60 sequences) are most closely related to the genus Syntrophus. Syntrophus spp. have been implicated in methanogenic alkane degradation in other studies (40, 44). Phylogenetic analysis of 148 archaeal clone

sequences yielded 17 OTUs (Figure 3b). Fifty-eight sequences comprised a single OTU classified within the Halobacteria, and 64 sequences comprised a single OTU within the class Thermococci. However, these classifications are not consistent with the culture conditions and are only VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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supported by very low identities (64-74%), indicating the presence of poorly described archaeal taxa in the analyzed consortium. Analogous observations were made using a pyrosequencing approach described by Hamady et al. (45), yielding equally low identities to known archaeal classes (data not shown). In the absence of metabolite information, assA was targeted to support the hypothesis that fumarate addition may be involved in anaerobic paraffin degradation. Five assA genotypes were detected in SDB using primer sets 5 and 9 (Figure 1, SI Table SI-3). OTU 1 was detected using both primer pairs, whereas OTUs 2, 3, 4, and 5 were unique to primer set 9. These observations suggest that fumarate addition may be involved in anaerobic paraffin degradation. However, in the absence of metabolite evidence, the possible role of assA genes in SDB remains to be demonstrated. Fort Lupton, CO. Archived sediment from a gascondensate contaminated aquifer near Fort Lupton, CO was interrogated with respect to the 16S rRNA gene and the assA and bssA genes. Phylogenetic analysis resulted in the resolution of 61 bacterial OTUs, 18 of which were classified as gammaproteobacteria (Figure 2c). Only 13 archaeal OTUs were resolved (89% of sequences were classified as Halobacteria) (Figure 3c). Archaeal classifications into Halobacteria, Thermoplasmata, and Thermococci were based on low sequence identities (59-74%), indicating the presence of poorly characterized archaeal taxa. Three assA and two bssA OTUs were detected in samples collected from Fort Lupton using primer sets 1 and 2, respectively (Figure 1, SI Table SI-3). Interestingly, two additional OTUs were detected with primer pair 2 that form a unique clade of putative glycyl radical enzymes not yet represented in the database. Sequences within this clade are 47%, 42%, and 31% identical at the amino acid level to ASSR, BSS-R, and pyruvate formate lyase (PFL), respectively, and contain the conserved cysteine residue found in all glycyl radical enzymes (46). The presence of assA and bssA genes in Fort Lupton sediment corroborates previous work showing that both alkylsuccinates and methylbenyzlsuccinates are present in site groundwater collected from the same location and at the same time as the sediment assayed in this study (SI Table SI-2) (6-8). These data represent the first example of the identification of functional genes associated with anaerobic alkane activation in conjunction with the in situ detection of alkylsuccinate metabolites. Casper, WY. Archived sediment from a contaminated aquifer in Casper, WY, was interrogated with respect to the 16S rRNA gene and the assA and bssA genes. Phylogenetic analysis of 166 bacterial clones yielded 15 OTUs, of which 103 sequences belong to a single gammaproteobacterial OTU most closely related to an unclassified genus within the order Rhodocyclales (Figure 2d). Rhodocyclales include the genera Azoarcus and Thauera, which are known to be metabolically very versatile with respect to carbon substrate utilization and capable of utilizing aromatic hydrocarbons as sole sources of carbon and energy (for review, see ref 47). Archaeal sequence reads (141) assembled into 13 OTUs (Figure 3d). The two dominant taxa were classified into Thermoplasmata and Archaeoglobi, but as with other samples described here, identities to well-described archaeal taxa were low (between 70 and 73%, respectively). Based on the detection of C5-alkylsuccinate, unsaturated alkylsuccinates (C5, C6, and C7), and benzylsuccinates (9) (SI Table SI-2), it was hypothesized the both assA and bssA should be present in the Casper sediment. Primer set 2 resulted in the positive amplification of bssA and the detection of six bssA OTUs (Figure 1, SI Table SI-3). However, assA was not detected despite the presence of n-pentylsuccinate and unsaturated alkylsuccinates in groundwater extracts. The failure to detect assA may be a reflection of primer specificity, 7292

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heterogeneity in sediment samples because of microniches, or the relative populations of alkanes degraders versus the total population. These results underscore the difficulty of characterizing anaerobic hydrocarbon degraders in complex systems and the need for combining multiple approaches in field investigations. Environmental Relevance. The recent discovery of alkylsuccinate synthase (17, 19) and its role in anaerobic alkane degradation have provided an opportunity to gain insight with respect to contaminated sites as well as uncharacterized enrichment cultures/isolates. Primer sets 1, 2, 5, and 9 resulted in the positive detection of assA and bssA genotypes in environmental samples and enrichments, whereas primer sets 3, 4, and 6-8 were only useful for the detection of assA in known isolates (SI Table SI-3). Phylogenetic analysis indicates that assA genes detected here are highly diverse. On the basis of known data, it was not possible to design a single primer pair that serves to amplify the full range of known assA-type genes. Additional complete sequences for assA would therefore be very valuable to iterative primer design efforts and may also shed light regarding the substrate specificity of ASS enzymes. An important tenet of monitored natural or enhanced attenuation is that the desired biological activity is not evident in nonimpacted environments. Similarly, the detection of catabolic genes is most useful when it coincides with observed trends in biodegradation (i.e., the in situ detection of genes in contaminated areas vs their absence in nonimpacted sites). The availability of environmental metagenome databases (NCBI, JGI IMG/M, and MG-RAST) (48-50) provides an opportunity for in silico interrogation of a large diversity of environmental conditions. For example, more than 415 metagenomes containing approximately 5,700 genome equivalents of sequence data were searched in the MG-RAST database for assA and bssA genes. Within these, only two sequence matches to assA or bssA of more than 100 amino acids were detected: one in the metagenome of the human gut microbiome and one in a whale fall bone metagenome (49). In addition, several shorter matches (