Anaerobic Degradation Pathways Research Articles

Comprehensive genomic and transcriptomic analyses of the anaerobic degradation of microcystin in Alcaligenes faecalis D04.

Microcystin LR (MC-LR) pollution is a serious threat to aquatic ecosystems and public health in China and is an environmental problem that urgently needs to be solved. However, few studies have investigated the anaerobic degradation pathway and related molecular biological mechanisms of MC-LR. In this study, a bacterium capable of degrading MC-LR with a degradation efficiency of 0.303 µg/mL/d under anaerobic conditions was isolated from water. The strain was identified as Alcaligenes and named Alcaligenes faecalis D04. Two new anaerobic degradation products, one pentapeptide (Adda-Glu-Mdha-Ala-Leu) and one tripeptide (Adda-Glu-Mdha), were identified by chromatography and mass spectrometry, and two new anaerobic degradation pathways for microcystins were proposed. This study revealed a new connection between related functional genes (mblH, ridA, paaA, livI, soxR, gltD, marR, etc.) and bacterial degradation functions through the analysis of multiomics data. Real-time quantitative PCR analysis verified that the expression trends of the differentially expressed genes were consistent with the transcriptomic data. Our study aimed to elucidate the anaerobic degradation pathway and molecular regulatory mechanism of MC-LR in Alcaligenes faecalis D04, which offers important practical significance for microbial strategies to prevent and regulate microcystin contamination.

A CoA-Transferase and Acyl-CoA Dehydrogenase Convert 2-(Carboxymethyl)cyclohexane-1-Carboxyl-CoA During Anaerobic Naphthalene Degradation.

The CoA thioester of 2-(carboxymethyl)cyclohexane-1-carboxylic acid has been identified as a metabolite in anaerobic naphthalene degradation by the sulfate-reducing culture N47. This study identified and characterised two acyl-CoA dehydrogenases (ThnO/ThnT) and an intramolecular CoA-transferase (ThnP) encoded within the substrate-induced thn operon, which contains genes for anaerobic degradation of naphthalene. ThnP is a CoA transferase belonging to the family I (Cat 1 subgroup) that catalyses the intramolecular CoA transfer from the carboxyl group of 2-(carboxymethyl)cyclohexane-1-carboxyl-CoA to its carboxymethyl moiety, forming 2-carboxycyclohexylacetyl-CoA. Neither acetyl-CoA nor succinyl-CoA functions as an exogenous CoA donor for this reaction. The flavin-dependent homotetrameric dehydrogenase ThnO is specific for (1R,2R)-2-carboxycyclohexylacetyl-CoA with an apparent Km value of 61.5 μM, whereas ThnT is a promiscuous enzyme catalysing the same reaction at lower rates. Identifying these three enzymes confirmed the involvement of the thn gene cluster in the anaerobic naphthalene degradation pathway. This study establishes a modified metabolic pathway for anaerobic naphthalene degradation upstream of 2-(carboxymethyl)cyclohexane-1-carboxyl-CoA and provides further insight into the subsequent second-ring cleavage reaction.

Disentangling the microbial genomic traits associated with aromatic hydrocarbon degradation in a jet fuel-contaminated aquifer.

Spills of petroleum or its derivatives in the environment lead to an enrichment of microorganisms able to degrade such compounds. The interactions taking place in such microbial communities are complex and poorly understood, since they depend on multiple factors, including diversity and metabolic potential of the microorganisms and a broad range of fluctuating environmental conditions. In our previous study, a complete characterization, based on high-throughput sequencing, was performed in a jet-fuel plume using soil samples and in in-situ microcosms amended with hydrocarbons and exposed for 120days. Herein, we propose a metabolic model to describe the monoaromatic hydrocarbon degradation process that takes place in such jet-fuel-contaminated sites, by combining genome-centered analysis, functional predictions, and flux balance analysis (FBA). In total, twenty high/medium quality MAGs were recovered; three of them assigned to anaerobic bacteria (Thermincolales, Geobacter and Pelotomaculaceace) and one affiliated to the aerobic bacterium Acinetobacter radioresistens, potentially the main players of hydrocarbon degradation in jet-fuel plumes. Taxonomic assignment of the genes indicated that a putative new species of Geobacteria has the potential for anaerobic degradation pathway, while the Pelotomaculaceae and Thermincolales members probably act via syntrophy oxidizing acetate and hydrogen (fermentation products of oil degradation) via sulfate and/or nitrate reduction.

Metagenomic survey reveals hydrocarbon biodegradation potential of Canadian high Arctic beaches

BackgroundDecreasing sea ice coverage across the Arctic Ocean due to climate change is expected to increase shipping activity through previously inaccessible shipping routes, including the Northwest Passage (NWP). Changing weather conditions typically encountered in the Arctic will still pose a risk for ships which could lead to an accident and the uncontrolled release of hydrocarbons onto NWP shorelines. We performed a metagenomic survey to characterize the microbial communities of various NWP shorelines and to determine whether there is a metabolic potential for hydrocarbon degradation in these microbiomes.ResultsWe observed taxonomic and functional gene evidence supporting the potential of NWP beach microbes to degrade various types of hydrocarbons. The metagenomic and metagenome-assembled genome (MAG) taxonomy showed that known hydrocarbon-degrading taxa are present in these beaches. Additionally, we detected the presence of biomarker genes of aerobic and anaerobic degradation pathways of alkane and aromatic hydrocarbons along with complete degradation pathways for aerobic alkane degradation. Alkane degradation genes were present in all samples and were also more abundant (33.8 ± 34.5 hits per million genes, HPM) than their aromatic hydrocarbon counterparts (11.7 ± 12.3 HPM). Due to the ubiquity of MAGs from the genus Rhodococcus (23.8% of the MAGs), we compared our MAGs with Rhodococcus genomes from NWP isolates obtained using hydrocarbons as the carbon source to corroborate our results and to develop a pangenome of Arctic Rhodococcus. Our analysis revealed that the biodegradation of alkanes is part of the core pangenome of this genus. We also detected nitrogen and sulfur pathways as additional energy sources and electron donors as well as carbon pathways providing alternative carbon sources. These pathways occur in the absence of hydrocarbons allowing microbes to survive in these nutrient-poor beaches.ConclusionsOur metagenomic analyses detected the genetic potential for hydrocarbon biodegradation in these NWP shoreline microbiomes. Alkane metabolism was the most prevalent type of hydrocarbon degradation observed in these tidal beach ecosystems. Our results indicate that bioremediation could be used as a cleanup strategy, but the addition of adequate amounts of N and P fertilizers, should be considered to help bacteria overcome the oligotrophic nature of NWP shorelines.

Transcriptional regulation of the anaerobic 3-hydroxybenzoate degradation pathway in Aromatoleum sp. CIB

Phenolic compounds are commonly found in anoxic environments, where they serve as both carbon and energy sources for certain anaerobic bacteria. The anaerobic breakdown of m-cresol, catechol, and certain lignin-derived compounds yields the central intermediate 3-hydroxybenzoate/3-hydroxybenzoyl-CoA. In this study, we have characterized the transcription and regulation of the hbd genes responsible for the anaerobic degradation of 3-hydroxybenzoate in the β-proteobacterium Aromatoleum sp. CIB. The hbd cluster is organized in three catabolic operons and a regulatory hbdR gene that encodes a dimeric transcriptional regulator belonging to the TetR family. HbdR suppresses the activity of the three catabolic promoters (PhbdN, PhbdE and PhbdH) by binding to a conserved palindromic operator box (ATGAATGAN4TCATTCAT). 3-Hydroxybenzoyl-CoA, the initial intermediate of the 3-hydroxybenzoate degradation pathway, along with benzoyl-CoA, serve as effector molecules that bind to HbdR inducing the expression of the hbd genes. Moreover, the hbd genes are subject to additional regulation influenced by the presence of non-aromatic carbon sources (carbon catabolite repression), and their expression is induced in oxygen-deprived conditions by the AcpR transcriptional activator. The prevalence of the hbd cluster among members of the Aromatoleum/Thauera bacterial group, coupled with its association with mobile genetic elements, suggests acquisition through horizontal gene transfer. These findings significantly enhance our understanding of the regulatory mechanisms governing the hbd gene cluster in bacteria, paving the way for further exploration into the anaerobic utilization/valorization of phenolic compounds derived from lignin.

Mechanisms and high-value applications of phthalate isomers degradation pathways in bacteria.

Phthalate isomers are key intermediates in the biodegradation of pollutants including waste polyethylene terephthalate (PET) plastics and plasticizers. So far, an increasing number of phthalate isomer-degrading strains have been isolated, and their degradation pathways show significant diversity. In this paper, we comprehensively review the current status of research on the degrading bacteria, degradation characteristics, aerobic and anaerobic degradation pathways, and degradation genes (clusters) of phthalate isomers, and discuss the current shortcomings and challenges. Moreover, the degradation process of phthalate isomers produces many important aromatic precursor molecules, which can be used to produce higher-value derivative chemicals, and the modification of their degradation pathways holds good prospects. Therefore, this review also highlights the current progress made in modifying the phthalate isomer degradation pathway and explores its potential for high-value applications.

Oxamic transcarbamylase of Escherichia coli is encoded by the three genes allFGH (formerly fdrA, ylbE, and ylbF).

Escherichia coli uses allantoin as the sole nitrogen source during anaerobic growth. In the final step of allantoin degradation, oxamic transcarbamylase (OXTCase) converts oxalurate to carbamoyl phosphate (CP) and oxamate. The activity of this enzyme was first measured in Streptococcus allantoicus in the 1960s, but no OXTCase enzyme or the encoding gene(s) have been found in any strain. This study discovered that allFGH (fdrA, ylbE, and ylbF) are the genes that encode the global orphan enzyme OXTCase. The three genes form an operon together with allK (ybcF), encoding catabolic carbamate kinase. The allFGHK operon is located directly downstream of the allECD operon that encodes enzymes for the preceding steps of OXTCase. The OXTCase kinetic parameters were analyzed using the purified protein composed of AllF-AllG-AllH (FdrA-YlbE-YlbF); for the substrate CP, KM and Vmax were 1.3 mM and 15.4 U/mg OXTCase, respectively, and for the substrate oxamate, they were 36.9 mM and 27.0 U/mg OXTCase. In addition, the OXTCase encoded by the three genes is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase, ornithine transcarbamylase, and YgeW transcarbamylase. The present study elucidated the anaerobic allantoin degradation pathway of E. coli. Therefore, we suggest that the genes fdrA, ylbE, and ylbF are renamed allF, allG, and allH, respectively.IMPORTANCEThe anaerobic allantoin degradation pathway of Escherichia coli includes a global orphan enzyme, oxamic transcarbamylase (OXTCase), which converts oxalurate to carbamoyl phosphate and oxamate. This study found that the allFGH (fdrA, ylbE, and ylbF) genes encode OXTCase. The OXTCase activity and kinetics were successfully determined with purified recombinant AllF-AllG-AllH (FdrA-YlbE-YlbF). This OXTCase is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase (ATCase), ornithine transcarbamylase (OTCase), and YgeW transcarbamylase (YTCase). In addition, OXTCase activity requires three genes, whereas ATCase is encoded by two genes, and OTCase and YTCase are encoded by a single gene. The current study discovered OXTCase, the last unknown step in allantoin degradation, and this enzyme is a new member of the transcarbamylase group that was previously unknown.

Degradation of Atrazine by an Anaerobic Microbial Consortium Enriched from Soil of an Herbicide-Manufacturing Plant.

Atrazine is an important herbicide that has been widely used for weed control in recent decades. However, with the extensive use of atrazine, its residue seriously pollutes the environment. Therefore, the microbial degradation and detoxification of atrazine have received extensive attention. To date, the aerobic degradation pathway of atrazine has been well studied; however, little is known about its anaerobic degradation in the environment. In this study, an anaerobic microbial consortium capable of efficiently degrading atrazine was enriched from soil collected from an herbicide-manufacturing plant. Six metabolites including hydroxyatrazine, deethylatrazine, N-isopropylammelide, deisopropylatrazine, cyanuric acid, and the novel metabolite 4-ethylamino-6-isopropylamino-1,3,5-triazine (EIPAT) were identified, and two putative anaerobic degradation pathways of atrazine were proposed: a hydrolytic dechlorination pathway is similar to that seen in aerobic degradation, and a novel pathway initiated by reductive dechlorination. During enrichment, Denitratisoma, Thiobacillus, Rhodocyclaceae_unclassified, Azospirillum, and Anaerolinea abundances significantly increased, dominating the enriched consortium, indicating that they may be involved in atrazine degradation. These findings provide valuable evidence for elucidating the anaerobic catabolism of atrazine and facilitating anaerobic remediation of residual atrazine pollution.

Genome-Centric Metatranscriptomics Reveals Multiple Co-occurring Routes for Hydrocarbon Degradation in Chronically Contaminated Marine Microbial Mats.

Long-term hydrocarbon pollution is a devious threat to aquatic and marine ecosystems. However, microbial responses to chronic pollution remain poorly understood. Combining genome-centric metagenomic and metatranscriptomic analyses of microbial mat samples that experienced chronic hydrocarbon pollution for more than 80 years, we analyzed the transcriptomic activity of alkane and aromatic hydrocarbon degradation pathways at the population level. Consistent with the fluctuating and stratified redox conditions of the habitat, both aerobic and anaerobic hydrocarbon degradation pathways were expressed by taxonomically and metabolically contrasted lineages including members of Bacteroidiales, Desulfobacteraceae, Pseudomonadales; Alcanivoraceae and Halieaceae populations with (photo)-heterotrophic, sulfur- and organohalide-based metabolisms, providing evidence for the co-occurrence and activity of aerobic and anaerobic hydrocarbon degradation pathways in shallow marine microbial mats. In addition, our results suggest that aerobic alkane degradation in long-term pollution involved bacterial families that are naturally widely distributed in marine habitats, but hydrocarbon concentration and composition were found to be a strong structuring factor of their intrafamily diversity and transcriptomic activities.

Anaerobic Degradation of Dicamba via a Novel Catabolic Pathway by a Consortium Enriched from Deep Paddy Soil.

Dicamba is widely used in the paddy field to control broadleaf weeds. Dicamba easily migrates to deep soil, which is anoxic; however, the anaerobic catabolism of dicamba in paddy soil is still unknown. In this study, an anaerobic dicamba-degrading consortium was enriched from deep paddy soil. The consortium completely degraded 0.83 mM dicamba within 7 days. Five metabolites were identified, one of which is a new metabolite, 2,5-dichlorophenol, and a novel anaerobic dicamba degradation pathway was proposed. 2.5 mM dicamba, 1.5-2.0% NaCl, and 20 mM electron acceptors Na2SO4, NaNO3, and FeCl3, and 0.5 mM or more of metabolites 3-CP and 2,5-DCP strongly inhibited the degradation efficiency. During enrichment, the microbial community of the consortium was significantly changed with OTU numbers, and diversity decreased. The study is valuable to elucidate the catabolism and ecotoxicology studies of dicamba in paddy soil and to facilitate the engineering application of anaerobic technology to treat dicamba-manufacturing wastewater.

Microbial electricity-driven anaerobic phenol degradation in bioelectrochemical systems

Microbial electrochemical technologies have been extensively employed for phenol removal. Yet, previous research has yielded inconsistent results, leaving uncertainties regarding the feasibility of phenol degradation under strictly anaerobic conditions using anodes as sole terminal electron acceptors. In this study, we employed high-performance liquid chromatography and gas chromatography-mass spectrometry to investigate the anaerobic phenol degradation pathway. Our findings provide robust evidence for the purely anaerobic degradation of phenol, as we identified benzoic acid, 4-hydroxybenzoic acid, glutaric acid, and other metabolites of this pathway. Notably, no typical intermediates of the aerobic phenol degradation pathway were detected. One-chamber reactors (+0.4 V vs. SHE) exhibited a phenol removal rate of 3.5 ± 0.2 mg L−1 d−1, while two-chamber reactors showed 3.6 ± 0.1 and 2.6 ± 0.9 mg L−1 d−1 at anode potentials of +0.4 and + 0.2 V, respectively. Our results also suggest that the reactor configuration certainly influenced the microbial community, presumably leading to different ratios of phenol consumers and microorganisms feeding on degradation products.

Metagenomic and Genomic Sequences from a Methanogenic Benzene-Degrading Consortium

Draft and complete metagenome assembled genomes (MAGs) were created from multiple metagenomic assemblies of DGG-B, a strictly anaerobic, stable mixed microbial consortium that degrades benzene completely to methane and CO2. Our objective was to obtain closed genome sequences of benzene-fermenting bacteria to enable the elucidation of their elusive anaerobic benzene degradation pathway.

Metagenomic Binning Revealed Microbial Shifts in Anaerobic Degradation of Phenol with Hydrochar and Pyrochar

Phenolic compounds, which are difficultly degraded, are one of the main toxic threats faced in the anaerobic digestion (AD) process. It has previously been reported that hydrochar/pyrochar produced by the hydrothermal liquefaction/pyrolysis of biomass can enhance AD by promoting direct interspecific electron transfer (DIET). The present study investigated the effects of different hydrochars and pyrochars on the anaerobic degradation of phenol and provided deep insights into the related micro-organisms at the species level through genome-centric metagenomic analysis. Compared with the control experiment, the addition of hydrochar and pyrochar shortened the lag time. However, hydrochar created a large increase in the maximum methane production rate (Rm) (79.1%) compared to the control experiments, while the addition of pyrochar decreased Rm. Metagenomic analysis showed that the addition of carbon materials affected the relative abundance of genes in the phenol anaerobic degradation pathway, as well as the species and relative abundance of phenol degrading micro-organisms. The relative abundance of key genes for phenol degradation, such as bsdB, bamB, oah, etc., under the action of hydrochar was higher than those under the action of pyrochar. In addition, hydrochar-enriched phenol degradation-related bacteria (Syntrophus aciditrophicus, etc.) and methanogen (Methanothrix soehngenii, etc.). These micro-organisms might improve the phenol degradation efficiency by promoting DIET. Therefore, hydrochar had a more significant effect in promoting anaerobic degradation of phenol.

Functional response of microbial communities in lab-controlled oil-contaminated marine sediment.

Crude oil contamination is one of the biggest problems in modern society. As oil enters into contact with the environment, especially if the point of contact is a body of water, it begins a weathering process by mixing and spreading. This is dangerous to local living organisms' communities and can impact diversity. However, despite unfavorable conditions, some microorganisms in these environments can survive using hydrocarbons as a nutrient source. Thus, understanding the local community dynamics of contaminated areas is essential. In this work, we analyzed the 16S rRNA amplicon sequencing and metatranscriptomic data of uncontaminated versus contaminated shallow marine sediment from publicly available datasets. We investigated the local population's taxonomic composition, species diversity, and fluctuations over time. Co-expression analysis coupled with functional enrichment showed us a prevalence of hydrocarbon-degrading functionality while keeping a distinct transcriptional profile between the late stages of oil contamination and the uncontaminated control. Processes related to the degradation of aromatic compounds and the metabolism of propanoate and butanoate were coupled with evidence of enhanced activity such as flagellar assembly and two-component system. Many enzymes of the anaerobic toluene degradation pathways were also enriched in our results. Furthermore, our diversity and taxonomical analyses showed a prevalence of the class Desulfobacteria, indicating interesting targets for bioremediation applications on marine sediment.

Subtle sequence differences between two interacting σ54 -dependent regulators lead to different activation mechanisms.

In the strictly anaerobic nitrate reducing bacterium Aromatoleum anaerobium, degradation of 1,3-dihydroxybenzene (1,3-DHB, resorcinol) is controlled by two bacterial enhancer-binding proteins (bEBPs), RedR1 and RedR2, which regulate the transcription of three σ54 -dependent promoters controlling expression of the pathway. RedR1 and RedR2 are identical over their length except for their N-terminal tail which differ in sequence and length (six and eight residues, respectively), a single change in their N-terminal domain (NTD), and nine non-identical residues in their C-terminal domain (CTD). Their NTD is composed of a GAF and a PAS domain connected by a linker helix. We show that each regulator is controlled by a different mechanism: whilst RedR1 responds to the classical NTD-mediated negative regulation that is released by the presence of its effector, RedR2 activity is constitutive and controlled through interaction with BtdS, an integral membrane subunit of hydroxyhydroquinone dehydrogenase carrying out the second step in 1,3-DHB degradation. BtdS sequesters the RedR2 regulator to the membrane through its NTD, where a four-Ile track in the PAS domain, interrupted by a Thr in RedR1, and the N-terminal tail are involved. The presence of 1,3-DHB, which is metabolized to hydroxybenzoquinone, releases RedR2 from the membrane. Most bEBPs assemble into homohexamers to activate transcription; we show that hetero-oligomer formation between RedR1 and RedR2 is favoured over homo-oligomers. However, either an NTD-truncated version of RedR1 or a full-length RedR2 are capable of promoter activation on their own, suggesting they should assemble into homohexamers in vivo. We show that promoter DNA behaves as an allosteric effector through binding the CTD to control ΔNTD-RedR1 multimerization and activity. Overall, the regulation of the 1,3-DHB anaerobic degradation pathway can be described as a novel mode of bEBP activation and assembly.

Elucidation of the biodegradation pathways of bis(2-hydroxyethyl) terephthalate and dimethyl terephthalate under anaerobic conditions revealed by enrichment culture and microbiome analysis

With the globally rising usage of plastics, including polyethylene terephthalate (PET), the environmental risk that disposal of waste plastics to landfills and discharge of microplastics to the marine environment pose have also increased. For example, observation of animal ingestion of fragmented waste plastics (micro- and nano-plastics) has driven awareness for the need of proper environmental risk assessment. In evaluating the biodegradability of PET-derived byproducts and their precursors, most work has focused on hydrolytic enzymes and aerobic organisms that possess such genes, but only few reports on biodegradation in the absence of oxygen (i.e., anaerobic) are available. Here, to elucidate the fate of PET-derived materials under anaerobic environments, a sludge-derived microbial community was cultured with bis(2-hydroxyethyl) terephthalate (BHET) as a model substrate for byproducts of PET degradation and dimethyl terephthalate (DMT) as a potential environmental pollutant discharged from the PET manufacturing process. Metagenome- and metabolome-informed microbiome analyses identified anaerobic BHET and DMT degradation pathways, uncultured organisms affiliated with Spirochaetota and Negativicutes predominant in the BHET-fed cultures, and Methanomethylovorans and Treponema_G predominant in the DMT-fed cultures. Metagenomic analyses newly identified three BHET-degrading and two DMT-degrading enzymes from the genomes of Spirochaeota. In addition, the Negativicutes in the BHET enrichment cultures possessed genes for acetogenically metabolizing EG and/or ethanol. Overall, this study successfully established anaerobic BHET- and DMT-degrading microbial consortia and newly proposed these degradation mechanisms under anaerobic conditions. This study indicated that the cultivation, microbiome, and metabolome analyses can be powerful tools for elucidating consortia capable of degrading plastics-associated waste compounds and the relevant metabolic mechanisms.

Anaerobic Hydroxyproline Degradation Involving C-N Cleavage by a Glycyl Radical Enzyme.

Hydroxyprolines are highly abundant in nature as they are components of many structural proteins and osmolytes. Anaerobic degradation of trans-4-hydroxy-l-proline (t4L-HP) was previously found to involve the glycyl radical enzyme (GRE) t4L-HP dehydratase (HypD). Here, we report a pathway for anaerobic hydroxyproline degradation that involves a new GRE, trans-4-hydroxy-d-proline (t4D-HP) C-N-lyase (HplG). In this pathway, cis-4-hydroxy-l-proline (c4L-HP) is first isomerized to t4D-HP, followed by radical-mediated ring opening by HplG to give 2-amino-4-ketopentanoate (AKP), the first example of a ring opening reaction catalyzed by a GRE 1,2-eliminase. Subsequent cleavage by AKP thiolase (OrtAB) yields acetyl-CoA and d-alanine. We report a crystal structure of HplG in complex with t4D-HP at a resolution of 2.7 Å, providing insights into its catalytic mechanism. Different from HypD commonly identified in proline-reducing Clostridia, HplG is present in other types of fermenting bacteria, including propionate-producing bacteria, underscoring the diversity of enzymatic radical chemistry in the anaerobic microbiome.

Pulling needles out of a haystack: Subtractive Community Metatranscriptomics retrieves anaerobic o-xylene degradation pathway genes out of a mixed microbial culture

For many contaminants, biomarker genes are unknown or assays are unavailable, and most biomarker assays target the first pathway step. Herein, we obtained sequences for all of the genes in a previously hypothesized o-xylene degradation pathway based on similarities to analogous genes in a known toluene degradation pathway. Comparative metatranscriptomics resulted in sequences for genes annotated as bssA, bbsEF, bbsCD, and bbsB, while genes for bbsG and bbsH were notably missing. Prokaryotic Suppressive Subtractive Hybridization PCR cDNA Subtraction (Prokaryotic SSH-PCR cDNA Subtraction) was applied for the first time to a mixed-species microbiome to enrich abundances of genes up-regulated during o-xylene degradation prior to metatranscriptomic sequencing. The subtracted metatranscriptome was sequenced using the MinION; this approach was highly effective at retrieving sequences for biodegradation genes including the previously missing bbsG and bbsH. Reverse transcription quantitative PCR (RT-qPCR) analysis confirmed up-regulation. Thus, data reported herein lend credence to the previously hypothesized anaerobic o-xylene degradation pathway, and new biomarker assays are presented. A novel biomarker development tool for mixed species systems, Subtractive Community Metatranscriptomics (SCM), is demonstrated.

Subsurface hydrocarbon degradation strategies in low- and high-sulfate coal seam communities identified with activity-based metagenomics

Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study—subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes—offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.

CANT-HYD: A Curated Database of Phylogeny-Derived Hidden Markov Models for Annotation of Marker Genes Involved in Hydrocarbon Degradation

Many pathways for hydrocarbon degradation have been discovered, yet there are no dedicated tools to identify and predict the hydrocarbon degradation potential of microbial genomes and metagenomes. Here we present the Calgary approach to ANnoTating HYDrocarbon degradation genes (CANT-HYD), a database of 37 HMMs of marker genes involved in anaerobic and aerobic degradation pathways of aliphatic and aromatic hydrocarbons. Using this database, we identify understudied or overlooked hydrocarbon degradation potential in many phyla. We also demonstrate its application in analyzing high-throughput sequence data by predicting hydrocarbon utilization in large metagenomic datasets from diverse environments. CANT-HYD is available at https://github.com/dgittins/CANT-HYD-HydrocarbonBiodegradation.