Advancing biomarkers for anaerobic o-xylene biodegradation via metagenomic analysis of a methanogenic consortium
Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.
KeywordsMetagenomics Methanogenic Biomarkers Fumarate-adding enzymes O-xylene Enzyme homology modeling
Thanks to Elizabeth Edwards and Fei Luo for supplying the enrichment culture and for assistance with culturing. Thanks to Jan Leach for the use of her qPCR machine while ours was repaired, and to Jillian Lang for assistance with their machine. Thanks to Diana Marcela Nuñez Hernandez for conducting some gas chromatography measurements. Thanks to Jennifer Steyaert for conducting preliminary protein structural modeling.
This project was funded by NSF CBET 1438660.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Alneberg JS, de Bruijn I, Hugerth L, Andersson A (2014) Mapping reads and quantifying genes. Metagenomics Workshop SciLifeLab. https://metagenomics-workshop.readthedocs.io/en/latest/annotation/quantification.html. Accessed Jan 11 2017
- Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402Google Scholar
- Beller HR, Kane SR, Legler TC, McKelvie JR, Lollar BS, Pearson F, Balser L, MacKay DM (2008) Comparative assessments of benzene, toluene, and xylene natural attenuation by quantitative polymerase chain reaction analysis of a catabolic gene, signature metabolites, and compound-specific isotope analysis. Environ Sci Technol 42(16):6065–6072. https://doi.org/10.1021/es8009666 Google Scholar
- Boyle B, Dallaire N, MacKay J (2009) Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR. BMC Biotechnol 9:75Google Scholar
- Bozinovski D, Taubert M, Kleinsteuber S, Richnow HH, von Bergen M, Vogt C, Seifert J (2014) Metaproteogenomic analysis of a sulfate-reducing enrichment culture reveals genomic organization of key enzymes in the m-xylene degradation pathway and metabolic activity of proteobacteria. Syst Appl Microbiol 37(7):488–501. https://doi.org/10.1016/j.syapm.2014.07.005 Google Scholar
- Edwards EA, Grbicgalic D (1994) Anaerobic degradation of toluene and o-xylene by a methanogenic consortium. Appl Environ Microbiol 60(1):313–322Google Scholar
- Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, M-y S, Pieper U, Sali A (2006) Comparative protein structure modeling using modeller current protocols in bioinformatics. WileyGoogle Scholar
- Harms G, Zengler K, Rabus R, Aeckersberg F, Minz D, Rossello-Mora R, Widdel F (1999) Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria. Appl Environ Microbiol 65(3):999–1004Google Scholar
- Jarling R, Kühner S, Basílio Janke E, Gruner A, Drozdowska M, Golding B, Rabus R, Wilkes H (2015) Versatile transformations of hydrocarbons in anaerobic bacteria: substrate ranges and regio- and stereo-chemistry of activation reactions. Front Microbiol 6(880). https://doi.org/10.3389/fmicb.2015.00880
- Kunapuli U, Jahn MK, Lueders T, Geyer R, Heipieper HJ, Meckenstock RU (2010) Desulfitobacterium aromaticivorans sp. nov. and Geobacter toluenoxydans sp. nov., iron-reducing bacteria capable of anaerobic degradation of monoaromatic hydrocarbons. Int J Syst Evol Microbiol 60:686–695. https://doi.org/10.1099/ijs.0.003525-0 Google Scholar
- Ledeker BM, De Long SK (2013) The effect of multiple primer-template mismatches on quantitative PCR accuracy and development of a multi-primer set assay for accurate quantification of pcrA gene sequence variants. J Microbiol Methods 94(3):224–231. https://doi.org/10.1016/j.mimet.2013.06.013 Google Scholar
- Leuthner B, Leutwein C, Schulz H, Horth P, Haehnel W, Schiltz E, Schagger H, Heider J (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(3):615–628Google Scholar
- Liang B, Wang LY, Zhou ZC, Mbadinga SM, Zhou L, Liu JF, Yang SZ, Gu JD, Mu BZ (2016) High frequency of Thermodesulfovibrio spp. and Anaerolineaceae in association with Methanoculleus spp. in a long-term incubation of n-alkanes-degrading methanogenic enrichment culture. Front Microbiol 7. https://doi.org/10.3389/fmicb.2016.01431
- Luo F (2016) PhD thesis, University of TorontoGoogle Scholar
- Markowitz VM, Chen IMA, Chu K, Szeto E, Palaniappan K, Pillay M, Ratner A, Huang J, Pagani I, Tringe S, Huntemann M, Billis K, Varghese N, Tennessen K, Mavromatis K, Pati A, Ivanova NN, Kyrpides NC (2014) IMG/M 4 version of the integrated metagenome comparative analysis system. Nucleic Acids Res 42(D1):D568–D573. https://doi.org/10.1093/nar/gkt919 Google Scholar
- McIlroy SJ, Kirkegaard RH, Dueholm MS, Fernando E, Karst SM, Albertsen M, Nielsen PH (2017) Culture-independent analyses reveal novel Anaerolineaceae as abundant primary fermenters in anaerobic digesters treating waste activated sludge. Front Microbiol 8(1134). https://doi.org/10.3389/fmicb.2017.01134
- Musat F, Galushko A, Jacob J, Widdel F, Kube M, Reinhardt R, Wilkes H, Schink B, Rabus R (2009) Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria. Environ Microbiol 11(1):209–219. https://doi.org/10.1111/j.1462-2920.2008.01756.x Google Scholar
- Rabus R, Boll M, Heider J, Meckenstock RU, Buckel W, Einsle O, Ermler U, Golding BT, Gunsalus RP, Kroneck PMH, Kruger M, Lueders T, Martins BM, Musat F, Richnow HH, Schink B, Seifert J, Szaleniec M, Treude T, Ullmann GM, Vogt C, von Bergen M, Wilkes H (2016) Anaerobic microbial degradation of hydrocarbons: from enzymatic reactions to the environment. J Mol Microbiol Biotechnol 26(1–3):5–28. https://doi.org/10.1159/000443997 Google Scholar
- Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75(23):7537–7541. https://doi.org/10.1128/aem.01541-09 Google Scholar
- Selesi D, Jehmlich N, von Bergen M, Schmidt F, Rattei T, Tischler P, Lueders T, Meckenstock RU (2010) Combined genomic and proteomic approaches identify gene clusters involved in anaerobic 2-methylnaphthalene degradation in the sulfate-reducing enrichment culture N47. J Bacteriol 192(1):295–306. https://doi.org/10.1128/jb.00874-09 Google Scholar
- Sipos R, Szekely AJ, Palatinszky M, Revesz S, Marialigeti K, Nikolausz M (2007) Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis. FEMS Microbiol Ecol 60(2):341–350. https://doi.org/10.1111/j.1574-6941.2007.00283.x Google Scholar
- von Netzer F, Pilloni G, Kleindienst S, Kruger M, Knittel K, Grundger F, Lueders T (2013) Enhanced gene detection assays for fumarate-adding enzymes allow uncovering of anaerobic hydrocarbon degraders in terrestrial and marine systems. Appl Environ Microbiol 79(2):543–552. https://doi.org/10.1128/aem.02362-12 Google Scholar
- von Netzer F, Kuntze K, Vogt C, Richnow HH, Boll M, Lueders T (2016) Functional gene markers for fumarate-adding and dearomatizing key enzymes in anaerobic aromatic hydrocarbon degradation in terrestrial environments. J Mol Microbiol Biotechnol 26(1–3):180–194. https://doi.org/10.1159/000441946 Google Scholar
- Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinf 13. https://doi.org/10.1186/1471-2105-13-134