Microbial diagnostic microarrays (MDMs) are highly parallel hybridization platforms containing multiple sets of immobilized oligonucleotide probes used for parallel detection and identification of many different microorganisms in environmental and clinical samples. Each probe is approximately specific to a given group of organisms. Here we describe the protocol used to develop and validate an MDM method for the semiquantification of a range of functional genes—in this case, particulate methane monooxygenase (pmoA)—and we give an example of its application to the study of the community structure of methanotrophs and functionally related bacteria in the environment. The development and validation of an MDM, following this protocol, takes ∼6 months. The pmoA MDM described in detail comprises 199 probes and addresses ∼50 different species-level clades. An experiment comprising 24 samples can be completed, from DNA extraction to data acquisition, within 3 d (12–13 h bench work).
This is a preview of subscription content, access via your institution
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Liu, W.T., Marsh, T.L., Cheng, H. & Forney, L.J. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63, 4516–4522 (1997).
Muyzer, G. & Smalla, K. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73, 127–141 (1998).
Muyzer, G., Teske, A., Wirsen, C. & Jannasch, H. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch. Microbiol. 164, 165–172 (1995).
Brown, M.V. et al. Microbial community structure in the North Pacific ocean. ISME J. 3, 1374–1386 (2009).
Sogin, M.L. et al. Microbial diversity in the deep sea and the underexplored 'rare biosphere'. Proc. Natl. Acad. Sci. USA 103, 12115–12120 (2006).
Brodie, E.L. et al. Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation. Appl. Environ. Microbiol. 72, 6288–6298 (2006).
Roh, S.W., Abell, G.C.J., Kim, K.-H., Nam, Y.-D. & Bae, J.-W. Comparing microarrays and next-generation sequencing technologies for microbial ecology research. Trends Biotechnol. 28, 291–299 (2010).
Sessitsch, A. et al. Diagnostic microbial microarrays in soil ecology. New Phytol. 171, 719–735 (2006).
Lee, D.Y., Shannon, K. & Beaudette, L.A. Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR. J. Microbiol. Methods 65, 453–467 (2006).
Wiesinger-Mayr, H. et al. Identification of human pathogens isolated from blood using microarray hybridisation and signal pattern recognition. BMC Microbiol. 7, 78 (2007).
Yoshida, C. et al. Methodologies towards the development of an oligonucleotide microarray for determination of Salmonella serotypes. J. Microbiol. Methods 70, 261–271 (2007).
Rich, V.I., Konstantinidis, K. & DeLong, E.F. Design and testing of 'genome-proxy' microarrays to profile marine microbial communities. Environ. Microbiol. 10, 506–521 (2008).
Kostic, T. et al. A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens. Anal. Biochem. 360, 244–254 (2007).
Strommenger, B. et al. DNA microarray for the detection of therapeutically relevant antibiotic resistance determinants in clinical isolates of Staphylococcus aureus. Mol. Cell Probes 21, 161–170 (2007).
Hemme, C.L. et al. Metagenomic insights into evolution of a heavy metal-contaminated groundwater microbial community. ISME J. 4, 660–672 (2010).
Yergeau, E. et al. Environmental microarray analyses of Antarctic soil microbial communities. ISME J. 3, 340–351 (2009).
Bodrossy, L. et al. Development and validation of a diagnostic microbial microarray for methanotrophs. Environ. Microbiol. 5, 566–582 (2003).
Stralis-Pavese, N. et al. Optimization of diagnostic microarray for application in analysing landfill methanotroph communities under different plant covers. Environ. Microbiol. 6, 347–363 (2004).
Taroncher-Oldenburg, G., Griner, E.M., Francis, C.A. & Ward, B.B. Oligonucleotide microarray for the study of functional gene diversity in the nitrogen cycle in the environment. Appl. Environ. Microbiol. 69, 1159–1171 (2003).
Wu, L. et al. Development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl. Environ. Microbiol. 67, 5780–5790 (2001).
He, Z. et al. GeoChip 3.0 as a high-throughput tool for analyzing microbial community composition, structure and functional activity. ISME J. 4, 1167–1179 (2010).
He, Z. et al. GeoChip: a comprehensive microarray for investigating biogeochemical, ecological and environmental processes. ISME J. 1, 67–77 (2007).
Gebert, J., Singh, B.K., Pan, Y. & Bodrossy, L. Activity and structure of methanotrophic communities in landfill cover soils. Environ. Microbiol. Reports 1, 414–423 (2009).
Bodrossy, L. et al. mRNA-based parallel detection of active methanotroph populations using a diagnostic microarray. Appl. Environ. Microbiol. 72, 1672–1676 (2006).
Fjellbirkeland, A., Torsvik, V. & Øvreås, L. Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences. Antonie van Leeuwenhoek 79, 209–217 (2001).
Horz, H.-P., Yimga, M.T. & Liesack, W. Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling. Appl. Environ. Microbiol. 67, 4177–4185 (2001).
Holmes, A.J., Costello, A., Lidstrom, M.E. & Murrell, J.C. Evidence that participate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol. Lett. 132, 203–208 (1995).
Abell, G.C.J., Stralis-Pavese, N., Sessitsch, A. & Bodrossy, L. Grazing affects methanotroph activity and diversity in an alpine meadow soil. Environ. Microbiol. Rep. 1, 457–465 (2009).
Han, B. et al. Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine. FEMS Microbiol. Ecol. 70, 40–51 (2009).
Moussard, H., Stralis-Pavese, N., Bodrossy, L., Josh, D.N. & Murrell, J.C. Identification of active methylotrophic bacteria inhabiting surface sediment of a marine estuary. Environ. Microbiol. Rep. 1, 424–433 (2009).
Chen, Y. et al. Diversity of the active methanotrophic community in acidic peatlands as assessed by mRNA and SIP-PLFA analyses. Environ. Microbiol. 10, 446–459 (2008).
Kip, N. et al. Global prevalence of methane oxidation by symbiotic bacteria in peat-moss ecosystems. Nat. Geosci. 3, 617–621 (2010).
Murrell, J.C. & Smith, T.J. Biochemistry and molecular biology of methane monooxygenase. Handbook of Hydrocarbon and Lipid Microbiology (ed. Timmis, K.N.) 1046–1055 (Springer-Verlag, 2010).
Bodrossy, L. Diagnostic oligonucleotide microarrays for microbiology. in A Beginner's Guide to Microarrays, Vol 1 (ed. Blalock, E.) 43–92 (Kluwer Academic Publishers, 2003).
Brown, T.J. & Anthony, R.M. The addition of low numbers of 3′ thymine bases can be used to improve the hybridization signal of oligonucleotides for use within arrays on nylon supports. J. Microbiol. Methods 42, 203–207 (2000).
Guo, Z., Guilfoyle, R.A., Thiel, A.J., Wang, R. & Smith, L.M. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res. 22, 5456–5465 (1994).
Shchepinov, M.S., Case-Green, S.C. & Southern, E.M. Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays. Nucleic Acids Res. 25, 1155–1161 (1997).
Peplies, J., Glockner, F.O. & Amann, R. Optimization strategies for DNA microarray-based detection of bacteria with 16S rRNA-targeting oligonucleotide probes. Appl. Environ. Microbiol. 69, 1397–1407 (2003).
Hughes, J.B., Hellmann, J.J., Ricketts, T.H. & Bohannan, B.J. Counting the uncountable: statistical approaches to estimating microbial diversity. Appl. Environ. Microbiol. 67, 4399–4406 (2001).
Costello, A.M. & Lidstrom, M.E. Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments. Appl. Environ. Microbiol. 65, 5066–5074 (1999).
Bourne, D.G., McDonald, I.R. & Murrell, J.C. Comparison of pmoA PCR primer sets as tools for investigating methanotroph diversity in three Danish soils. Appl. Environ. Microbiol. 67, 3802–3809 (2001).
Ramette, A. Multivariate analyses in microbial ecology. FEMS Microbiol. Ecol. 62, 142–160 (2007).
Clarke, K.R. Non-parametric multivariate analyses of changes in community structure. Australian J. Ecol. 18, 117–143 (1993).
Anderson, M.J., Gorley, R.N. & Clarke, K.R. PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods (Primer-E, Plymouth, UK, 2008).
Cebron, A. et al. Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing. FEMS Microbiol. Ecol. 62, 12–23 (2007).
Cebron, A. et al. Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity. Appl. Environ. Microbiol. 73, 798–807 (2007).
Chen, Y. et al. Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands. Environ. Microbiol. 10, 2609–2622 (2008).
Chen, Y. et al. The impact of burning and Calluna removal on below-ground methanotroph diversity and activity in a peatland soil. Appl. Soil Ecol. 40, 291–298 (2008).
Gebert, J., Stralis-Pavese, N., Alawi, M. & Bodrossy, L. Analysis of methanotrophic communities in landfill biofilters using diagnostic microarray. Environ. Microbiol. 10, 1175–1188 (2008).
Hery, M. et al. Effect of earthworms on the community structure of active methanotrophic bacteria in a landfill cover soil. ISME J. 2, 92–104 (2008).
Kumaresan, D., Abell, G.C.J., Bodrossy, L., Stralis-Pavese, N. & Murrell, J.C. Spatial and temporal diversity of methanotrophs in a landfill cover soil are differentially related to soil abiotic factors. Environ. Microbiol. Rep. 1, 398–407 (2009).
Research at AIT was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (FWF), Austria (project number P15044) and by the European Science Foundation EuroDiversity microbial methane oxidation as a modelsystem for microbial ecology (METHECO) program (Nr. FP018, national funding agency: FWF, Austria, project number I40-B06).
The authors declare no competing financial interests.
About this article
Cite this article
Stralis-Pavese, N., Abell, G., Sessitsch, A. et al. Analysis of methanotroph community composition using a pmoA-based microbial diagnostic microarray. Nat Protoc 6, 609–624 (2011). https://doi.org/10.1038/nprot.2010.191
This article is cited by
A new cell morphotype among methane oxidizers: a spiral-shaped obligately microaerophilic methanotroph from northern low-oxygen environments
The ISME Journal (2016)
The ISME Journal (2013)
One millimetre makes the difference: high-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic–anoxic interface in a flooded paddy soil
The ISME Journal (2012)