E-mail: anna.synnove.nordgard@biotech.ntnu.no Detection of methanogenic archaea in DGGE by the use of primers for Methyl-Coenzyme M Reductase A (mcrA) genes Anna Synnøve Røstad Nordgård1, Ingrid Bakke1, Odd Gunnar Brakstad2, Kjetill Østgaard1 Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim Marine Environmental Biotechnology, SINTEF Materials and Chemistry, Brattørkaia 17, 7010 Trondheim E-mail: anna.synnove.nordgard@biotech.ntnu.no Phone: 0047 73 59 16 47 Department of Biotechnology Abstract Denaturing gradient gel electrophoresis (DGGE) is a fingerprinting technique that enables the researcher to separate the PCR products amplified from different methanogenic archaea, but attaching the GC-clamp to the PCR product for use in DGGE has often proved to be problematic. In order to solve this issue, the gene was first amplified from DNA extracted from cow manure using the ML primers provided by Luton et al. (2002). This PCR product was then used as template for the same primer with the GC-clamp attached. This nested protocol gave a positive result and the PCR products were succesfully separated using DGGE. Introduction Each biogas fermenter is a unique system defined through its substrate and process conditions. The role of many of the microorganisms involved is still unknown or poorly investigated. Knowledge about the microbial community in biogas fermentors could help not only in design, but equally important in proper operation to optimize reactor performance. The enzyme Methyl-coenzyme M reductase (MCR) catalysing the last step in methanogenesis is present in all methanogens (Friedrich 2005) and should therefore be a good indicator for detection of methanogenic archaea. DGGE (Muyzer 1993) enables the researcher to separate the PCR products amplified from different methanogenic archaea, thereby providing an overview of the microbial community. PCR primers targeting mcrA have been available for about a decade (Lueders et al., 2001; Luton et al. 2002), but attaching the GC-clamp to the PCR product for use in DGGE has often proved to be problematic. Fig. 1. Batch reactor placed at Vestråt Farm, Ørland, Norway, running on manure from diary cows at 39 °C. Total volume of 6m3 with 10 % headspace. The reactor was run for 32 weeks. Samples were collected weekly for the first eight weeks and every other week thereafter. Methods The samples used in this experiment originated from a batch reactor running on dairy cow manure, see figure 1. The total DNA was extracted using FastDNA® SPIN Kit for Soil from MP Biomedicals. The primer pair ML (MLf; 5’-GGTGGTGTMGGA-TTCACACARTAYGCWACAGC-3’, MLr; 5’-TTCATTGCRTAG-TTWGGRTAGTT-3’, Luton et al. 2002; Nettmann et al. 2008) which targets the functional gene mcrA was chosen for studying the composition of methanogenic archaea in the reactor. PCR reactions were run for 40 cycles (95 °C 30 s, 48 °C 30 s and 72 °C 60 s) with 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.3 µM of each primer, 0.4 M bovine serum albumin (BSA, New England BioLabs Inc.) and Taq polymerase and buffer from TaqPCR Core Kit (QIAGEN). Fig. 2. PCR product amplified using ML-GC. PCR product amplified from total DNA isolated from sample no. 0-15 using the ML primer without the GC-clamp served as template. L: GeneRuler™ 1kb DNA Ladder (0.5 μg/μl, Fermentas). Negative template control (NTC) was empty. Results and discusion The PCR protocol gave clear bands of 440 bp in length when analyzed on a regular agarose gel (results not included). The same ML primers with a GC-clamp attached to the 5’-end of the forward primer (5’-CGCCCGCCGCGCGCGGCGGGCGGGGC-GGGGGCACGGGGGG-3’, Teske et al. 1996) were then tested on the same total DNA from the samples. This gave no bands when analyzed on agarose gel. The GC-clamp might have hindered the primers in annealing to the template. To avoid this, the mcrA gene was first amplified using the ML primers without the GC-clamp. This PCR product was then diluted 1:20 and used as template for the ML-GC primers. This nested protocol gave a band of 480 bp when analyzed on agarose gel as illustrated in figure 2. DGGE was subsequently run using a standard protocol (BioRad).

References Friedrich, M. W. (2005). «Unique functional markers for methanogenic and anaerobic methane-oxidizing Archaea.» Environ. Microbiol. 397: 428-442 Lueders, T., Chin, K.J., Conrad, R. and Friedrich, M. (2001). «Molecular analyses of methyl-coenzyme M reductase alpha-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage.» Environ. Microbiol. 3(3): 194-204 Luton, P. E., Wayne, J. M., Sharp, R. J. and Riley, P. W. (2002). «The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill.» Microbiology-(UK) 148: 3521-3530 Muyzer, G., Dewaal, E. C., Uitterlinden, A. G. (1993). «Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S ribosomal RNA.» Appl. Environ. Microbiol. 59: 695-700 Malin, C. and Illmer, P. (2008). «Ability of DNA content and DGGE analysis to reflect the performance condition of an anaerobic biowaste fermenter.» Microbiol. Res. 163(5): 503-511 Nettmann, E., Bergmann, I., Mundt, K., Linke, B. and Klocke, M. (2008). «Archaea diversity within a commercial biogas plant utilizing herbal biomass determined by 16S rDNA and mcrA analysis.» J. Appl. Microbiol. 105(6): 1835-1850 Teske, A., Wawer, C., Muyzer, G. and Ramsing, N. B. (1996). «Distribution of sulphate-reducing bacteria in a stratified fjord (Mariager fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments.» Appl. Environ. Microbiol. 62(4): 1405-1415 Wang, Y., Qian, P. Y. (2009). «Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.» PLoS ONE 4 Fig. 3. DGGE gel (6 % acrylamide, denaturing gradient 50-80%) with PCR product obtained from total DNA isolated from sample no. 0-15 using the nested ML protocol. L: GeneRuler™ 1kb DNA Ladder (0.5 μg/μl, Fermentas). Two distinct rows of bands denatured at approximately 55 % and 65 %, and a clear separation of bands was obtained, see figure 3. Researchers have been using DGGE analysis to detect community shifts in both Bacteria and Archaea for a long time, but analysis of Archaea in general is not enough to give a good picture of methane-producing microorganisms. It can be tricky to design 16S rDNA primers that amplify only the target group of interest. Often other species will be amplified in addition, or the 16S rDNA primers fail to cover the entire target group (Wang 2009). Using functional genes to target a group of species might be better than just using the 16S rRNA gene. The activity of methane-producing Archaea needs to be investigated in addition to monitoring species diversity and abundance. Using functional genes such as genes coding for enzymes in methane formation should suit that purpose (Malin and Illmer 2007). Conclusions These results imply that the GC-clamp can be attached even if the template site is problematic by using a nested PCR protocol.