title: New DGGE strategies for the analyses of methanotrophic microbial communities using different combinations of existing 16S rRNA-based primers.
abstract: Methane-oxidising microbial communities are studied intensively because of their importance for global methane cycling. A suite of molecular microbial techniques has been applied to the study of these communities. Denaturing gradient gel electrophoresis (DGGE) is a diversity screening tool combining high sample throughput with phylogenetic information of high resolution. The existing 16S rRNA-based DGGE assays available for methane-oxidising bacteria suffer from low-specificity, low phylogentic information due to the length of the amplified fragments and/or from lack of resolving power. In the present study we developed new combinations of existing primers and applied these on methane-oxidising microbial communities in a freshwater wetland marsh. The designed strategies comprised nested as well as direct amplification of environmental DNA. Successful application of direct amplification using combinations of universal and specific primers circumvents the nested designs currently used. All developed assays resulted in identical community profiles in wetland soil cores with Methylobacter sp. and Methylocystis sp.-related sequences. Changes in the occurrence of Methylobacter-related sequences with depth in the soil profile may be related to the decrease in methane-oxidizing activity.
authors.affiliation: Netherlands Institute of Ecology (NIOO-KNAW), Centre for Limnology, Department of Microbial Ecology, Rijksstraatweg 6, NL3631 AC Nieuwersluis, The Netherlands. p.bodelier@nioo.knaw.nl
authors.forename: Paul L E
authors.initials: PL
authors.lastname: Bodelier
authors.forename: Marion
authors.initials: M
authors.lastname: Meima-Franke
authors.forename: Gabriel
authors.initials: G
authors.lastname: Zwart
authors.forename: Hendrikus J
authors.initials: HJ
authors.lastname: Laanbroek
mesh_terms: D017931:DNA Primers / Q000235:genetics*; D017753:Ecosystem; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005618:Fresh Water; D014644:Genetic Variation; D008697:Methane / Q000378:metabolism; D042082:Methylobacteriaceae / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D010802:Phylogeny; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008697:Methane