Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil

Abstract

Stable isotope probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (< 22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of 'heavy' bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

“…Methanol-utilizing methylotrophs can be metabolically stimulated by adding millimolar concentrations of methanol to aerated and flooded soil (Radajewski et al, 2002;Lueders et al, 2004). However, the methylotrophs responsible for in situ methanol consumption are unresolved.…”

“…Knowledge of the in situ diversity of methanol-utilizing methylotrophs in soils is limited despite the fact that many soil-derived methanol-utilizing methylotrophs have been isolated or detected by molecular analyses by employing high and likely not in situ-relevant methanol concentrations (Radajewski et al, 2002;Lueders et al, 2004;Lidstrom, 2006;Kolb, 2009a). Methanolutilizing Burkholderiaceae, Beijerinkiaceae and Bradyrhizobiacea occur in aerated soils (Radajewski et al, 2002).…”

Effect of earthworms on the community structure of active methanotrophic bacteria in a landfill cover soil

“…Lueders et al (2004) combined RNAand DNA-SIP to monitor activation and temporal dynamics of methylotrophs in soil. The authors suggested that after 6 days of incubation with labelled substrate, RNA-SIP recovered the initially active bacteria, whereas a specific enriched methylotroph was identified after 42 days of incubation Ecology of methanotrophs in a landfill soil M Héry et al using DNA-SIP.…”

Bacterial community structure corresponds to performance during cathodic nitrate reduction

“…Application of the PhyloChip (Affymetrix, Santa Clara, CA, USA) to cathode biofilm communities offered a higherresolution analysis of the microbial community composition than previously reported methods Wrighton et al, 2008), and thus the possibility of uncovering more diversity than was previously observed in these systems. We restricted our analysis to the active members of the communities by monitoring 16S rRNA (rather than the 16S rRNA gene), which is a more responsive biomarker and a better surrogate for microbial activity in bacterial communities (Lueders et al, 2004). Populations identified as dominant by PhyloChip were verified with fluorescence in situ hybridization (FISH) analysis.…”