Ribosome analysis reveals prominent activity of an uncultured member of the class Actinobacteria in grassland soils

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Abstract
Summary: A 16S rRNA-based molecular ecological study was performed to search for dominant bacterial sequences in Drentse A grassland soils (The Netherlands). In the first step, a library of 165 clones was generated from PCR-amplified 16S rDNA. By sequence comparison, clone DA079 and two other identical clones could be affiliated to a group of recently described uncultured Actinobacteria. This group contained 16S rDNA clone sequences obtained from different environments across the world. To determine whether such uncultured organisms were part of the physiologically active population in the soil, ribosomes were isolated from the environment and 16S rRNA was partially amplified via RT-PCR using conserved primers for members of the domain Bacteria. Subsequent sequence-specific separation by temperature-gradient gel electrophoresis (TGGE) generated fingerprints of the amplicons. Such community fingerprints were compared with the TGGE pattern of PCR-amplified rDNA of clone DA079 which was generated with the same set of primers. One of the dominant fingerprint bands matched with the band obtained from the actinobacterial clone. Southern blot hybridization with a probe made from clone DA079 confirmed sequence identity of clone and fingerprint band. This is the first report that a member of the novel actinobacterial group may play a physiologically active role in a native microbial community.