2 results for Addison, Sarah Louise, 2010

  • Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients

    Addison, Sarah Louise; McDonald, Ian R.; Lloyd-Jones, Gareth (2010-01)

    Journal article
    University of Waikato

    RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments.

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  • Identifying diazotrophs by incorporation of nitrogen from ¹⁵N₂ into RNA

    Addison, Sarah Louise; McDonald, Ian R.; Lloyd-Jones, Gareth (2010)

    Journal article
    University of Waikato

    The diversity and abundance of active diazotrophs was investigated in a New Zealand pulp and paper wastewater by enrichment with ¹⁵N₂. Purified ¹⁵N-RNA was analysed by reverse transcription, molecular cloning and sequence analysis of 16S rRNA to reveal a diverse community of bacteria as indicated by a Shannon Weaver Index value of > 2.8. The major class represented in the enriched culture were the γ-Proteobacteria at 85% with a secondary group of the phylum Firmicutes present at 8.2%, the remaining sequences were affiliated with the α- and β-Proteobacterial classes (1.4% and 4.3%, respectively). Three dominant genera, Aeromonas, Pseudomonas and Bacillus, were identified by comparison with published sequences and phylogenetic analysis. To confirm that representatives of the taxonomic groups identified from the active enriched nitrogen-fixing community were capable of fixing nitrogen Aeromonas and Pseudomonas species were cultivated and shown to possess nifH genes. In wastewater, fluorescence in situ hybridisation probing revealed that the dominant nitrogen-fixing population identified in this study were present in the population, but at lower levels. The population is, therefore, reliant on a small sub-population of diazotrophs to supply the community's nitrogen needs above that already present in the wastewater.

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