Browsing by Author "Galada, Ncebakazi"

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    Exploring diversity and ecology of nonarchaea in hydrothermal biotopes
    (University of the Western Cape, 2005) Galada, Ncebakazi; Cowan, Donald A.; Dept. of Biotechnology; Faculty of Science
    The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with “universal” archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of “universal” archaeal primers A571F (5’-GCYTAA AGSRIC CGT AGC-3’) and UA1204R (5’-TTM GGG GCA TRCIKA CCT-3’) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermaystems. Upon sequencing of amplicons it was discovered that Chinese and. New Zealandsamples contained novel nanoarchaeal phylotypes. The preliminary nanoarchaeal phylotypes were used to design nanoarchaeal-specific primer N989R (5'-GGT TTC CGG TGT CAG TTC-3'), which was coupled with A571F and used in screening of nanoarchaeotes. The nanoarchaeal phylotypes identified with these primers were further screened by amplified ribosomal DNA restriction analysis (ARDRA), which was used to explore the diversity of these phylotypes. The novel nanoarchaeotes cluster into 9 cosely related clades which may represent separate species. Three of the New Zealand phylotypes form one separate clade which is closely related to the published nanoarchaeotes. The following nanoarchaeal sequences were submitted to the GenBank, TC9F (AY572420), TC11-5 (AY571283), TC11-B6 (AY727890), TC11-B7 (AY727887), TC11-C4 (AY727886), TC11-C6 (AY727889), TC11-C8 (AY727888), AND TC11-D4 (AY727891). Fluorescence in situhybridization was also used to simultaneously visualize, identify and localize nanoarchaeotes.
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    Metagenomic analysis and characterization of microbial diversity from hydrothermal samples of El Tatio geyser field, Chile
    (University of the Western Cape, 2012) Galada, Ncebakazi; Cowan, D. A.
    The El Tatio geyser field (ETGF) is the largest known geothermal field in Chile, forming part of a wide spectrum of extremophilic habitats in the country. The ETGF is NaCl rich, with high concentrations of toxic elements such as Li, As and Cs, which are contributed mainly by volcanic activities in the region. Most previous studies in the area have focused on the geology and geochemistry for mining purposes, as well as on the search for geothermal resources for power generation. Very little is currently known about the composition of the microbial communities of the ETGF, which makes the study reported here of particular novelty.A metagenomic approach, involving the amplification of 16S rRNA gene phylogenetic markers from metagenomic DNA was used to investigate seven different sites within the geyser field. The sample sites were characterized by high temperatures (80-85 °C) and a range of pH values (6.3-8). Various molecular methods, including clone library construction and PCR-DGGE analyses were used to target a wide range of microbial populations within the ETGF sites. Multivariate analysis was also applied to assess differences in the microbial diversity from different sites and to correlate microbial diversity with environmental conditions. Culture-dependent screening of novel nanoarchaeal species was also undertaken.These were coupled with PCR and other detection methods such as fluorescent in situ hybridization (FISH) to trace the presence of nanoarchaeal signals from enriched cultures.The results have shown that the ETGF encompasses a limited microbial diversity represented by only 30 dominant phylotypes, and most likely due to the toxic chemical content of the geyser field. The microbial representatives identified were assigned to OTUs from archaeal,nanoarchaeal and bacterial taxonomic groups. The dominant microbial taxa included members of the Proteobacteria, Firmicutes, Aquificae, Actinobacteria, Euryarchaeota(Halobacteriales, Archaeoglobales), Crenarchaeota (Thermoproteales, Desulfurococcales),together with uncultured representatives of the bacteria, archaea and nanoarchaeota. Notably,representatives of mesophilic, thermophilic and hyperthermophilic taxonomic groups were all detected in ETGF samples. This is attributed to various factors such as temperature gradients and dispersal mechanisms (e.g. natural forces such as rain and volcanic activities). Principal component analysis (PCA) showed significant differences (P < 0.05) in the microbial diversity of the ETGF samples, with principal components (based on the sequenced species from both 16S rRNA clone libraries and PCR-DGGE profiles) explaining up to 62.7% of variance. Furthermore, CCA showed that the differences in phylogenetic diversity were most influenced by temperature and salinity. This was also confirmed by the sequencing results,which showed that hyperthermophilic and haloarchaeal taxa were dominant in the ETGF sites. However, conductivity and pH were also found to contribute to variations in the microbial diversity of the experimental samples, with TDS (total dissolved solids) being a less influential factor. Attempts to generate nanoarchaeal-host co-cultures, and to recover sufficient nanoarchaeal genomic DNA for fosmid and/or large insert cloning for comparative genome analysis, were unsuccessful.This study is the first to employ metagenomic approaches to analyse the microbial diversity of sites in the ETGF, and has expanded our knowledge of microbiota present in this geyser field.