Xanthogenate nucleic acid isolation from cultured and environmental cyanobacteria

Tillett, Daniel; Neilan, Brett A.

doi:10.1046/j.1529-8817.2000.99079.x

Cited by 311 publications

(262 citation statements)

References 43 publications

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“…; 21 1C) in the dark. At the endpoint of each SIP incubation-defined as the time when the extent of mineralisation of the respective 14 C-labelled substrate began to approach an asymptote-whole DNA from the total volume in the paired flasks amended with the [U-13 C] hydrocarbon and the corresponding paired set with unlabelled hydrocarbon was extracted using the method of Tillett and Neilan (2000).…”

Section: Sip Incubationsmentioning

confidence: 99%

“…Barcoded amplicon pyrosequencing DNA was extracted from surface (PE5) and water column (B1, B3, B6, B11) frozen samples using the method of Tillett and Neilan (2000), and barcoded 16S rRNA gene pyrosequencing was performed to analyse the bacterial community of these samples. Ten-fold dilutions of extracted DNA in water were used as a template for triplicate PCR reactions for each sample.…”

Section: Real-time Quantitative Pcrmentioning

confidence: 99%

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Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

et al. 2013

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The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly 13 C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)-and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbondegrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.

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Section: Sip Incubationsmentioning

confidence: 99%

Section: Real-time Quantitative Pcrmentioning

confidence: 99%

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

et al. 2013

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“…Spring water was concentrated via a Millipore amicon 50 kDa cutoff centrifugal filter (Millipore, Billerica, MA, USA), according to manufacturer's specifications before undergoing DNA extraction as described previously (Tillett and Neilan, 2000;Moissl-Eichinger, 2011). Concentrations of doublestranded DNA in the samples were determined using Qubit Quantitation Platform (Invitrogen, Carlsbad, CA, USA).…”

Section: Sample Collectionmentioning

confidence: 99%

Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm

et al. 2012

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Archaea are usually minor components of a microbial community and dominated by a large and diverse bacterial population. In contrast, the SM1 Euryarchaeon dominates a sulfidic aquifer by forming subsurface biofilms that contain a very minor bacterial fraction (5%). These unique biofilms are delivered in high biomass to the spring outflow that provides an outstanding window to the subsurface. Despite previous attempts to understand its natural role, the metabolic capacities of the SM1 Euryarchaeon remain mysterious to date. In this study, we focused on the minor bacterial fraction in order to obtain insights into the ecological function of the biofilm. We link phylogenetic diversity information with the spatial distribution of chemical and metabolic compounds by combining three different state-of-the-art methods: PhyloChip G3 DNA microarray technology, fluorescence in situ hybridization (FISH) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy. The results of PhyloChip and FISH technologies provide evidence for selective enrichment of sulfate-reducing bacteria, which was confirmed by the detection of bacterial dissimilatory sulfite reductase subunit B (dsrB) genes via quantitative PCR and sequence-based analyses. We further established a differentiation of archaeal and bacterial cells by SR-FTIR based on typical lipid and carbohydrate signatures, which demonstrated a co-localization of organic sulfate, carbonated mineral and bacterial signatures in the biofilm. All these results strongly indicate an involvement of the SM1 euryarchaeal biofilm in the global cycles of sulfur and carbon and support the hypothesis that sulfidic springs are important habitats for Earth's energy cycles. Moreover, these investigations of a bacterial minority in an Archaea-dominated environment are a remarkable example of the great power of combining highly sensitive microarrays with label-free infrared imaging.

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“…DNA isolation and PCR amplification of 16S rRNA genes Genomic DNA extraction of isolates was performed with an extraction protocol utilizing xanthogenate (Tillett and Neilan, 2000). The total genomic DNA from ground stromatolite was extracted using the TNE buffer extraction protocol (Neilan et al, 2002).…”

Section: Culturing and Isolationmentioning

confidence: 99%

Determining the specific microbial populations and their spatial distribution within the stromatolite ecosystem of Shark Bay

et al. 2008

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The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were a-and c-proteobacteria (58%), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology.

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Xanthogenate nucleic acid isolation from cultured and environmental cyanobacteria

Cited by 311 publications

References 43 publications

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm

Determining the specific microbial populations and their spatial distribution within the stromatolite ecosystem of Shark Bay

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