Simple and Safe Method for Simultaneous Isolation of Microbial RNA and DNA from Problematic Populations

McIlroy, Simon Jon; Porter, Kate; Seviour, Robert J.; Tillett, Daniel

doi:10.1128/aem.01047-08

Cited by 18 publications

(24 citation statements)

References 19 publications

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“…From AMV sediment incubations, RNA was extracted according to McIlroy et al (2008), whereas from GB sediments DNA was extracted according to Zhou et al (1996), because RNA extraction did not result in the required amounts of high-quality rRNA.…”

Section: Card-fishmentioning

confidence: 99%

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

et al. 2014

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Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with 13 C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA-and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short-and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent b-oxidation and the reverse Wood-Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.

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Section: Card-fishmentioning

confidence: 99%

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

et al. 2014

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“…Three different DNA extraction methods were used in attempts to minimize any potential biases associated with each, and the extracts were combined. These three methods included those of McVeigh et al (38) and McIlroy et al (36) and a FASTDNA spin kit (Qbiogene, Melbourne, Australia). The former two were selected because they had performed best in comparative trials at recovering DNA from marker activated sludge populations known to resist many DNA extraction protocols (S. McIlroy, K. Porter, S. Schroeder, R. J. Seviour, and D. Tillett, unpublished data).…”

Section: Methodsmentioning

confidence: 99%

Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

Speirs

Nittami

McIlroy

et al. 2009

Appl Environ Microbiol

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Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removalactivated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-␤-hydroxyalkanoates or polyphosphate.Most activated sludge plants suffer from the operational disorders of bulking and foaming, both of which are caused by excessive growth of certain filamentous bacteria. Several different filament morphotypes have been described from systems treating domestic and industrial wastes (17, 18) but, in the absence of pure cultures, many of these have never been characterized sufficiently to resolve their taxonomy or provide them with valid names. Hence, they are often still referred to as numerical types persisting from the study of Eikelboom (18). Success has been achieved with some cultured and uncultured filaments in elucidating their phylogeny from 16S rRNA sequence analyses (6, 9, 10, 31, 50) and providing them with valid names (37, 49). Furthermore, with such sequence information, rRNA targeted oligonucleotide probes have been designed for their in situ identification and, together with microautoradiography (MAR) and other techniques (29,40), their ecophysiology may be elucidated (25,27,28). Type 0092, originally described by Eikelboom (18), appears prominently in many filament surveys carried out on plants around the world, where microscopy was used to identify them (35, 48). These morphotypes have been associated especially with long sludge age (Ͼ15-day) operational conditions (22) and thus frequently appear in enhanced biological phosphate removal (EBPR) systems (see, for example, reference 11), where the biomass is recycled repeatedly through anaerobic: aerobic zones. Consequently, this filament morphotype was classified as an "all-zone" grower by Wanner and Grau (52), able in their view to grow under aerobic, anoxic, and anaerobic conditions. However, its physiology from pure culture studies was described as being strictly aerobic (13, 21). These isolates were never deposited in recognized culture collections, and so confirmation of their identity is difficult. Similarly, the precise identification of the type 0092 filaments claimed to have been cultured by Ramothokang et al. (43) is unclear.Type 0092 has ver...

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“…The analysis of 16S rRNA genes through clone libraries and fingerprinting approaches such as denaturing gradient gel electrophoresis (DGGE) has greatly extended our knowledge about the phylogenetic richness of sponge-associated bacteria and archaea (Webster et al, 2001(Webster et al, , 2004Hentschel et al, 2002;Taylor et al, 2004Taylor et al, , 2007bHolmes and Blanch, 2006;Longford et al, 2007;Schmitt et al, 2007Schmitt et al, , 2008Thiel et al, 2007;Mohamed et al, 2008b;Zhu et al, 2008). Researchers in other systems have taken this approach one step further, yielding insights into both richness and activity by comparing 16S rRNA gene-and 16S rRNA-derived sequences, respectively (Moeseneder et al, , 2005Winter et al, 2001;Troussellier et al, 2002;Mills et al, 2005;Gentile et al, 2006;Martinez et al, 2006;Brinkmann et al, 2008;McIlroy et al, 2008;West et al, 2008;Rodriguez-Blanco et al, 2009). In general, cellular concentrations of rRNA are correlated with growth rate and activity (DeLong et al, 1989;Poulsen et al, 1993), hence-with acknowledgement of certain caveats (e.g., for ammoniaoxidizing bacteria; Morgenroth et al, 2000)-the rRNA itself can yield useful information about which community members are active.…”

Section: Introductionmentioning

confidence: 99%

Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons

2010

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The phylogenetic diversity of microorganisms in marine sponges is becoming increasingly well described, yet relatively little is known about the activities of these symbionts. Given the seemingly favourable environment provided to microbes by their sponge hosts, as indicated by the extraordinarily high abundance of sponge symbionts, we hypothesized that the majority of spongeassociated bacteria are active in situ. To test this hypothesis we compared, for the first time in sponges, 16S rRNA gene-vs 16S rRNA-derived bacterial community profiles to gain insights into symbiont composition and activity, respectively. Clone libraries revealed a highly diverse bacterial community in Ancorina alata, and a much lower diversity in Polymastia sp., which were identified by electron microscopy as a high-and a low-microbial abundance sponge, respectively. Substantial overlap between DNA and RNA libraries was evident at both phylum and phylotype levels, indicating in situ activity for a large fraction of sponge-associated bacteria. This active fraction included uncultivated, sponge-specific lineages within, for example, Actinobacteria, Chloroflexi and Gemmatimonadetes. This study shows the potential of RNA vs DNA comparisons based on the 16S rRNA gene to provide insights into the activity of sponge-associated microorganisms.

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Simple and Safe Method for Simultaneous Isolation of Microbial RNA and DNA from Problematic Populations

Cited by 18 publications

References 19 publications

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

Activity profiles for marine sponge-associated bacteria obtained by 16S rRNA vs 16S rRNA gene comparisons

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