Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale

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Abstract

Investigations into the deep marine environment have demonstrated the presence of a significant microbial biomass buried deep within sediments on a global scale. It is now believed that this deep biosphere plays a major role in the global cycling of elements and contains a large reservoir of organic carbon. This paper reports the development of a DNA extraction protocol that addresses the particular problems faced in applying molecular ecological techniques to samples containing very low biomass. Sediment samples were collected from different geographical locations within the Pacific Ocean and include the Ocean Drilling Program (ODP) Leg 190, Nankai Trough Accretionary Prism. Seven DNA extraction protocols were tested and a commercially available DNA extraction kit with modifications was shown to produce higher yields of polymerase chain reaction (PCR)-amplifiable DNA than standard laboratory methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene diversity revealed that template DNA from these extremely low biomass sediment samples was susceptible to PCR bias and random amplification. We propose that it is essential to screen 16S rRNA gene products for bacterial diversity by DGGE or other rapid fingerprinting methods, prior to their use in establishing a representative clone library of deep sub-seafloor bacteria. This represents a cautionary approach to analysis of microbial diversity in such sub-seafloor ecosystems.

Introduction

Recent studies on the deep sub-seafloor biosphere Parkes et al., 1994, Parkes et al., 2000 have shown that microbial populations can be found buried within sediments as deep as 800 mbsf (metres below the seafloor) and as old as 15 million years (Wellesbury et al., 2002). Deep marine sediments have also been estimated to contain a bacterial mass ≥10% of the total surface biosphere Parkes et al., 1994, Whitman et al., 1998. Microbial ecology studies over the last decade have revealed low culturability (Cragg et al., 1990), activity, cell density and productivity (Parkes et al., 2000) in these deep sediments. So culture independent molecular methods are needed to investigate the diversity of prokaryotes that are likely to be important in biogeochemical cycles in this habitat.

Molecular analyses of microbial diversity in complex environmental samples such as marine sediments require efficient and unbiased DNA extraction procedures (for review, see Roose-Amsaleg et al., 2001). Direct DNA extraction from environmental samples by either mechanical or chemical methods yields more DNA, and is more representative, than methods that employ cell removal from the environmental matrix before DNA extraction von Wintzingerode et al., 1997, Li et al., 1999b. In addition, direct DNA extraction methods facilitate rapid processing and allow higher sample throughput. Detergent lysis and freeze–thaw procedures Rochelle et al., 1992a, Marchesi et al., 1998, Juniper et al., 2001 have been used for direct DNA extraction from subsurface sediments. Bead beating with and without detergent lysis Kuske et al., 1998, Teske et al., 2002, and in the presence of phenol Webster et al., 2002, Griffiths et al., 2000, Stephen et al., 1996, have been used effectively to extract good quality DNA from sediments and soil. Recently, commercially available bead-beating-based DNA extraction kits, developed for soil, have also been used on samples from the deep biosphere Orphan et al., 2001, Reed et al., 2002, Takai et al., 2001.

In addition to DNA extraction, other steps in nucleic acid-based community analyses are also subject to bias and error. For example, sample handling and/or storage (Rochelle et al., 1994), DNA template concentration (Chandler et al., 1997), DNA purity Roose-Amsaleg et al., 2001, Juniper et al., 2001 and PCR conditions (Chandler et al., 1997) can result in the selective amplification of marker genes.

In this report, we describe the empirical development of a protocol which aims to overcome the major potential problems faced when analysing bacterial 16S rRNA gene diversity in the deep sub-seafloor biosphere. The paper will detail a method of DNA extraction suitable for samples with extremely low biomass, organic matter and DNA template followed by a series of steps to produce representative clone libraries of deep sediment samples. We also recommend the use of denaturing gradient gel electrophoresis (DGGE) as a time saving step to screen PCR products for 16S rRNA sequence diversity prior to cloning.

Section snippets

Sample description and handling

Sediment samples were collected as whole round cores (WRC) from locations in the Pacific Ocean, South East of Shikoku Island, Japan by the Ocean Drilling Program (ODP) Leg 190 (Nankai Trough) and along the Chilean Continental Margin by the German research vessel SONNE during cruise SO156, Peru und Chile Kontinentalhang (PUCK). Nankai Trough, Site 1173 (32° 14.663′N 135° 1.509′E) at 4790.7 mbsl (metres below sea level), sample depths 4.15, 98.3 and 193.3 mbsf (metres below seafloor); Site 1174

DNA recovery from deep sub-seafloor sediments

A methodological comparison of different DNA extraction techniques and commercially available kits found that previous laboratory methods used on deep sediments (JRM), humic rich soils (GW, RIG and MB) and ancient DNA (ANC) were not reliable for the extraction of PCR-amplifiable DNA from sediments of the Nankai Trough site 1173 (Table 1). In contrast, DNA was readily extracted from the control Cardiff Bay sediment by all methods (data not shown). The best method tested for obtaining DNA from

Discussion

Analysis of microbial community structure in the deep sub-seafloor biosphere using nucleic acid techniques requires efficient and unbiased DNA extraction procedures, and because samples are usually rare and material is limited, it is essential that investigators optimise DNA extraction protocols before undertaking comprehensive studies. In our study we found that the most suitable DNA extraction protocol for sediments tested was a commercially available kit, reported to lyse a wide range of

Acknowledgments

The authors would like to thank the ODP and Barry Cragg, University of Bristol for Leg 190 samples, Jens Kallmeyer, Tim Ferdelman and Bo Barker Jørgenson, MPI, Bremen for supplying samples from the PUCK SO156 Cruise. The authors would also like to acknowledge Barry Cragg, Julian Marchesi (University of Cork) and Laurent Toffin (Institut Universitaire Européen de la Mer, Plouzané) for Leg 190 WRC sub-sampling and additionally Julian Marchesi for preliminary investigations. GW was funded by the

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