Mar Biotechnol DOI 10.1007/s10126-010-9259-1

ORIGINAL ARTICLE

Construction and Preliminary Analysis of a Deep-Sea Sediment Metagenomic Fosmid Library from Qiongdongnan Basin, South China Sea

Yongfei Hu & Chengzhang Fu & Yeshi Yin & Gong Cheng & Fang Lei & Xi Yang & Jing Li & Elizabeth Jane Ashforth & Lixin Zhang & Baoli Zhu

Received: 13 October 2009 /Accepted: 19 January 2010 # Springer Science+Business Media, LLC 2010

Abstract Preliminary characterization of the microbial annotations) highlighted the existence of ‘one-carbon’ metab- phylogeny and metabolic potential of a deep-sea sediment olism within this community as well as identifying functional sample from the Qiongdongnan Basin, South China Sea, was genes involved in methanogenesis. Furthermore, diverse carried out using a metagenomic library approach. An genes involved in the biodegradation of xenobiotics were effective and rapid method of DNA isolation, purification, found using Kyoto Encyclopedia of Genes and Genomes and library construction was used resulting in approximately metabolic pathway analysis. 200,000 clones with an average insert size of about 36 kb. End sequencing of 600 individual clones from the fosmid Keywords Deep-sea sediment . Metagenomics . library generated 1,051 sequences with an average sequence Fosmid library . End sequences . Methane hydrate length of 619 bp. Phylogenetic ascription indicated that this library was dominated by Bacteria, predominately Proteobacteria, though Planctomycetes were also relatively Introduction abundant. Sulfate-reducing and anaerobic ammonium- oxidizing bacteria, which play important roles in the cycling Marine sediments cover more than two-thirds of Earth’s of sedimentary nutrients, were abundant in the library. surface. Intact cells (Parkes et al. 2000)andintact Cluster of orthologous groups category analysis showed that membrane lipids (Zink et al. 2003) provide evidence of most of the genes contained in the end sequences were related prokaryotic populations in sediments as deep as 800 m to metabolism, and with cellular processes and signaling. below the seafloor and recently in sediments down to Functional groups assigned by SEED (subsystems-based 1,626 mbsf (Roussel et al. 2008). The prokaryotes of subseafloor sediments have been estimated to constitute as much as one-third of Earth’s total living biomass (Whitman Yongfei Hu and Chengzhang Fu contributed equally to this article. et al. 1998). Despite this, microbial community structure Electronic supplementary material The online version of this article and metabolic activity in subseafloor environments have (doi:10.1007/s10126-010-9259-1) contains supplementary material, been the subject of fewer studies than their terrestrial which is available to authorized users. counterparts (Llobet-Brossa et al. 1998). : : : : : : : Y. Hu C. Fu :Y. Yin G. Cheng: F. Lei X. Yang J. Li The paradigm that more than 99% of terrestrial bacteria E. J. Ashforth L. Zhang (*) B. Zhu (*) cannot be cultured by conventional means also holds true Institute of Microbiology, Chinese Academy of Sciences, for marine environments (Kennedy et al. 2008). DNA- No.1 West Beichen Road, Chaoyang District, Beijing 100101, People’s Republic of China based molecular methods have been developed to overcome e-mail: [email protected] many of the difficulties and limitations associated with e-mail: [email protected] cultivation techniques. 16S rRNA gene analysis, for example, provides important information on the taxonomy Y. Hu : C. Fu : Y. Yin : G. Cheng : F. Lei Graduate University of Chinese Academy of Sciences, of bacteria present in marine sediments (Edlund et al. Beijing 100049, People’s Republic of China 2008); however, there are problems associated with this Mar Biotechnol technique. PCR bias can be a problem especially in core was then transported back to the laboratory and environments where only low concentrations of extractable dissected into 5-cm sediment subsamples and stored at nucleic acids can be obtained, i.e., due to low numbers of −80°C. The top 5 to 10 cm layer was used for the prokaryotic cells (Chandler et al. 1997; Webster et al. 2003). construction of metagenomic fosmid library. 16S rRNA gene analysis also provides little information about the total genome of microbes within the community Microbial DNA Extraction and the functional role they play. By contrast, metagenomics (also referred to as environmental and community genomics), Prior to the total microbial DNA extraction, a sediment is a rapid and effective culture-independent approach to washing step, modified from Fortin et al. (2004), was used understanding and accessing the genetic information from to remove contaminants. Marine sediment (10 g wet microbial community within a specific environment by weight) was washed three times with 100 ml washing direct extraction and cloning of bacterial community DNA buffer (50 mM Tris–HCl, pH9.0, 100 mM Na2EDTA, 1.0% (Riesenfeld et al. 2004). This new and rapidly developing PVP, 100 mM NaCl, 0.05% Triton X-100) by vortexing for field has great potential in the study of marine micro- 1 min, incubating in a 55°C water bath for 3 min, and then organisms and in the past few years, has been applied centrifuged at 3,000×g for 5 min. The phosphate-buffered worldwide in the study of bacterial assemblages in marine saline (PBS) buffer was used as control with the same water and sediment (Beja et al. 2000; Hallam et al. 2004; washing procedures as described above. After sediment Venter et al. 2004;DeLongetal.2006; Martin-Cuadrado et washing, a 5 g pellet was mixed by vortexing with 13.5 ml al. 2007; Rusch et al. 2007; Biddle et al. 2008). of extraction buffer (100 mM Tris–HCl, pH8.0; 100 mM The South China Sea, one of the largest marginal seas in sodium EDTA, pH8.0; 100 mM sodium phosphate, pH8.0; the western Pacific, was formed by oceanic spreading along 1.5 M NaCl; and 1% CTAB). Three cycles of freezing in a WSW-ENE axis during the Oligo-Miocene, and has over liquid nitrogen and thawing in a 65°C water bath were then 4 km of organic-rich Cenozoic deposits in its sedimentary applied to the suspensions (Kauffmann et al. 2004). After basins (Ludmann et al. 2001). The microbial abundances the samples had cooled to 37°C, 50 μl of proteinase K in the surface sediment of this area were approximately (20 mg/ml) was added and the samples were incubated at 107cells/g sediment (Jiang et al. 2007), which indicates this 37°C with horizontal shaking at 225 rpm for 30 min. The is a huge ecological environment inhabited by large remaining DNA extraction procedures were performed as numbers of microbes. Previous studies using isolation of described by Zhou et al. (1996). pure cultures and 16S rRNA gene-based community diversity analysis have been carried out in this region (Xu Metagenomic Library Construction et al. 2004; Guo et al. 2007; Jiang et al. 2007; Liu et al. 2008; Tao et al. 2008; Tian et al. 2009); however, little is The extracted DNA was fractionated in 1% agarose (pulse known about the indigenous metagenome. In this study, a field-certified agarose, Bio-Rad) by pulsed-field gel electro- fosmid library from Qiongdongnan Basin sediment, a phoresis using a CHEF-DRIII system (Bio-Rad). Pulsed-field potential methane hydrate-bearing basin on the northwest- gel electrophoresis (PFGE) gels were run in 0.5× Tris–borate- ern continental shelf of the South China Sea (Wu et al. EDTA buffer for 16 h at 5 V/cm at an angle of 120°. Ramping 2003; Su et al. 2005) was constructed. The community was carried out from an initial switch time of 0.1 s to a final diversity and metabolic profile were preliminarily analyzed switch time of 40 s at 14°C. DNA fragments of approximately through the fosmid end sequences. To our knowledge, this 36 to 48 kb were cut from the gel and recovered by is the first insight into the function of this microbial electroelution (Sambrook et al. 1989). The DNA concentra- community using a metagenomic library approach. tion and purity was measured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). 16S rRNA genes were amplified using bacterial Materials and Methods primers 519F (5′-CAGCMGCCGCGGTAATAC-3′, posi- tions 519 to 536 in the Escherichia coli 16S rRNA gene) Marine Sediment Sample Collection and 1492R (5′-GGTTACCTTGTTACGACTT-3′,positions 1510 to 1492 in the E. coli gene). Pure E. coli DNA was The deep-sea sediment core was collected by gravity piston used as control template for 16S rRNA PCR amplification. corer in March 2006 at the BD7-2 station (110°28′47.231″ E PCR was performed as follows: one cycle of 94°C for 5 min, and 17°34′11.603″ N according to the global positioning followed by 30 cycles of denaturation at 94°C for 1 min, system) in the Qiongdongnan Basin, South China Sea. After annealing at 55°C for 1 min and elongation at 72°C for collection, the sediment core was stored onboard at 4°C 2 min, with a final extension at 72°C for 10 min. The (approximate in situ temperature) for about 2 weeks. The metagenomic library named IMCAS-F003 was constructed Mar Biotechnol using CopyControl™ HTP fosmid library production kit Results (Epicentre) according to the manufacturer's instructions. The repaired DNA was ligated into the pCC2 FOS fosmid vector Fosmid Library Construction and End Sequencing (Epicentre). Ligated DNA mixtures were then packaged using the supplied lambda packaging extracts and were Prior to the DNA extraction, a washing step was used to transformed into an EPI300-T1R phage T1-resistant E. coli remove contaminants (see Materials and Methods). After host. The infected bacterial cells were stored in glycerol at the washing buffer treatment, the supernatant was brown in −80°C, final glycerol concentration 20%. To determine the color compared to the clear PBS buffer treatment using the inserted fragment size, 64 randomly selected fosmids were same procedure. The crude DNA extract ranged in size extracted using Plasmid Mini Kit I (Omega, Doraville, GA, from 6 to 100 kb as shown in Fig. 1 and its yield was USA) and digested with EcoRI. approximately 1µg/g (wet weight) marine sediment. The UV (ultraviolet) absorption spectra showed that, without Fosmid End Sequencing and Sequence Analysis the washing step, contaminants in the crude DNA extract showed strong absorption in the UV range, masking any A total of 600 randomly selected fosmid clones were absorption of nucleic acids at 260 nm (Fig. 2a). After the subjected to bi-directional end sequencing at Open lab of washing step, the absorption interference was greatly Institute of Crop Sciences, Chinese Academy of Agricultural reduced and the absorption of DNA at 260 nm could be Sciences, using T7 promoter primer (5′-TAATACGACT detected clearly (Fig. 2b), indicating that humic and fulvic CACTATAGGG-3′) and pCC2 reverse sequencing primer substance contamination was removed to some degree, (5′-CAGGAAACAGCCTAGGAA-3′). All fosmid end however, the low A260/A230 value (0.9) suggested that sequences were revised and trimmed using Lasergene some contaminants remained. The following purification package, version 7.10 (DNA star, USA), and compared with sequences in the NCBI nr (non-redundant) database Fig. 1 Pulse field gel using BLASTX program (Ye et al. 2006)underacut-off electrophoresis of extracted value of 1e-5. Taxonomic binning analysis was based on DNA. Lane 1 PFGE Ladder. MEtaGenome Analyzer (MEGAN, version 3.0.2) (Huson Lane 2 the environmental DNA extracted from the sediment et al. 2007), which allows dissection of large datasets washed with washing buffer without the need for assembly or the targeting of specific (see Materials and Methods) phylogenetic markers. In a preprocessing step to using this program, sequences were compared against NCBI nr data- bases using BLASTX under default parameters. MEGAN was then used to compute and interactively explore the taxonomical content of the dataset, employing the NCBI taxonomy to summarize and order the results (absolute cut- off, BLASTX bitscore 75; relative cut-off, 10% of the top hit). For cluster of orthologous groups (COG) assignments, sequences were compared with the COG database using rpsBLAST (-p F) with a cut-off value of 1e-5. Other analysis including protein-based general taxonomy, SEED subsystem based metabolic profile and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway investigation, were per- formed with the help of the Meta Genome Rapid Annotation using Subsystem Technology (MG-RAST) server (Meyer et al. 2008) using an e-value cut-off of 1e-5 and a minimum alignment length of 50 bp. MG-RAST program is a fully automated service for annotating metagenomic data (http:// metagenomics.theseed.org/metagenomics.cgi?).

Nucleotide Sequence Accession Numbers

Nucleotide sequences of the 1,051 fosmid ends have been deposited into the GenBank GSS database with accession numbers of FI496080-FI497130. Mar Biotechnol

Fig. 2 UV absorption spectra of extracted and purified DNA. a Crude DNA extracted from the sediment washed with PBS buffer. b Crude DNA extracted from the sediment washed with washing buffer (see Materials and Methods). c DNA purified after PFGE and electroelution step was combined with size selection using PFGE, and MEGAN software, a program that assigns read to lowest- DNA fragments of 36–48 kb were cut from the gel and common ancestor of all its BLAST hits. By using this recovered by electroelution. After these steps, approximate- program, 660 sequences were assigned to different taxo- ly 70% of the DNA was recovered and showed a very good nomic categories, 389 had no hits, and two sequences were absorption profile with an A260/A280 value of 1.83 and an not assigned to any taxa (Fig. 3). The results showed that A260/A230 value of 1.85 (Fig. 2c). This DNA had a high the IMCAS-F003 metagenomic library was dominated by enough purity for restriction endonucleases EcoR I and Bacteria with a total of 583 sequences assigned to discrete Hind III digestion and PCR amplification of 16S rRNA taxa and 383 sequences assigned to different definite genes (data not shown). subcategories. The metagenomic library (IMCAS-F003) contained The largest group of sequences were from the bacterial approximately 200,000 clones. EcoRI digestion of 64 phylum Proteobacteria (164 sequences, accounting for randomly selected fosmid clones indicated that the library 42.8% of the total sequences assigned to definite subcate- possessed a rich diversity (data not shown). The average gories), and within this the Deltaproteobacteria were most insert size was 36 kb and the total size of this fosmid library abundant followed by Alpha- and Gammaproteobacteria. was estimated to be 7.2 Gb of metagenomic DNA. Given Within the Deltaproteobacteria group, sulfate-reducing an average prokaryotic genome is approximately 5 Mb, the bacteria (SRB) Desulfobacterales, Desulfuromonadales, library theoretically was the size of over 1,400 prokaryotic and some Syntrophobacter species accounted for the genomes. largest proportion. The second largest group following the Sixhundredrandomlychosenfosmidcloneswere Proteobacteria was Planctomycetes, with 96 sequences subjected to bi-directional end sequencing. After vector (25.1%) assigned to this phyla. Sequences related to trimming, low quality and shorter sequences were removed Planctomyces maris, Blastopirellula marina, and Candida- and a total of 1,051 fosmid end sequences with read lengths tus Kuenenia stuttgartiensis were in relatively high numb- from 300 to 800 bp were obtained. These sequences were ers within this group. Other taxa were also detected, namely of high quality with an average read length of 619 bp, and a Chloroflexi, Cyanobacteria, Acidobacteria, Verrucomicrobia, sequence length >450 bp accounted for 93.0% of the total Actinobacteria, and Firmicutes. Furthermore, 12 sequences end sequences. The GC content of the sequences ranged were assigned to Archaea among which the methanogenic from 24% to 73%, with an average content of 52.9%. More Euryarchaeota orders Methanomicrobiales, Methanosarcinales, than 65% of these sequences exhibited high GC content Methanopyrales, and two uncultured methanogenic archaeon (>55%). from environmental samples were detected.

Community Composition Metabolic Potential

MG-RAST analysis revealed 758 hits against the SEED Based on the BLASTX search against the NCBI nr protein non-redundant database and 0 hits against the database, 579 of the 1,051 end sequences were assigned ribosomal RNA database Greengenes. Most of the sequen- to functional genes; 203 were classified as hypothetical ces, 701 (92.48%) were of bacteria origin, 33 were archaea, proteins; 20 sequences were genes with unknown function; and 15 sequences were of eukaryota origin, nine sequences and 249 sequences did not match any hits (for detail see were not assigned to any group. supplementary material Table 1). COG category analysis To obtain detailed taxonomic category information from showed that 42.2% of the genes were related to metabolism all the fosmid ends, the sequences were analyzed using the and 25.2% to cellular processes and signaling, whereas only Mar Biotechnol

Fig. 3 Phylogenetic diversity of the IMCAS-F003 fosmid end number means number of reads summarized, that is number of reads sequences computed by MEGAN. Each circle in the figure represents assigned to the corresponding taxa, or to any that contained in the a taxon in the NCBI taxonomy and is labeled by its name and two subtree (no summarized number for the last taxon). The size of the numbers. The first number means number of reads assigned, that is circle is scaled logarithmically to represent the number of reads number of reads assigned to the corresponding taxa. The second summarized

12.6% corresponded to housekeeping genes involved in subsystems (12.01%); carbohydrate (11.8%); and amino information-related processes. In addition, 20% of the acids and derivatives (10.35%). The clustering-based sub- genes fell into a poorly characterized group (Fig. 4). The systems included a total of 20 subcategories, such as most abundant metabolic type was amino acid transport biosynthesis of galactoglycans and related lipopolysacharides and metabolism (11.3%), followed by energy production (seven sequences), cytochrome biogenesis (five sequences), and conversion (9.6%) and then carbohydrate transport and fatty acid metabolic cluster (three sequences), etc. (for detail metabolism (6.2%; for detail see supplementary material see supplementary material Table 3). The carbohydrate Table 2). subsystem was dominated by central carbohydrate metabo- Further analysis of functional groups was carried out lism (nearly 28% of the total for the category). One-carbon using SEED subsystems. A subsystem was defined as a set metabolism and monosaccharides metabolism subcategories of functional roles that together implement a specific were also very common, accounting for 17.54% and 14.04% biological process, such as the genes whose products are of the identified carbohydrate subsystem, respectively. In involved in a metabolic pathway, or the group of genes addition, three hits to methanogenesis were detected in whose products make a cellular structure (Overbeek et al. this subsystem, namely N5-methyltetrahydromethanopterin: 2005). A total of 483 sequences were classed into SEED coenzyme M methyltransferase subunit H (29% identity to subsystems using an e-value cut-off of 1e-5 (Fig. 5). The top Archaeoglobus fulgidus DSM 4304), formylmethanofuran- three functional categories determined were: clustering-based tetrahydromethanopterin N-formyltransferase (50% identity Mar Biotechnol

Fig. 4 Distribution of fosmid ends in COG categories. Name of subcategories in COG database are listed on the left, and corresponding major categories are list on the right. The number of reads assigned to each major category and their ratio are shown to Methanopyrus kandleri AV19), and dimethylamine naphthalene, anthracene, styrene, tetrachloroethene, and methyltransferase corrinoid protein (46% identity to gamma-hexachlorocyclohexane (for detail see supplemen- Methanosarcina acetivorans C2A; for detail see supple- tary material Table 4). This result reinforces the previous mentary material Table 3). It is unsurprising, due to the observations that microorganisms living in deep-sea deep-sea origin of the sample, that genes related to environment are adapted to degrade recalcitrant pools of photosynthesis were not detected. In addition, the distri- organic matter (Martin-Cuadrado et al. 2007). bution of the fosmid ends in KEGG metabolic pathway indicated that genes involved in the biodegradation of xenobiotics were very diverse, including the degradation Discussion of DL-dithiothreitol, dichloroethane, chloroacrylic acid, benzoate, biphenyl, caprolactam, ethylbenzene, fluorene, Construction of large insert environmental metagenomic libraries depend on efficient recovery and purification of high-quality microbial DNA. Many extraction procedures have been developed to isolate nucleic acids directly from environmental samples; however, the post-extraction puri- fication procedures, such as the use of silica gel/membrane, ion exchange chromatography etc., are often time- consuming and laborious (Schneegurt et al. 2003). In the present study, a washing step prior to SDS-based DNA extraction was used. This step removed the contaminants to a great extent as was shown in the UV spectral profile. After this treatment, the extracted crude DNA was directly subjected to size selection by using pulsed-field gel electrophoresis and recovered by electroelution. Both the PFGE and electroelution are also considered as effective strategies to separate DNA from humic materials (Rajendhran and Gunasekaran 2008). Since size selection was a necessary step for fosmid library construction, the combination of purification and size selection in one step here reduced the number of operation steps and saved time, thus avoiding of DNA loss and Fig. 5 Distribution of fosmid ends in SEED subsystems degeneration. It should be mentioned that some microbes Mar Biotechnol may be lost from the washing step, thus causing the DNA indicates their potentially important role in the flux of extraction bias. Therefore, the recovery rate of microbial nutrients in this sediment environment. cells during the washing process and to what extent it Functional groups assigned by SEED subsystems indi- influence the final DNA yield deserves further investigation. cated that the most dominant category in this library was The phylogenetic ascription of random sequenced fosmid clustering-based subsystems. A clustering-based subsystem ends allowed us to compare the magnitude of Bacteria and is described as “one in which there is functional coupling Archaea in this sediment. Analysis indicated that Bacteria evidence that genes belong together, but we do not yet were approximately 20- and 50-fold more abundant than know what they do”. http://www.nmpdr.org/FIG/wiki/view. Archaea according to protein-based taxonomy in SEED and cgi/FIG/ClusteringBasedSubsystem. Hence we cannot com- MEGAN phylogenetic ascription, respectively. This sug- ment on the majority of the genes assigned to these gested that Bacteria dominated the prokaryotes in this subsystems or speculate as to the roles that they play. subsurface sediment, with Archaea contributing only a small The sampling site located at slope of Qiongdongnan proportion. Similar results have also been found using Basin is one of potential methane hydrate-bearing basins quantitative molecular techniques, indicating Archaea are on the northwestern continental shelf. The presence of 10–1,000-fold less abundant than Bacteria in sediment methanehydratesinthisareahasbeenconfirmedby environments (Schippers and Neretin 2006). Recent lipid- geological and geophysical evidence (Wu et al. 2003; based evidence, however, has indicated that in marine Su et al. 2005). In this study, one-carbon metabolisms subsurface sediments Archaea are more abundant than were found very common. This metabolic type convert Bacteria (Lipp et al. 2008). It is possible, therefore, that complex organic matter to simple one-carbon compounds archaeal biomass may have been underestimated in this which play important roles in the process of methano- study due to poor extraction efficiency of Archaea DNA. genesis and are dominant in the methanogenic Archaea The Bacteria group in this library was dominated by the (Ferry 1999). More important, three enzymes closely related Proteobacteria and within this the Deltaproteobacteria were to those involved in methanogenesis were detected in this most abundant (68 sequences, nearly 41% of the total library according to SEED subsystems. The first one N5- sequences assigned to Proteobacteria). The Deltaproteobac- methyltetrahydromethanopterin: coenzyme M methyltransfer- teria was also the majority in Nasha and Xisha surface ase is an important enzyme in the process of methanogenesis sediments of South China Sea (38% and 64% of 16S rRNA from H2 and CO2, as well as from acetate. The second one genes belonging to Proteobacteria, respectively) of 16S formylmethanofuran-tetrahydromethanopterin N-formyltrans- rRNA genes belonging to Proteobacteria. The Deltaproteo- ferase is a key enzyme of the pathway for dismutation of bacteria contain most of the known sulfate-reducing methanol to methane. The last one dimethylamine (DMA) bacteria. The abundance of these microorganisms may well methyltransferase methylates the DMA corrinoid protein relate to the relatively high sulfate content (Jiang et al. during a methylamine initiated methanogenesis process. In 2007) in this area. SRB use sulfate as a terminal electron addition to this, seven sequences were assigned to Euryarch- acceptor for the degradation of organic compounds, result- aeota, an archaeal kingdom that most of marine origin ing in the production of sulfide. SRB have been found to methanogens belonged to. These methanogenic archaea have play a dominant role in the bacterial communities of deep- one common attribute in they all use a methane-generating sea sediments (Fry et al. 2008) and have been linked to pathway for growth (Sowers 2004). This data provides the anaerobic carbon cycling, in fact it has been estimated that initial microbiological evidence for methanogenesis in this sulfate reduction can account for more than 50% of the area. Further efforts on screening and shotgun sequencing of organic carbon mineralization in marine sediments (Muyzer fosmid clones containing key genes for methanogenesis are and Stams 2008). under way in order to identify complete methane-generating The Planctomycetes were the next most abundant group pathways. in this library, and have also been found in high numbers in Since the metagenomic fosmid library presented here the Xisha Trough (20.3% of the bacterial 16S rRNA gene was constructed from an extreme environment, novel library) and Nansha sediments (10% of the bacterial 16S biotechnological biocatalysts with diverse biochemical and rRNA gene library) of the South China Sea (Xu et al. 2004; physiological characteristics were expected. The search for Tao et al. 2008). Planctomycetes have been recently novel enzymes, lipolytic enzymes, for example, is now in identified as the “missing lithotroph” responsible for the progress. anaerobic oxidation of ammonium (anammox process; In conclusion, high-quality microbial DNA was isolated Strous et al. 1999), and this has led to the discovery of from Qiongdongnan Basin sediment from the South China other autotrophic Planctomycetes with similar activities Sea, and a metagenomic fosmid library was constructed (Schmid et al. 2000). Taken together, with the relatively with the purified DNA. A total of 1,051 fosmid end high abundance of these microbes observed in the library, sequences were obtained from random selected clones. Mar Biotechnol

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