Isolation of High Molecular Weight DNA from Marine Sponge Bacteria for BAC Library Construction

Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.     

A novel retrieval system for nearly complete microbial genomic fragments from soil samples

The construction of a complex genomic library is one of the comprehensive ways to study a complex bacterial community and to access the variety of metabolic pathways present in the rich soil environment. In this report, we developed a new protocol whereby we are able to retrieve nearly complete microbe genomic fragments from soil samples, which are employed to generate a metagenomic library for visualizing the basic scaffolding of the soil microbial community. The use of direct cell lysis within soil-embedded agarose plugs, along with a double-size selection, enabled us to successfully isolate pure and high-molecular weight DNA (0.1-1 Mb) without the need for any further purification. A metagenomic library containing 1.2 Gbp of DNA in total was constructed. Furthermore, analysis of the microbial community structure using 16S rDNA partial sequences found the dominant phylotypes to consist of alpha-Proteobacteria and Actinobacteria, which are similar to those seen in forest and agricultural soils, and numerous uncultured microbes from a wide variety of bacterial taxa as well. In conclusion, this study presents a novel protocol for generating a metagenomic library that carries much larger and diverse DNA fragments from soil bacteria that will be applied for the reconstruction of soil microbial genomes and the discovery of novel habitat-specific pathways.     

Construction of a metagenomic library for the marine sponge Halichondria okadai

Symbionts of the marine sponge Halichondria okadai are promising as a source of natural products. Metagenomic technology is a powerful tool for accessing the genetic and biochemical potential of bacteria. Hence, we established a method of recovering bacterial-enriched metagenomic DNA by stepwise centrifugation. The metagenomic DNA was analyzed by ultrafast 454-pyrosequencing technology, and the results suggested that more than three types of bacterial DNA, Alphaproteobacteria, Actinobacteria, and Cyanobacteria, had been recovered, and that eukaryotic genes comprised only 0.02% of the metagenomic DNA. These results indicate that stepwise centrifugation and real-time quantitative PCR were effective for separating sponge cells and symbiotic bacteria, and that we constructed a bacteria-enriched metagenomic library from a marine sponge, H. okadai, selectively for the first time.     

Community level physiological profile of soil bacteria unaffected by extraction method.

Extraction and purification of bacteria from soil by the Nycodenz gradient centrifugation procedure described by Bakken and Lindahl (1995; Recovery of bacterial cells from soil. In: van Elsas, J.D., Trevors, J.T. (Eds.), Nucleic Acids in the Environment: Methods and Applications. Springer Verlag, Berlin, pp. 9-27) were compared to soil slurry extractions. Bacterial communities from four different soils were described by the bacterial abundance, CTC-reducing capacity, culturability and the community level physiological profiles (CLPP) in BIOLOG GN plates. A significant loss of both total and culturable number of bacteria g(-1) soil dry weight were found after extraction and purification of cells. The origin of soil influenced the yield of cells and a difference between the four soils and an interaction between the soils and extraction procedure were found. The culturability and the CLPP were different between the four soils but were unaffected by the extraction procedure. The bacterial community obtained after extraction and purification thus represented the same fraction of the indigenous bacterial community.     

Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.     

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.     

Bacterial diversity of metagenomic and PCR libraries from the Delaware River

To determine whether metagenomic libraries sample adequately the dominant bacteria in aquatic environments, we examined the phylogenetic make-up of a large insert metagenomic library constructed with bacterial DNA from the Delaware River, a polymerase chain reaction (PCR) library of 16S rRNA genes, and community structure determined by fluorescence in situ hybridization (FISH). The composition of the libraries and community structure determined by FISH differed for the major bacterial groups in the river, which included Actinobacteria, beta-proteobacteria and Cytophaga-like bacteria. Beta-proteobacteria were underrepresented in the metagenomic library compared with the PCR library and FISH, while Cytophaga-like bacteria were more abundant in the metagenomic library than in the PCR library and in the actual community according to FISH. The Delaware River libraries contained bacteria belonging to several widespread freshwater clusters, including clusters of Polynucleobacter necessarius, Rhodoferax sp. Bal47 and LD28 beta-proteobacteria, the ACK-m1 and STA2-30 clusters of Actinobacteria, and the PRD01a001B Cytophaga-like bacteria cluster. Coverage of bacteria with > 97% sequence identity was 65% and 50% for the metagenomic and PCR libraries respectively. Rarefaction analysis of replicate PCR libraries and of a library constructed with re-conditioned amplicons indicated that heteroduplex formation did not substantially impact the composition of the PCR library. This study suggests that although it may miss some bacterial groups, the metagenomic approach can sample other groups (e.g. Cytophaga-like bacteria) that are potentially underrepresented by other culture-independent approaches.     

Evaluation of quantitative recovery of bacterial cells and DNA from different lake sediments by Nycodenz density gradient centrifugation

While purified bacterial cells and DNA – the signature of life – from soil and sediment matrices have been extensively studied in a wide range of environments and in different microbial ecosystems, the paucity of data on DNA extraction from contaminated sediments emphasizes the need for further research on the isolation and quantification of bacterial cells and DNA in sediments. Consequently, the Nycondez gradient centrifugation method was applied to extract bacterial cells from contaminated and uncontaminated sediments. Quantitative estimates of recovered bacterial cells were obtained from direct counts performed using DAPI (4′,6′-diamino-2-phenylindole hypochloride) staining couples with fluorescence microscopy and indirect counts (colony-forming units). The estimation was improved by using an efficient method of comparing sediment types composed of quantifying bacterial densities in three steps: S₁ the initial freshwater sediments; S₂ the first supernatant recovered after mixing the sediments with sodium hexametaphosphate solution followed by centrifugation; and S₃ the extracted cells. Total and extracellular DNA were extracted and quantified in each of the three steps. Additional analysis of faecal indicator bacteria (FIB) including E. coli and Enterococcus (ENT) was also performed in each step. The results display considerable variability in the quantity of bacteria cells depending on sediment type, ranging from 1.2 × 10⁵ to 6.2 × 10⁹ cell g^?1 dry sediments. The treatment with sodium hexametaphosphate solution (2%) leads to the desorption of bacterial populations which were firmly adsorbed on contaminated sediment surfaces resulting in more than 90% of the FIB being recovered. The Nycondez density gradient centrifugation method makes it possible to extract bacterial cells from freshwater sediments without extracellular DNA so it is ideal for metagenomic analysis of bacteria.     

Nonlinear electrophoresis for purification of soil DNA for metagenomics

Purification of microbial DNA from soil is challenging due to the co-extraction of humic acids and associated phenolic compounds that inhibit subsequent cloning, amplification or sequencing. Removal of these contaminants is critical for the success of metagenomic library construction and high-throughput sequencing of extracted DNA. Using three different composite soil samples, we compared a novel DNA purification technique using nonlinear electrophoresis on the synchronous coefficient of drag alteration (SCODA) instrument with alternate purification methods such as direct current (DC) agarose gel electrophoresis followed by gel filtration or anion exchange chromatography, Wizard DNA Clean-Up System, and the PowerSoil DNA Isolation kit. Both nonlinear and DC electrophoresis were effective at retrieving high-molecular weight DNA with high purity, suitable for construction of large-insert libraries. The PowerSoil DNA Isolation kit and the nonlinear electrophoresis had high recovery of high purity DNA suitable for sequencing purposes. All methods demonstrated high consistency in the bacterial community profiles generated from the DNA extracts. Nonlinear electrophoresis using the SCODA instrument was the ideal methodology for the preparation of soil DNA samples suitable for both high-throughput sequencing and large-insert cloning applications.     

A procedure for the metagenomics exploration of disease-suppressive soils

The microbiota of, in particular, disease-suppressive soils contains a wealth of antibiotic biosynthetic loci that are inaccessible by traditional cultivation-based techniques. Hence, we developed a methodology based on soil microbial DNA, which allowed the metagenomics-based unlocking of the relevant genes. Here, a streamlined soil metagenomics protocol is presented. The protocol consists of an optimized method to extract bacterial cells from a Rhizoctonia solani AG3 suppressive loamy sand soil followed by DNA extraction and purification, and the preparation of a clone library in an efficient host/vector system. Methods for the functional and genetic screening of the library for antibiotic production loci are also described. Using the suppressive soil, we thus produced, screened and tested an approximate 15,000-membered metagenomic library of fosmids in an Escherichia coli host. Functional screens, based on dual culturing of clone arrays with R. solani AG3 and Bacillus subtilis 168, were largely negative. Genetic screens, based on hybridizations with soil-generated probes for polyketide biosynthesis, non-ribosomal protein synthesis and gacA, revealed several inserts, of around 40-kb in size, with potential antibiotic production capacity. We present the full sequences of three selected clones. We further examine the challenges that still impinge on the metagenomic exploration of disease-suppressive soil.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

Construction, Analysis, and β-Glucanase Screening of a Bacterial Artificial Chromosome Library from the Large-Bowel Microbiota of Mice

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for β-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with β-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.     

Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.     

结合宏基因组末端随机测序和16S rDNA技术分析温室黄瓜根围土壤细菌多样性

土壤细菌在温室土壤环境中具有十分重要的生态功能,与温室作物以及微生物内部存在互作关系。研究土壤细菌的群落结构组成,有助于了解土地利用变化与生态环境效应之间的关系。结合16S rRNA基因克隆文库和宏基因组末端测序对温室黄瓜根围土壤细菌的多样性进行了分析。在16S文库中,根据97%的序列相似性水平划分OTU,共有35个OTU,其中优势菌群是γ-Proteobacteria,其次为Firmicutes,Bacillus为优势细菌。在纲分类水平上,16S文库和宏基因组末端测序结果均包含γ-Proteobacteria、α-Proteobacteria、δ-Proteobacteria、β-Proteobacteria、Actinomycetales和Firmicutes,各纲比例有差别;在优势种群属水平上,末端测序的结果包含的属多于16S文库(4035);在优势细菌种类上,两者反映的结果一致,均为Bacillus。但是,宏基因组末端测序包含了大多数的弱势种群,更能反映细菌多样性的真实水平。与露地土壤细菌16S文库相比较,土壤细菌多样性降低,这可能与温室多年连作,种植蔬菜种类单一直接相关。     

Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.     

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen-suppressive soil

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF¹⁰³ of the isolate Streptomyces mutomycini and/or Streptomyces clavifer . There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.     

A procedure for the metagenomics exploration of disease-suppressive soils

The microbiota of, in particular, disease-suppressive soils contains a wealth of antibiotic biosynthetic loci that are inaccessible by traditional cultivation-based techniques. Hence, we developed a methodology based on soil microbial DNA, which allowed the metagenomics-based unlocking of the relevant genes. Here, a streamlined soil metagenomics protocol is presented. The protocol consists of an optimized method to extract bacterial cells from a Rhizoctonia solani AG3 suppressive loamy sand soil followed by DNA extraction and purification, and the preparation of a clone library in an efficient host/vector system. Methods for the functional and genetic screening of the library for antibiotic production loci are also described. Using the suppressive soil, we thus produced, screened and tested an approximate 15,000-membered metagenomic library of fosmids in an Escherichia coli host. Functional screens, based on dual culturing of clone arrays with R. solani AG3 and Bacillus subtilis 168, were largely negative. Genetic screens, based on hybridizations with soil-generated probes for polyketide biosynthesis, non-ribosomal protein synthesis and gacA, revealed several inserts, of around 40-kb in size, with potential antibiotic production capacity. We present the full sequences of three selected clones. We further examine the challenges that still impinge on the metagenomic exploration of disease-suppressive soil.     

A Simple Silica-based Method for Metagenomic DNA Extraction from Soil and Sediments

A new method is described for extraction of metagenomic DNA from soil and sediments which is based on DNA adsorption to silica without the use of phenol, ethanol precipitation or a cesium chloride gradient. High-quality DNA was obtained, and PCR inhibition was overcome by adding bovine serum albumin and adjusting magnesium concentration. By using PCR-DGGE with Firmicutes and lactic acid bacteria-specific primers the extracted metagenomic DNA was shown to contain a mixture of bacterial genomes. This method can be used for screening bacterial diversity in soil and sediment samples.     

Enriching plant microbiota for a metagenomic library construction

Plant microbiota (the microorganisms that live in any associations with plant tissues) represents a rather unexplored area of metagenomic research compared with soils and oceans. Constructing a metagenomic library for plant microbiota is technically challenging. Using all the biomass without pre-enrichment could lead to vast proportions of the host plant DNA in the metagenomic library, doubtless obliterating the microbial contribution. Therefore, the first and essential step is to enrich for the constituent microorganisms from plant tissues. Here, a strong enrichment for plant microbiota was achieved by coupling SDS (sodium dodecyl sulfate) with NaCl, creating a predominantly microbial metagenomic library that contains 88% bacterial inserts. 16S rDNA sequence analysis revealed that the metagenomic DNA of enrichments originates from very diverse microorganisms. At least 74 distinct ribotypes (at a 97% threshold) from seven different bacterial phyla were identified and mainly distributed among Actinobacteria and Proteobacteria. Additionally, a simplified version of Amplified Ribosomal DNA Restriction Analysis (ARDRA) was developed for a quick and efficient assessment of the enriching procedures. This work opens further insight into the great biotechnical potential of plant microbiota, holding more potential for drug discovery through a metagenomic strategy, and paving the way for recovery and biochemical characterization of functional gene repertoire from plant microbiota.