Preservation, origin and genetic imprint of extracellular DNA in permanently anoxic deep-sea sediments

Molecular approaches that target the total DNA pool recovered from permanently anoxic marine ecosystems have revealed an extraordinary diversity of prokaryotes and unicellular eukaryotes. However, the presence of gene sequences contained within the extracellular DNA pool is still largely neglected. We have investigated the preservation, origin and genetic imprint of extracellular DNA recovered from permanently anoxic deep-sea sediments of the Black Sea. Despite high DNase activities, huge amounts of total extracellular DNA were found in both the surface and subsurface sediment layers, suggesting reduced availability of the extracellular DNA pool to nuclease degradation. The reduced degradation of the total extracellular DNA was confirmed by its low decay rate and the high accumulation in the deeper sediment layers. The copy numbers of 16S and 18S rDNA contained within the extracellular DNA pool in both the surface and subsurface sediment layers was very high, indicating that permanently anoxic sediments of the deep Black Sea are hot spots of preserved extracellular gene sequences. The extracellular DNA recovered from these sediment layers also contained highly diversified 18S rDNA sequences. These were not only representative of the major protistan lineages, but also of new very divergent lineages, branching as independent clades at the base of the tree. Our findings indicate that the extracellular DNA pool is a major archive of present/past eukaryotic gene sequences, and they highlight the importance of integrating molecular cell-oriented approaches with molecular analyses of the extracellular DNA pool, for a better assessment of microbial diversity and temporal changes in marine benthic ecosystems.     

Persistence of transgenic and not transgenic extracellular DNA in soil and bacterial transformation.

The study of the fate of transgenic and not transgenic extracellular DNA in soil is of extreme relevance because the soil extracellular DNA pool represents a genetic reservoir that could be utilized as a source of food by any heterotrophic microorganism or genetic information by recipient eukaryotic and prokaryotic cells. Several data have clearly evidenced that extracellular DNA could persist in soil for long time maintaining a sufficient integrity of the molecule. Recent microcosm studies under laboratory conditions have evidenced that extracellular DNA molecule could be leached or raised up by capillarity. The persistence and movement of extracellular DNA molecule in soil suggest that the genetic information of extracellular DNA could be taken up by microorganisms temporarily and spatially separated. Several authors have studied the persistence and transformation efficiency of the extracellular DNA in soil demonstrating that there is a sharp discrepancy between its biological efficiency and its persistence; fragments of target DNA were detected after a long time in soil but no transformations were determined probably because the genetic information originally present in the complete DNA molecule could be lost by degradation. It is also important to underline that the frequency of gene transfer in soil is markedly limited by the few number of bacteria able to develop competence and that this physiological state is reached only under certain conditions. Furthermore the dilution of the transgene in the soil extracellular DNA pool drastically decreases chances for the uptake of the transgene. Anyway the importance of transformation in evolutionary terms, represents a valid reason to continue the investigation on the fate of extracellular DNA in soil.     

Metabolism of organic compounds in anaerobic,hydrothermal sulphate-reducing marine sediments

Previous studies of hot (>80 degrees C) microbial ecosystems have primarily relied on the study of pure cultures or analysis of 16S rDNA sequences. In order to gain more information on anaerobic metabolism by natural communities in hot environments, sediments were collected from a shallow marine hydrothermal vent system in Baia di Levante, Vulcano, Italy and incubated under strict anaerobic conditions at 90 degrees C. Sulphate reduction was the predominant terminal electron-accepting process in the sediments. The addition of molybdate inhibited sulphate reduction in the sediments and resulted in a linear accumulation of acetate and hydrogen over time. [U-14C]- acetate was completely oxidized to 14CO2, and the addition of molybdate inhibited 14CO2 production by 60%. [U-14C]-glucose was oxidized to 14CO2, and this was inhibited when molybdate was added. When the pool sizes of short-chain fatty acids were artificially increased, radiolabel from [U-14C]-glucose accumulated in the acetate pool. L-[U-14C]-glutamate, [ring-14C]-benzoate and [U-14C]-palmitate were also anaerobically oxidized to 14CO2 in the sediments, but molybdate had little effect on the oxidation of these compounds. These results demonstrate that natural microbial communities living in a hot, microbial ecosystem can oxidize acetate and a range of other organic electron donors under sulphate-reducing conditions and suggest that acetate is an important extracellular intermediate in the anaerobic degradation of organic matter in hot microbial ecosystems.     

Degradation and turnover of extracellular DNA in marine sediments: ecological and methodological considerations

Degradation rates of extracellular DNA determined in marine sediments were much higher than those in the water column. However, due to the high sediment DNA content, turnover times were much shorter in seawater. Results reported here provide new insights into the role of extracellular DNA in P cycling in marine ecosystems.     

Degradation and Turnover of Extracellular DNA in Marine Sediments: Ecological and Methodological Considerations

Degradation rates of extracellular DNA determined in marine sediments were much higher than those in the water column. However, due to the high sediment DNA content, turnover times were much shorter in seawater. Results reported here provide new insights into the role of extracellular DNA in P cycling in marine ecosystems.     

Non-Enzymatic Depurination of Nucleic Acids: Factors and Mechanisms

Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC). Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius’ law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA) was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination.     

Specific 16S rDNA Sequences Associated with Naphthalene Degradation under Sulfate-Reducing Conditions in Harbor Sediments

Previous studies have demonstrated that naphthalene and other polycyclic aromatic hydrocarbons (PAHs) can be anaerobically oxidized with the reduction of sulfate in PAH-contaminated marine harbor sediments, including those in San Diego Bay. In order to learn more about the microorganisms that might be involved in anaerobic naphthalene degradation, the microorganisms associated with naphthalene degradation in San Diego Bay sediments were evaluated. A dilution-to-extinction enrichment culture strategy, designed to recover the most numerous culturable naphthalene-degrading sulfate reducers, resulted in the enrichment of microorganisms with 16S rDNA sequences in the d-Proteobacteria, which were closely related to a previously described pure culture of a naphthalene-degrading sulfate reducer, NaphS2, isolated from sediments in Germany. A more traditional enrichment culture approach, expected to enrich for the fastest-growing naphthalene-degrading sulfate reducers, yielded 16S rDNA sequences closely related to those found in the dilution-to-extinction enrichments and NaphS2. Analysis of 16S rDNA sequences in sediments from two sites in San Diego Bay that had been adapted for rapid naphthalene degradation by continual amendment with low levels of naphthalene suggested that the microbial community composition in the amended sediments differed from that present in the unamended sediments from the same sites. Most significantly, 6-8% of the sequences recovered from 100 clones of each of the naphthalene-amended sediments were closely related to the 16S rDNA sequences in the enrichment cultures as well as the sequence of the pure culture, NaphS2. No sequences in this NaphS2 phylotype were recovered from the sediments that were not continually exposed to naphthalene. A PCR primer, which was designed based on these phylotype sequences, was used to amplify additional 16S rDNA sequences belonging to the NaphS2 phylotype from PAH-degrading sediments from Island End River (Boston), MA, and Liepaja Harbor, Latvia. Closely related sequences were also recovered from highly contaminated sediment from Tampa Bay, FL. These results suggest that microorganisms closely related to NaphS2 might be involved in naphthalene degradation in harbor sediments. This finding contrasts with the frequent observation that the environmentally relevant microorganisms cannot be readily recovered in pure culture and suggests that further study of the physiology of NaphS2 may provide insights into factors controlling the rate and extent of naphthalene degradation in marine harbor sediments.     

Genetic characterization of microbial communities living at the surface of building stones

Aims:  The aim of the present study was to reveal the microbial genetic diversity of epilithic biofilms using a DNA-based procedure.
Methods and Results:  A DNA extraction protocol was first selected to obtain PCR-amplifiable metagenomic DNA from a limestone biofilm. Extracted DNA was used to amplify either 16S rRNA genes or ITS regions from prokaryotic and eukaryotic genomes, respectively. Amplified DNAs were subsequently cloned, amplified by colony PCR and screened by restriction analysis [restriction analyses of amplified ribosomal DNA (ARDRA)] for DNA sequencing. Phylogenetic analysis using 16S rDNA sequences showed that predominating bacteria were Alphaproteobacteria belonging to the genera Sphingomonas , Erythrobacter , Porphyrobacter , Rhodopila and Jannashia ; Cyanobacteria and Actinobacteria were also identified. Analysis of ITS rDNA sequences revealed the presence of algae of the Chlorophyceae family and fungi related either to Rhinocladiella or to a melanized ascomycete. Statistical analysis showed that the specific richness evidenced was representative of the original sampled biofilm.
Conclusions:  The molecular methodology developed here constitutes a valuable tool to investigate the genetic diversity of microbial biofilms from building stone.
Significance and Impact of the Study:  The easy-to-run molecular method described here has practical importance to establish microbiological diagnosis and to define strategies for protection and restoration of stone surfaces.     

Identification of acquired DNA in Neisseria lactamica

Anomalous DNA (aDNA) in prokaryotic genomes, identified by its aberrant nucleotide composition, generally represents horizontally acquired DNA. Previous studies showed that frequent DNA transfer occurs between commensal Neisseriae and Neisseria meningitidis. Currently, it is unknown whether aDNA regions are also transferred between these species. The genome of Neisseria lactamica strain 892586 was assessed by a strategy that enables the selective isolation of aDNA, using endonucleases with recognition sites that are overrepresented in aDNA. Of eight regions with aDNA, five displayed similarity to virulence-associated meningococcal sequences. Of three aDNA fragments with limited or no similarity to neisserial sequences, one encodes a novel putative autotransporter/adhesin. The remaining two fragments are adjacent in the N. lactamica genome, and encode a novel putative ATPase/subtilisin-like protease operon. A similar operon is present in the genomes of different respiratory tract pathogens. The identification of aDNA from N. lactamica with similarity to meningococcal aDNA shows that genetic exchange between the Neisseriae is not limited to the neisserial core genome. The discovery of aDNA in N. lactamica similar to a locus in other pathogens substantially expands the neisserial gene pool.     

凝胶条带内DNA回收测序评估DGGE可靠性

为了评估DGGE的可靠性,对DGGE条带中回收的DNA片段进行了测序比较分析,并引入了DGGE可靠性指数的概念评价其可靠性。结果显示同一条DGGE条带回收的DNA来自同一属的概率为64.7%,相同位置的DGGE条带可以被认为是同一OTU;不同的DGGE条带回收到类似的DNA序列（16S rDNA V3区差异小于4 bp）的概率为10.5%;DGGE可靠性指数为74.8%。以上结果表明尽管DGGE技术与理论预期存在一定的差距,但是DGGE技术基本能够反映微生物群落的多样性。       

Rarity associated with specific ecological niches in the bacterial world: the 'Synergistes' example

The 'Synergistes' group, which apparently represents an as yet unnamed division of the bacteria, was explored in 93 anaerobic environments (guts, soils, digestors, etc.). From 16S rDNA gene-targeted polymerase chain reaction (PCR) assays, this group appeared to be present in 90% of the anaerobic microbial ecosystems analysed. The phylogeny of 103 16S rDNA sequences from 30 ecosystems showed a strong link between 16S rDNA sequences and given ecosystems. 'Synergistes' 16S rDNA sequences from animal sources (termites, guinea pigs, pigs, birds, etc.) formed clustered phylogenetical groups. 'Synergistes' groups were also associated either with anaerobic digestors and soils or with thermophilic conditions. Sequences available from the DNA database were consistent with the results. These results show the wide diversity of the 'Synergistes' division as well as the specific ecological niche of each 16S rDNA sequences.     

Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition

Serial analysis of ribosomal sequence tags (SARST) is a novel technique for characterizing microbial community composition. The SARST method captures sequence information from concatemers of short 16S rDNA polymerase chain reaction (PCR) amplicons from complex populations of DNA. Here, we describe a similar method, serial analysis of V6 ribosomal sequence tags (SARST-V6), which targets the V6 hypervariable region of bacterial 16S rRNA genes. The SARST-V6 technique exploits internal primer sequences to generate compatible restriction digest overhangs, thereby improving upon the efficiency of SARST. Serial analysis of V6 ribosomal sequence tags of bacterial community composition in hydrothermal marine sediments from Guaymas Basin resembled results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identified the same major bacterial groups, but only SARST-V6 recovered thermodesulfobacteria and gamma-proteobacteria sequences, while only full-length PCR product cloning recovered candidate division OP11 se-quences. There were differences in the relative frequencies of some phylotypes. The disparities reflect differences in the amplicon pool obtained during initial amplification that may result from different primer affinities or DNA degradation. These results demonstrate the utility of SARST-V6 in collecting taxonomically informative data for high-throughput analysis of microbial communities.     

Crenarchaeota and Euryarchaeota in temperate estuarine sediments

AIMS: Application of molecular techniques to ecological studies has unveiled a wide diversity of micro-organisms in natural communities, previously unknown to microbial ecologists. New lineages of Archaea were retrieved from several non-extreme environments, showing that these micro-organisms are present in a large variety of ecosystems. The aim was therefore to assess the presence and diversity of Archaea in the sediments of the river Douro estuary (Portugal), relating the results obtained to ecological data. METHODS AND RESULTS: Total DNA was extracted from sediment samples obtained from an estuary deprived of vegetation, amplified by PCR and the resulting DNA fragments cloned. The archaeal origin of the cloned inserts was checked by Southern blot, dot blot or colony blot hybridization. Recombinant plasmids were further analysed by restriction with AvaII and selected for sequencing. Phylogenetic analyses of 14 sequences revealed the presence of members of the domain Archaea. Most of the sequences could be assigned to the kingdom Crenarchaeota. CONCLUSION: Most of these sequences were closely related to those obtained from non-extreme Crenarchaeota members previously retrieved from diverse ecosystems, such as freshwater and marine environments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of archaeal 16S rDNA sequences in temperate estuarine sediments emerges as a valuable contribution to the understanding of the complexity of the ecosystem.     

Simultaneous Recovery of Extracellular and Intracellular DNA Suitable for Molecular Studies from Marine Sediments

The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes.     

Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments

The occurrence of high extracellular DNA concentrations in aquatic sediments (concentrations that are 3 to 4 orders of magnitude greater than those in the water column) might play an important role in biogeochemical cycling, as well as in horizontal gene transfer through natural transformation. Since isolation of extracellular DNA from sediments is a difficult and unsolved task, in this study we developed an efficient procedure to recover simultaneously DNA associated with microbial cells and extracellular DNA from the same sediment sample. This procedure is specifically suitable for studying extracellular DNA because it avoids any contamination with DNA released by cell lysis during handling and extraction. Applying this procedure to different sediment types, we obtained extracellular DNA concentrations that were about 10 to 70 times higher than the intracellular DNA concentrations. Using specific targeted prokaryotic primers, we obtained evidence that extracellular DNA recovered from different sediments did not contain amplifiable 16S rRNA genes. By contrast, using DNA extracted from microbial cells as the template, we always amplified 16S rRNA genes. Although 16S rRNA genes were not detected in extracellular DNA, analyses of the sizes of extracellular DNA indicated the presence of high-molecular-weight fragments that might have contained other gene sequences. This protocol allows investigation of extracellular DNA and its possible participation in natural transformation processes.     

Identification of bacteriophage K20 binding regions of OmpF and lipopolysaccharide in Escherichia coli K-12

Methane hydrates represent an enormous carbon and energy source in many low temperature deep marine sediments. However, little information is available concerning the nature of the microbial communities associated with these structures. Here, we describe a phylogenetic analysis based on ribosomal DNA (rDNA) sequences obtained from sediment and fluid samples present in a region of gas hydrate formation in shallow sediments within the Cascadia margin in and around Ocean Drilling Program (ODP) Site 892B. Our studies detected diverse sulfur-utilizing microbes, methanogens, methanotrophs, and non-thermophilic members of the kingdom Crenarchaeota. This is the first culture-independent phylogenetic analysis of a gas hydrate habitat.     

Aromatic hydrocarbon degradation genes from chronically polluted Subantarctic marine sediments

Aim:  The goal of this study was to identify functional targets to detect polycyclic aromatic hydrocarbon (PAH)-degrading bacterial populations in cold marine ecosystems.
Methods and Results:  We designed a degenerate primer set targeting genes encoding the α subunit of PAH-dioxygenases from Gram-positive bacteria. This primer set was used to amplify gene fragments from metagenomic DNA isolated from Subantarctic marine sediments (Ushuaia Bay, Argentina). These gene fragments were cloned and sequenced. We identified 14 distinct groups of genes, most of them showing significant relatedness with dioxygenases from Gram-positive bacteria of the genera Rhodococcus , Mycobacterium , Nocardioides , Terrabacter and Bacillus . The level of identity with these genes, however, was low to moderate (33–62% at the amino acid level).
Conclusion:  These results indicate the presence of a high diversity of hitherto unidentified dioxygenase genes in this cold polluted environment.
Significance and Impact of the Study:  Subantarctic marine ecosystems are particularly vulnerable to hydrocarbon pollution, and the development of environmental restoration strategies for these environments is pressing. The information obtained in this work will be the starting point for the design of quantitative molecular tools to analyse the abundance and dynamics of these aromatic hydrocarbon-degrading bacterial populations in the marine environment.     

Eukaryotic diversity in late Pleistocene marine sediments around a shallow methane hydrate deposit in the Japan Sea

Marine sediments contain eukaryotic DNA deposited from overlying water columns. However, a large proportion of deposited eukaryotic DNA is aerobically biodegraded in shallow marine sediments. Cold seep sediments are often anaerobic near the sediment–water interface, so eukaryotic DNA in such sediments is expected to be preserved. We investigated deeply buried marine sediments in the Japan Sea, where a methane hydrate deposit is associated with cold seeps. Quantitative PCR analysis revealed the reproducible recovery of eukaryotic DNA in marine sediments at depths up to 31.0 m in the vicinity of the methane hydrate deposit. In contrast, the reproducible recovery of eukaryotic DNA was limited to a shallow depth (8.3 m) in marine sediments not adjacent to the methane hydrate deposit in the same area. Pyrosequencing of an 18S rRNA gene variable region generated 1,276–3,307 reads per sample, which was sufficient to cover the biodiversity based on rarefaction curves. Phylogenetic analysis revealed that most of the eukaryotic DNA originated from radiolarian genera of the class Chaunacanthida, which have SrSO₄ skeletons, the sea grass genus Zostera, and the seaweed genus Sargassum. Eukaryotic DNA originating from other planktonic fauna and land plants was also detected. Diatom sequences closely related to Thalassiosira spp., indicative of cold climates, were obtained from sediments deposited during the last glacial period (MIS‐2). Plant sequences of the genera Alnus, Micromonas, and Ulmus were found in sediments deposited during the warm interstadial period (MIS‐3). These results suggest the long‐term persistence of eukaryotic DNA from terrestrial and aquatic sources in marine sediments associated with cold seeps, and that the genetic information from eukaryotic DNA from deeply buried marine sediments associated with cold seeps can be used to reconstruct environments and ecosystems from the past.     

Bacterial and Archaeal 16S rRNA Genes in Late Pleistocene to Holocene Muddy Sediments from the Kanto Plain of Japan

Microbial communities in ancient marine sediments composed of clay and silt obtained from the terrestrial subsurface were phylogenetically analyzed based on their 16S rRNA gene sequences. Chloroflexi and Miscellaneous Crenarchaeotic Group were predominant in bacterial and archaeal clone libraries, respectively. Of 44 operational taxonomic units (OTUs) that had close relatives in the database, 30 were close to sequences obtained from marine environments. Some sequences belonged to the candidate groups JS1, ANME-I, and Marine Benthic Group-C, which are typically found in marine sediments. Low chloride concentrations in the sediments suggest that these marine-affiliated sequences may not reflect currently active microbial communities. Our results indicate the existence of long-term preserved DNA or descendants of ancient oceanic microbial components in subsurface muddy sediments in a temperate region, which may reflect indigenous population of paleoenvironments.     

Review and re-analysis of domain-specific 16S primers

The Polymerase Chain Reaction (PCR) has facilitated the detection of unculturable microorganisms in virtually any environmental source and has thus been used extensively in the assessment of environmental microbial diversity. This technique relies on the assumption that the gene sequences present in the environment are complementary to the "universal" primers used in their amplification. The recent discovery of new taxa with 16S rDNA sequences not complementary to standard universal primers suggests that current 16S rDNA libraries are not representative of true prokaryotic biodiversity. Here we re-assess the specificity of commonly used 16S rRNA gene primers and present these data in tabular form designed as a tool to aid simple analysis, selection and implementation. In addition, we present two new primer pairs specifically designed for effective "universal" Archaeal 16S rDNA sequence amplification. These primers are found to amplify sequences from Crenarchaeote and Euryarchaeote type strains and environmental DNA.