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PubMed2001

Screening of environmental DNA libraries for the presence of genes conferring Na(+)(Li(+))/H(+) antiporter activity on Escherichia coli: characterization of the recovered genes and the corresponding gene products.

Environmental DNA libraries prepared from three different soils were screened for genes conferring Na(+)(Li(+))/H(+) antiporter activity on the antiporter-deficient Escherichia coli strain KNabc. The presence of those genes was verified on selective LK agar containing 7.5 mM LiCl. Two positive E. coli clones were obtained during the initial screening of 1,480,000 recombinant E. coli strains. Both clones harbored a plasmid (pAM1 and pAM3) that conferred a stable Li(+)-resistant phenotype. The insert of pAM2 (1,886 bp) derived from pAM1 contained a gene (1,185 bp) which encodes a novel Na(+)/H(+) antiporter belonging to the NhaA family. The insert of pAM3 harbored the DNA region of E. coli K-12 containing nhaA, nhaR, and gef. This region is flanked by highly conserved insertion elements. The sequence identity with E. coli decreased significantly outside of the insertion sequence elements, indicating that the unknown organism from which the insert of pAM3 was cloned is different from E. coli. The products of the antiporter genes located on pAM2 and pAM3 revealed functional homology to NhaA of E. coli and enabled the antiporter-deficient E. coli mutant to grow on solid media in the presence of up to 450 mM NaCl or 250 mM LiCl at pH 8.0. The Na(+)/H(+) antiporter activity in everted membrane vesicles that were derived from the E. coli strains KNabc/pAM2 and KNabc/pAM3 showed a substantial increase between pHs 7 and 8.5. The maximal activity was observed at pHs 8.3 and 8.6, respectively. The K(m) values of both antiporters for Na(+) were approximately 10-fold higher than the values for Li(+).

Amino Acid Sequence↗
PubMed2003

Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries.

A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.

Amino Acid Sequence↗
PubMed2001

Recovery of new integron classes from environmental DNA.

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.

Amino Acid Sequence↗
PubMed2006

Exploration of genes that encode a carbocycle-forming enzyme involved in biosynthesis of aminoglycoside antibiotics from the environmental DNA.

2-Deoxy-scyllo-inosose (DOI) synthase is the enzyme participating in biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The gene which encodes the enzyme can be a marker for screening of DOS-containing aminoglycoside-producer and exploration of its biosynthetic gene. Further, this enzyme is expected to be of use in industry, because it converts sugar into 6-membered carbocycle. In the present study, we identified 21 clones encoding DOI synthase from environmental DNA by degenerate PCR. They were clearly divided into two groups. One appeared to derive from actinomycetes, and the other from non-actinomycetes. The latter group was larger (17 clones) than the former (four clones) despite the fact that only one strain of non-actinomycete was identified for DOS-containing aminoglycoside production. This result indicates that there are still many unidentified non-actinomycetes for DOS-containing aminoglycoside biosynthesis. We showed the possibility of identification of novel aminoglycoside-producing non-actinomycete from soil, and for development of more efficient enzymes for industrial use.

Actinobacteria↗
PubMedDate not supplied

Ozonation of DNA forms adducts: a 32P-DNA labeling and thin-layer chromatography technique to measure DNA environmental biomarkers.

Little direct documented evidence of ozone's genotoxicity exists. Deoxyribonucleic acid (DNA) adducts are produced by environmental toxic agents, including ozone. We have described a modified thin-layer chromatography (TLC) technique that can assess adduct formation as a biomarker of ozone injury. This requires 32P-labeling DNA, digestion of deoxynucleotides (dNMPs), and separation in two-dimensional PEI-cellulose TLC. We have applied this technique to control DNAs, to control DNA in solution exposed to acute ambient ozone, and to control DNA exposed to acute bubbled-through ozone (2 ppm for 24 h). We detected stable DNA adducts, including hydroxymethyluracil (HMU), thymine glycol (TG), 8-hydroxyguanine (8-OHG), and demonstrated, as yet, unidentified adducts that may serve as a "fingerprint" pattern of DNA adduction. This technique quantifies low-molecular-mass DNA adducts, both in vivo and in vitro, with potential applications to environmental toxicology.

Animals↗
PubMed2006

Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library.

There is a great interest in xylanases due to the wide variety of industrial applications for these enzymes. We cloned a xylanase gene (xyn8) from an environmental genomic DNA library. The encoded enzyme was predicted to be 399 amino acids with a molecular weight of 45.9 kD. The enzyme was categorized as a glycosyl hydrolase family 8 member based on sequence analysis of the putative catalytic domain. The purified enzyme was thermolabile, had an activity temperature optimum of 20 degrees C on native xylan substrate, and retained significant activity at lower temperatures. At 4 degrees C, the apparent K (m) was 3.7 mg/ml, and the apparent k (cat) was 123/s.

Agriculture↗
PubMed2003

Synthesis of long-chain fatty acid enol esters isolated from an environmental DNA clone.

[structure: see text] Long-chain fatty acid enol ester 1 is the major metabolite of a new family of small molecules isolated from the heterologous expression of environmentally derived DNA. A versatile synthesis of 1, in which an aromatic acetaldehyde is O-acylated with a long-chain acyl chloride allowed for the rapid construction of both the isolated product (1) and a number of structural analogues (including 8, 17, and 18).

Acyl Carrier Protein↗
PubMed2004

Phylum-specific environmental DNA analysis reveals remarkably high global biodiversity of Cercozoa (Protozoa).

This study presents the first 18S rRNA multi-library environmental PCR survey of a single protozoan phylum, Cercozoa Cavalier-Smith 1998, from a range of different habitats. Phylogenetic analysis reveals at least nine novel clades within the phylum, several possibly at the level of order or above. Further experiments are described to ascertain the true ecological and geographical distributions of some clades that might be inferred from the tree to be restricted in either or both ways. These results suggest that the diversity of cercozoan taxa may run into thousands of lineages, making it comparable in diversity to the largest better-characterized protozoan phyla, e.g. Ciliophora (ciliates and suctorians) and Foraminifera. New sequences of cultured Spongomonas, Metromonas and Metopion are also presented. In the light of these additions, and the increased taxon sampling from the environmental libraries, some revisions of cercozoan classification are made: the transfer of Spongomonadea from Reticulofilosa to Monadofilosa; the removal of Metopiida from Sarcomonadea; and the creation of the new order Metromonadida, currently containing the single genus Metromonas. Although Metromonas groups with weak to moderate support with Chlorarachnea, it is here placed in superclass Monadofilosa, to which it is morphologically more similar.

Animals↗
PubMed2005

Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts.

BACKGROUND: The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set. RESULTS: Numerous sequences for PCR primers were tested to develop PCR assays with a wide range of fungal compatibility and high discrimination from plant DNA. A nested set of 4 primers was developed that reflected these criteria and performed well amplifying ITS regions of fungal rDNA. Primers in the 5.8S sequence were also developed that would permit separate amplifications of ITS1 and ITS2. A range of basidiomycete fruiting bodies and ascomycete cultures were analyzed with the nested set of primers and Restriction Fragment Length Polymorphism (RFLP) fingerprinting to demonstrate the specificity of the assay. Single ectomycorrhizal root tips were similarly analyzed. These primers have also been successfully applied to Quantitative PCR (QPCR), Length Heterogeneity PCR (LH-PCR) and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analyses of fungi. A set of wide-range plant-specific primers were developed at positions corresponding to one pair of the fungal primers. These were used to verify that the host plant DNA was not being amplified with the fungal primers. CONCLUSION: These plant primers have been successfully applied to PCR-RFLP analyses of forest plant tissues from above- and below-ground samples and work well at distinguishing a selection of plants to the species level. The complete set of primers was developed with an emphasis on discrimination between plant and fungal sequences and should be particularly useful for studies of fungi where samples also contain high levels of background plant DNA, such as verifying ectomycorrhizal morphotypes or characterizing phylosphere communities.

DNA Primers↗
PubMed2006

Molecular analysis of the nitrogen cycle in deep-sea microorganisms from the Nankai Trough: genes for nitrification and denitrification from deep-sea environmental DNA.

Nitrification and denitrification are bacterial functions, which are important for the global nitrogen cycle. Thus, it is important to study the diversity and distribution of bacteria in the environment, which are involved in the nitrogen cycle on the earth. Ammonia monooxygenase encoded by the amoA gene and nitrite reductase encoded by nirK or nirS are essential enzymes for nitrificaton and denitrification, respectively. These genes can be used as markers for the identification of organisms in the nitrogen cycle. In this study, we identified amoA (42 clones) and nirS (98 clones) genes in parallel from samples recovered from the deep-sea of the Nankai Trough. Genes for nirK could not be amplified from these samples. The obtained amoA sequences were not so closely related to those of amoA genes from previously isolated environmental organisms and those of genes from environmental DNAs. On the other hand, the nirS genes sequenced showed some relationship to some extent with the latter genes. However, some of the newly sequenced genes formed clusters, which contained no previously identified genes on a phylogenetic tree. These are likely present in specific denitrifiers from the deep-sea. The results of this study further suggest that nitrifiers and denitrifiers live in the same area of the Nankai Trough and the nitrogen cycle exists even in the deep-sea.

DNA↗
PubMed2025

Bone Adhered Sediments as a Source of Target and Environmental DNA and Proteins.

In recent years, sediments from cave environments have provided invaluable insights into ancient hominids, as well as past fauna and flora. Unfortunately, however, sediments are not always collected during excavation. In this study, we analyzed an overlooked but abundant resource in archaeological collections - sediments adhered to bone. We performed metagenomics and metaproteomics analysis on sediment from several human skeletal elements, originating from Neolithic to Medieval sites in England. We were able to reconstruct a partial human genome, the genetic profile of which matches that recovered from the original skeletal element. Additionally, aDNA sequences matching the genomes of endogenous gut microbiome bacteria were identified. We also found the presence of genetic sequences corresponding to animals and plants. In particular, we managed to retrieve the partial genome and proteome of a Black Rat (Rattus rattus), sharing close genetic affinities to other medieval Rattus rattus. Our results demonstrate that material that is usually ignored or discarded, can be used to reveal information about the individual and the environmental conditions at the time of their death.

Animals↗
PubMed1998

Mms4, a putative transcriptional (co)activator, protects Saccharomyces cerevisiae cells from endogenous and environmental DNA damage.

mms4-1 is one of several Saccharomyces cerevisiae mutants that exhibit an increased sensitivity to methyl methanesulfonate (MMS), but not to UV or X-rays. We have isolated the MMS4 gene by functional complementation of the MMS-sensitive phenotype in the mms4-1 strain. The MMS4 gene encodes a 691-amino acid, 78.7-kDa protein. The deduced Mms4 protein does not show significant homology to any of the known proteins in the database. However, several putative functional domains suggest that it may be a nuclear protein capable of interacting with other proteins. Examination of the mms4delta mutant phenotype indicates that the mutation not only sensitizes DNA to methylating and ethylating agents, but also to other DNA damage that blocks DNA replication. However, the mms4delta mutant appears to be more sensitive to chronic treatment than to acute treatment by DNA-damaging agents. Furthermore, the spontaneous mutation rate increases significantly in the mms4delta mutant. Mms4 alone, when fused to a Gal4 DNA-binding domain, is able to activate P(GAL1)-lacZ and P(GAL1)-HIS3 reporter genes in a two-hybrid system; the Mms4 transactivation domain maps to the highly acidic N-terminal region. These results collectively suggest that Mms4 may function as a transcriptional (co)activator and play an important role in DNA repair and/or synthesis.

Amino Acid Sequence↗
PubMedDate not supplied

Freshwater foraminiferans revealed by analysis of environmental DNA samples.

Sediment-dwelling protists are among the most abundant meiobenthic organisms, ubiquitous in all types of aquatic ecosystems. Yet, because their isolation and identification are difficult, their diversity remains largely unknown. In the present work, we applied molecular methods to examine the diversity of freshwater Foraminifera, a group of granuloreticulosan protists largely neglected until now. By using specific PCR primers, we detected the presence of Foraminifera in all sediment samples examined. Phylogenetic analysis of amplified SSU rDNA sequences revealed two distinct groups of freshwater foraminiferans. All obtained sequences branched within monothalamous (single-chambered), marine Foraminifera, suggesting a repeated colonization of freshwater environments. The results of our study challenge the traditional view of Foraminifera as essentially marine organisms, and provide a conceptual framework for charting the molecular diversity of freshwater granuloreticulosan protists.

Animals↗
PubMed2006

Characterization of a novel cyclomaltodextrinase expressed from environmental DNA isolated from Bor Khleung hot spring in Thailand.

A novel gene belonging to the alpha-amylase family was isolated directly from community DNA obtained from soil sediments collected from Bor Khleung hot spring in Thailand. Partial sequences harboring four conserved regions of the alpha-amylase family were amplified by PCR using degenerate primers. Upstream and downstream sequences of these fragments were obtained by a genome walking approach to identify a full-length gene (Env cda13A) encoding 619 amino acids. Amino acid sequence alignments of Env Cda13A with other enzymes suggested that this enzyme was a cyclomaltodextrinase. The Env cda13A gene was expressed in Pichia pastoris as a secreted functional protein of 68 kDa. The partially purified enzyme was shown to be monomeric and hydrolyzed various maltodextrins from maltotriose to maltoheptaose and cyclomaltodextrins to give maltose and glucose as the main products. The enzyme also hydrolyzed pullulan and soluble starch to yield glucose, but the rate of hydrolysis was slow. This study demonstrated the possibility of isolating potentially novel enzymes directly from natural environments and opens an unexplored biodiversity resource in Thailand for future novel gene discoveries.

Amino Acid Sequence↗
PubMed1997

Infrared spectral models demonstrate that exposure to environmental chemicals leads to new forms of DNA.

Environmental chemicals are known to induce a high degree of hydroxyl radical-mediated damage in DNA. Accordingly, we tested the hypothesis that this exposure leads to new forms of DNA using principal components analysis of Fourier transform infrared spectra. The hepatic DNA of English sole (controls) from an essentially clean environment was compared with that of sole inhabiting a chemically contaminated environment. All livers studied were cancer-free; however, a high incidence of liver cancer has been found in the exposed population. The exposed sole were sampled twice, 2 years apart, while the sediments in which they live were under remediation. After obtaining infrared spectra, the first three principal components (PC1, PC2, and PC3) were calculated and found to represent 97% of the total spectral variance. When the principal component scores were plotted in 3-dimensional space, clusters of points were obtained that represented the DNA from the control and exposed groups. Each of the points was derived from approximately 10(6) wavenumber-absorbance correlations. The spatial location of a point was a highly discriminating measure of DNA structure. The clusters of points were completely separated, demonstrating that the three groups could be 100% correctly classified. The points from the control group were tightly clustered whereas those from the exposed groups were highly diverse. The findings demonstrate that exposure to environmental chemicals results in new, structurally diverse forms of DNA that likely play an important role in carcinogenesis.

Animals↗
PubMed2001

[Extraction of DNA from environmental samples and construction of mixed genomic DNA libraries].

A method has been developed for extracting and purifying genomic DNA from environmental samples. In this method, an environmental sample is treated first by grinding and freezing/thawing and subsequently by SDS/proteinase K-based DNA extraction. The yields of purified DNA from three samples used in this study ranged from 2 to 16 micrograms per gram of dry sample. Mixed genomic DNA libraries for two of the environmental samples were constructed by inserting restriction fragments (3-8 kb) of the purified DNAs into plasmid pUC18 and transforming E. coli DH5 alpha with the resultant plasmids. Approximately 10(3) to 10(4) insert-containing clones were obtained from 1 g of each sample. Clone libraries were analyzed by DNA sequencing and gene annotation. Among 20 randomly-selected clones, 14 contained an insert whose sequence had not been reported while the rest had an insert of either E. coli or vector origin. A search of sequence databases using the end sequences of each of the foreign inserts showed that each sequence was part of a gene encoding, in most cases, a predictable function. Our results are of significance to the collection, investigation and exploitation of the genes of uncultured microorganisms.

Amino Acid Sequence↗