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

Majerník, Alan I.; Gottschalk, Gerhard; Daniel, Rolf

doi:10.1128/jb.183.22.6645-6653.2001

Cited by 101 publications

(47 citation statements)

References 47 publications

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“…The potential for application of metagenomics to biotechnology seems endless. Functional screens have identified new enzymes (39,52,53,67,68,72,73,88,93,104,118,122,124,144) and antibiotics (22,23,39,61,69,91,110,118,146) and other reagents in libraries from diverse environments. A number of barriers have limited the discovery of new genes that provide insight into microbial community structure and function or that can be used to solve medical, agricultural, or industrial problems.…”

Section: Discussionmentioning

confidence: 99%

“…3). The clones can be screened for phylogenetic markers or "anchors," such as 16S rRNA and recA, or for other conserved genes by hybridization or multiplex PCR (136) or for expression of specific traits, such as enzyme activity or antibiotic production (39,44,61,78,87,88,91,93,118,125), or they can be sequenced randomly (141,142). Each approach has strengths and limitations; together …”

Section: Approaches To Metagenomic Analysismentioning

confidence: 99%

“…For example, the Daniel group designed a clever selection for Na ϩ (Li ϩ )/H ϩ antiporters that requires complementation of an E. coli mutant deficient in the three Na ϩ /H ϩ antiporters (nhaA, nhaB, and chaA) enabling growth on medium containing 7.5 mM LiCl (93). This powerful selection facilitated the discovery of two novel antiporter proteins in a library of 1,480,000 clones containing DNA isolated from soil.…”

Section: Functional Metagenomics Heterologous Expressionmentioning

confidence: 99%

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Metagenomics: Application of Genomics to Uncultured Microorganisms

Handelsman

2004

Microbiol Mol Biol Rev

2,023

1,038

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Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na+(Li+)/H+ antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies

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Section: Discussionmentioning

confidence: 99%

Section: Approaches To Metagenomic Analysismentioning

confidence: 99%

Section: Functional Metagenomics Heterologous Expressionmentioning

confidence: 99%

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Metagenomics: Application of Genomics to Uncultured Microorganisms

Handelsman

2004

Microbiol Mol Biol Rev

2,023

1,038

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“…Na ϩ /H ϩ antiporters play a primary role in these homeostatic mechanisms (16,20,28,45) and are ubiquitous proteins inserted in cytoplasmic membranes of cells and in membranes of many organelles. In Escherichia coli, genes for the three distinct antiporters nhaA (18), nhaB (32), and chaA (17) have been characterized already.…”

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confidence: 99%

Roles of NhaA, NhaB, and NhaD Na ⁺ /H ⁺ Antiporters in Survival of Vibrio cholerae in a Saline Environment

et al. 2003

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Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na ؉ /H ؉ antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc-nhaA gene encoding the V. cholerae homolog of Escherichia coli. Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E. coli. We showed that both Vc-NhaA and Vc-NhaB confer Na ؉ resistance and that Vc-NhaA displays an antiport activity in E. coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E. coli NhaA. To determine the roles of the Na ؉ /H ؉ antiporters in V. cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants). In contrast to E. coli, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na ؉ concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li ؉ -sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na ؉ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na ؉ /H ؉ antiporter activity becomes essential for the resistance of V. cholerae to Na ؉ at alkaline pH. Since the ion pump NQR is Na ؉ specific, we suggest that its activity masks the Na ؉ /H ؉ but not the Li ؉ /H ؉ antiporter activities. Our results indicate that the Na ؉ resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.

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“…In addition, genomic fragments that are Ͼ100 kb long can be obtained, and they provide significant functional and taxonomic information about the organisms from which they were derived. Such metagenomic libraries have been used to identify novel genes from uncultivated species of archaea, bacteria, and viruses that are responsible for significant ecosystem processes (4,5,8,12) and to isolate enzymes that are involved in biosynthesis of novel pharmaceuticals (7,15,24,41,42) or have other industrial uses (11,16,17,26,33).Given the immense uncultivated and uncharacterized metabolic diversity in the environment, one would need to sequence relatively few bacterial artificial chromosome (BAC) or cosmid clones to discover fundamentally interesting sets of genes. If modern genomic techniques can be used to carry out more comprehensive surveys of metagenomic libraries, our understanding of natural genetic diversity should be greatly enhanced.…”

mentioning

confidence: 99%

Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library

Sebat

Colwell

Crawford

2003

Appl Environ Microbiol

122

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Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ϳ1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.Microorganisms contribute significantly to the earth's biological diversity, yet relatively few of the microorganisms present in nature have been cultured and characterized. It is generally accepted that less than 1% of bacteria and fungi present in most habitats have been cultivated for study in pure culture (2). Although direct analysis of environmental DNA samples by PCR is effective for showing the presence of uncultured microorganisms, biases in primer specificity and amplification of different targets prevent full recognition of microbial diversity (31,37,40,41). Thus, new approaches to examination of community genomes are needed.The use of large-insert genomic libraries is a powerful approach for isolating DNA sequences from complex mixtures of uncultured microorganisms. Direct cloning of DNA from environmental samples makes it possible to avoid some of the biases of cultivation and PCR. In addition, genomic fragments that are Ͼ100 kb long can be obtained, and they provide significant functional and taxonomic information about the organisms from which they were derived. Such metagenomic libraries have been used to identify novel genes from uncultivated species of archaea, bacteria, and viruses that are respons...

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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

Cited by 101 publications

References 47 publications

Metagenomics: Application of Genomics to Uncultured Microorganisms

Metagenomics: Application of Genomics to Uncultured Microorganisms

Roles of NhaA, NhaB, and NhaD Na ⁺ /H ⁺ Antiporters in Survival of Vibrio cholerae in a Saline Environment

Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library

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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

Cited by 101 publications

References 47 publications

Metagenomics: Application of Genomics to Uncultured Microorganisms

Metagenomics: Application of Genomics to Uncultured Microorganisms

Roles of NhaA, NhaB, and NhaD Na + /H + Antiporters in Survival of Vibrio cholerae in a Saline Environment

Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library

Contact Info

Product

Resources

About

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

Roles of NhaA, NhaB, and NhaD Na ⁺ /H ⁺ Antiporters in Survival of Vibrio cholerae in a Saline Environment