Recovery, Purification, and Cloning of High-Molecular-Weight DNA from Soil Microorganisms

Liles, Mark R.; Williamson, Lillie D.; Rodbumrer, Jitsupang; Torsvik, Vigdis; Goodman, Robert M.; Handelsman, Jo

doi:10.1128/aem.02630-07

Cited by 69 publications

(47 citation statements)

References 24 publications

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“…High-molecular-weight metagenomic DNA was extracted from 100 g of sieved soil using an indirect extraction method as described by to Liles et al (55). Pulse-field gel electrophoresis (PFGE) was used to size fractionate the DNA onto a 1% agarose gel.…”

Section: Methodsmentioning

confidence: 99%

Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming

Lau

Engelen

Gordon

et al. 2017

Appl Environ Microbiol

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Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized as accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are "hot spots" for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the "resistome" of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline, and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPP AZI 4 , encoded within an alternative open reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPP AZI 4 can promote resistance against different macrolides but not other ribosome-targeting antibiotics, implicating a new macrolidespecific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.IMPORTANCE Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remains limited. Our discovery of several previously unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and "repurposed" by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomicsdriven gene discovery.

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Section: Methodsmentioning

confidence: 99%

Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming

Lau

Engelen

Gordon

et al. 2017

Appl Environ Microbiol

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“…Metagenomic libraries were constructed as previously described (21), with the following modifications. Agarose plugs containing lysed bacterial cells were embedded in a 1% low-melt agarose gel and electrophoresed to size select the DNA.…”

Section: Methodsmentioning

confidence: 99%

Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins

Donato

Moe

Converse

et al. 2010

Appl Environ Microbiol

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143

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To gain insight into the diversity and origins of antibiotic resistance genes, we identified resistance genes in the soil in an apple orchard using functional metagenomics, which involves inserting large fragments of foreign DNA into Escherichia coli and assaying the resulting clones for expressed functions. Among 13 antibioticresistant clones, we found two genes that encode bifunctional proteins. One predicted bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and a ␤-lactamase. Sequence analysis of the entire metagenomic clone encoding the predicted bifunctional ␤-lactamase revealed a gene potentially involved in chloramphenicol resistance as well as a predicted transposase. A second clone that encodes a predicted bifunctional protein confers resistance to kanamycin and contains an aminoglycoside acetyltransferase domain fused to a second acetyltransferase domain that, based on nucleotide sequence, was predicted not to be involved in antibiotic resistance. This is the first report of a transcriptional regulator fused to a ␤-lactamase and of an aminoglycoside acetyltransferase fused to an acetyltransferase not involved in antibiotic resistance.

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“…CSML presents opportunities for both success and trouble-shooting. Protocols are well-established, but need to be optimized to each soil type (6,9). CSML involves a variety of techniques, and allows the development of multiple research skills of different complexities.…”

Section: Metagenomics As a Teaching Toolmentioning

confidence: 99%

Summer Workshop in Metagenomics: One Week Plus Eight Students Equals Gigabases of Cloned DNA

Rı́os-Velázquez

Williamson

Cloud-Hansen

et al. 2011

J Microbiol Biol Educ.

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We designed a week-long laboratory workshop in metagenomics for a cohort of undergraduate student researchers. During this course, students learned and utilized molecular biology and microbiology techniques to construct a metagenomic library from Puerto Rican soil. Pre-and postworkshop assessments indicated student learning gains in technical knowledge, skills, and confidence in a research environment. Postworkshop construction of additional libraries demonstrated retention of research techniques by the students.

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Recovery, Purification, and Cloning of High-Molecular-Weight DNA from Soil Microorganisms

Cited by 69 publications

References 24 publications

Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming

Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming

Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins

Summer Workshop in Metagenomics: One Week Plus Eight Students Equals Gigabases of Cloned DNA

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