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Development of an inducible lytic system for functional metagenomic screening

Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. During functional screenings of enzymes, the ability of the substrate to enter the surrogate host or the ability of this bacterium to export heterologous extracellular enzymes may hamp...

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Autores principales: Cárcel-Márquez, Jara, Flores, Amando, Martín-Cabello, Guadalupe, Santero, Eduardo, Camacho, Eva M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405747/
https://www.ncbi.nlm.nih.gov/pubmed/30846762
http://dx.doi.org/10.1038/s41598-019-40470-4
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author Cárcel-Márquez, Jara
Flores, Amando
Martín-Cabello, Guadalupe
Santero, Eduardo
Camacho, Eva M.
author_facet Cárcel-Márquez, Jara
Flores, Amando
Martín-Cabello, Guadalupe
Santero, Eduardo
Camacho, Eva M.
author_sort Cárcel-Márquez, Jara
collection PubMed
description Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. During functional screenings of enzymes, the ability of the substrate to enter the surrogate host or the ability of this bacterium to export heterologous extracellular enzymes may hamper the technique. Here we have used an inducible autolysis system that lyses bacteria thus releasing its content in both, liquid and solid cultures, in response to anhydrotetracycline. The lytic cluster is tightly regulated to prevent impaired bacterial growth in absence of the inducer and produced very efficient though not complete bacterial lysis upon induction, which allowed the recovery of live bacteria. The system can be used in combination with specialised fosmids and E. coli strains that maximize transcription of metagenomic DNA. Our results show that colony-lysis on plates allows detection of an endogenous intracellular amylase activity naturally present in E. coli and clearly increased detection of clones coding for cellulase activities in a metagenomic screening, while allowing recovery of survivor positive clones from the lysed colonies in all cases. Therefore, this tool represents an important step towards the effective access to the extraordinary potential of the uncultivated bacteria genetic resources.
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spelling pubmed-64057472019-03-11 Development of an inducible lytic system for functional metagenomic screening Cárcel-Márquez, Jara Flores, Amando Martín-Cabello, Guadalupe Santero, Eduardo Camacho, Eva M. Sci Rep Article Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. During functional screenings of enzymes, the ability of the substrate to enter the surrogate host or the ability of this bacterium to export heterologous extracellular enzymes may hamper the technique. Here we have used an inducible autolysis system that lyses bacteria thus releasing its content in both, liquid and solid cultures, in response to anhydrotetracycline. The lytic cluster is tightly regulated to prevent impaired bacterial growth in absence of the inducer and produced very efficient though not complete bacterial lysis upon induction, which allowed the recovery of live bacteria. The system can be used in combination with specialised fosmids and E. coli strains that maximize transcription of metagenomic DNA. Our results show that colony-lysis on plates allows detection of an endogenous intracellular amylase activity naturally present in E. coli and clearly increased detection of clones coding for cellulase activities in a metagenomic screening, while allowing recovery of survivor positive clones from the lysed colonies in all cases. Therefore, this tool represents an important step towards the effective access to the extraordinary potential of the uncultivated bacteria genetic resources. Nature Publishing Group UK 2019-03-07 /pmc/articles/PMC6405747/ /pubmed/30846762 http://dx.doi.org/10.1038/s41598-019-40470-4 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Cárcel-Márquez, Jara
Flores, Amando
Martín-Cabello, Guadalupe
Santero, Eduardo
Camacho, Eva M.
Development of an inducible lytic system for functional metagenomic screening
title Development of an inducible lytic system for functional metagenomic screening
title_full Development of an inducible lytic system for functional metagenomic screening
title_fullStr Development of an inducible lytic system for functional metagenomic screening
title_full_unstemmed Development of an inducible lytic system for functional metagenomic screening
title_short Development of an inducible lytic system for functional metagenomic screening
title_sort development of an inducible lytic system for functional metagenomic screening
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405747/
https://www.ncbi.nlm.nih.gov/pubmed/30846762
http://dx.doi.org/10.1038/s41598-019-40470-4
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