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Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC...

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Autores principales: Montenegro, Eduarda Morgana da Silva, Delabary, Gabriela Scholante, da Silva, Marcus Adonai Castro, Andreote, Fernando Dini, Lima, André Oliveira de Souza
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590472/
https://www.ncbi.nlm.nih.gov/pubmed/28952531
http://dx.doi.org/10.3390/bioengineering4020052
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author Montenegro, Eduarda Morgana da Silva
Delabary, Gabriela Scholante
da Silva, Marcus Adonai Castro
Andreote, Fernando Dini
Lima, André Oliveira de Souza
author_facet Montenegro, Eduarda Morgana da Silva
Delabary, Gabriela Scholante
da Silva, Marcus Adonai Castro
Andreote, Fernando Dini
Lima, André Oliveira de Souza
author_sort Montenegro, Eduarda Morgana da Silva
collection PubMed
description The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)—a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.
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spelling pubmed-55904722017-09-21 Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria Montenegro, Eduarda Morgana da Silva Delabary, Gabriela Scholante da Silva, Marcus Adonai Castro Andreote, Fernando Dini Lima, André Oliveira de Souza Bioengineering (Basel) Article The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)—a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity. MDPI 2017-05-25 /pmc/articles/PMC5590472/ /pubmed/28952531 http://dx.doi.org/10.3390/bioengineering4020052 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Montenegro, Eduarda Morgana da Silva
Delabary, Gabriela Scholante
da Silva, Marcus Adonai Castro
Andreote, Fernando Dini
Lima, André Oliveira de Souza
Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
title Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
title_full Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
title_fullStr Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
title_full_unstemmed Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
title_short Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
title_sort molecular diagnostic for prospecting polyhydroxyalkanoate-producing bacteria
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590472/
https://www.ncbi.nlm.nih.gov/pubmed/28952531
http://dx.doi.org/10.3390/bioengineering4020052
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