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Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation

Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to...

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Autores principales: Liu, Chieh-Lun, Place, Allen R., Jagus, Rosemary
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577597/
https://www.ncbi.nlm.nih.gov/pubmed/28763019
http://dx.doi.org/10.3390/md15080242
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author Liu, Chieh-Lun
Place, Allen R.
Jagus, Rosemary
author_facet Liu, Chieh-Lun
Place, Allen R.
Jagus, Rosemary
author_sort Liu, Chieh-Lun
collection PubMed
description Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 μg/mL), carbenicillin (100 μg/mL), and streptomycin sulfate (50 μg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments.
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spelling pubmed-55775972017-09-05 Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation Liu, Chieh-Lun Place, Allen R. Jagus, Rosemary Mar Drugs Article Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 μg/mL), carbenicillin (100 μg/mL), and streptomycin sulfate (50 μg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments. MDPI 2017-08-01 /pmc/articles/PMC5577597/ /pubmed/28763019 http://dx.doi.org/10.3390/md15080242 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
Liu, Chieh-Lun
Place, Allen R.
Jagus, Rosemary
Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation
title Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation
title_full Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation
title_fullStr Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation
title_full_unstemmed Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation
title_short Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation
title_sort use of antibiotics for maintenance of axenic cultures of amphidinium carterae for the analysis of translation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577597/
https://www.ncbi.nlm.nih.gov/pubmed/28763019
http://dx.doi.org/10.3390/md15080242
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