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Mar. Drugs 2017, 15(8), 242; doi:10.3390/md15080242

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

Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, 701 E. Pratt Street, Baltimore, MD 21202, USA
Author to whom correspondence should be addressed.
Received: 17 April 2017 / Revised: 17 July 2017 / Accepted: 27 July 2017 / Published: 1 August 2017
(This article belongs to the Special Issue Advances and New Perspectives in Marine Biotechnology II 2016)
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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. View Full-Text
Keywords: Amphidinium carterae; axenic cultures; bacterized cultures; antibiotics; protein synthesis inhibitors Amphidinium carterae; axenic cultures; bacterized cultures; antibiotics; protein synthesis inhibitors

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Liu, C.-L.; Place, A.R.; Jagus, R. Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation. Mar. Drugs 2017, 15, 242.

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