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Toxin analysis in Alexandrium minutum infected with parasite Parvilucifera infectans

ABSTRACT: Semi targeted metabolic profiling from microalgal cells in acid-water methanolic extracts using UHPLC-HRMS (Orbitrap Q Exactive). Dinoflagellate Alexandrium minutum were either healthy or infected with alveolate parasite Parvilucifera infectans, and cultivated under 20 PSU or 32 PSU. Samples were unpaired and extracted on Day 14 and 21. The samples were processed using ZIC-HILIC chromatography followed by PRM aquisition in the positive polarity mode for target PSP toxins. The supplementary files include, the study and result files from Compound Discoverer, the metadata table with normalization factor, sample code and conditions. The raw files of standards used confirm retention time and MS2 fragments of samples are included as well. Comparative toxin levels of Paralytic shellfish toxin was monitored in the enodmetabolome.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Alexandrium Minutum Parvilucifera Infectans (ncbitaxon:103983)

SUBMITTER: Dr. Marine Vallet Ruchicka Oniel Georg Pohnert

PROVIDER: MSV000100057 | MassIVE | Sun Nov 30 15:03:00 GMT 2025

REPOSITORIES: MassIVE

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Project description:Bacterial toxin-antitoxin systems help bacteria to reduce their metabolism in various stressful conditions and also play a part in generating antibiotic tolerant bacterial subpopulation called persisters. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. Current libraries are a part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We were also interested in the activity of promoters during toxin overexpression. We used a targeted RNA-sequencing approach that allowed us to map the distinct 5- and 3-ends of toxin-cleaved RNA and also the beginnings of transcripts. We extracted total RNA from cultures where the expression of MazF or MqsR was induced; RNA from log phase culture was used as the control. In addition, RNA extracted from stationary phase culture was used to test for the possible toxin cleavage in natural stress conditions. The cleaved RNA is rapidly recycled making it possible that we miss important cleavage sites due to RNA degradation during the prolonged stationary phase. Therefore, in addition to wt E. coli we also used stationary phase RNA from exoribonuclease deficient strain where RNA cleavage products accumulate. Identification of the 5 ends is based on ligation of RNA adapters to the cellular RNA molecules, which requires 5 -monophosphates. Mapping of the 3 -ends is based on poly(A) tailing of RNA, which requires 3 -OH groups. Transcription initiation, ordinary cellular RNases, and toxin endonucleases all produce different types of RNA ends. These can be enzymatically converted to 5 -phosphates and 3 -OHs, which allows one to separately quantify the RNA ends produced by each of these processes from a single biological sample. Briefly, (i) primary transcripts have 5 -triphosphate ends that can be converted to monophosphate by Tobacco Acid Pyrophosphatase (TAP), and 3 -hydroxyl ends that can be directly polyadenylated by poly(A) polymerase. (ii) Most cellular RNases (excluding RNase I and the toxins) produce 5 -monophosphates and 3 -hydroxyls, which are directly usable for ligation/polyadenylation. (iii) RNase I, MazF, and several other toxins produce 5 -hydroxyl ends that need to be phosphorylated by T4 Polynucleotide Kinase (PNK) prior to ligation, and 2,3 -cyclic phosphates that need to be dephosphorylated and converted to 3 -hydroxyls, also by T4 PNK. In short, for each of our RNA samples three 5 end and two 3 end libraries were made. The PNK treated libraries contain reads mapping specifically to 5- or 3-ends left by toxin (or RNaseI) cleavage and TAP treated libraries have extra reads mapping to beginnings of the transcripts.

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Toxin analysis in Alexandrium minutum infected with parasite Parvilucifera infectans

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