Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i>
In non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products—namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), <i>N</i>-propargyloxy-carbo...
Saved in:
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-06-01
|
| Series: | Toxins |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2072-6651/17/6/278 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850164524153307136 |
|---|---|
| author | Rainer Kurmayer Rubén Morón Asensio |
| author_facet | Rainer Kurmayer Rubén Morón Asensio |
| author_sort | Rainer Kurmayer |
| collection | DOAJ |
| description | In non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products—namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), <i>N</i>-propargyloxy-carbonyl-L-lysine (Prop-Lys), or <i>O</i>-propargyl-L-tyrosine (Prop-Tyr). Subsequently, chemo-selective labeling is used to visualize the clickable cyanopeptides using Alexa Fluor 488 (A488). In this study, the time-lapse build up or decline of azide- or alkyne-modified MCs or APs was visualized during maximum growth, specifically MC biosynthesis in <i>Microcystis aeruginosa</i> and AP biosynthesis in <i>Planktothrix agardhii</i>. Throughout the time-lapse build up or decline, the A488 signal occurred with heterogeneous intracellular distribution. There was a fast increase or decrease in the A488 signal for either Prop-Tyr or Prop-Lys, while a delayed or unobservable A488 signal for Phe-Az was related to increased cell size as well as a reduction in growth and autofluorescence. The proportion of clickable MC/AP in peptide extracts as recorded by a chemical–analytical technique correlated positively with A488 labeling intensity quantified via laser-scanning confocal microscopy for individual cells or via flow cytometry at the population level. It is concluded that chemical modification of MC/AP can be used to track intracellular dynamics in biosynthesis using both analytical chemistry and high-resolution imaging. |
| format | Article |
| id | doaj-art-4fb76fa2392e4c20843c0a578f0cdaa3 |
| institution | OA Journals |
| issn | 2072-6651 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj-art-4fb76fa2392e4c20843c0a578f0cdaa32025-08-20T02:21:58ZengMDPI AGToxins2072-66512025-06-0117627810.3390/toxins17060278Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i>Rainer Kurmayer0Rubén Morón Asensio1Research Department for Limnology, University of Innsbruck, Mondseestrasse 9, 5310 Mondsee, AustriaResearch Department for Limnology, University of Innsbruck, Mondseestrasse 9, 5310 Mondsee, AustriaIn non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products—namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), <i>N</i>-propargyloxy-carbonyl-L-lysine (Prop-Lys), or <i>O</i>-propargyl-L-tyrosine (Prop-Tyr). Subsequently, chemo-selective labeling is used to visualize the clickable cyanopeptides using Alexa Fluor 488 (A488). In this study, the time-lapse build up or decline of azide- or alkyne-modified MCs or APs was visualized during maximum growth, specifically MC biosynthesis in <i>Microcystis aeruginosa</i> and AP biosynthesis in <i>Planktothrix agardhii</i>. Throughout the time-lapse build up or decline, the A488 signal occurred with heterogeneous intracellular distribution. There was a fast increase or decrease in the A488 signal for either Prop-Tyr or Prop-Lys, while a delayed or unobservable A488 signal for Phe-Az was related to increased cell size as well as a reduction in growth and autofluorescence. The proportion of clickable MC/AP in peptide extracts as recorded by a chemical–analytical technique correlated positively with A488 labeling intensity quantified via laser-scanning confocal microscopy for individual cells or via flow cytometry at the population level. It is concluded that chemical modification of MC/AP can be used to track intracellular dynamics in biosynthesis using both analytical chemistry and high-resolution imaging.https://www.mdpi.com/2072-6651/17/6/278microcystinanabaenopeptintime-lapse experimentspulse-feedingchemo-selective labelinghigh-resolution microscopy |
| spellingShingle | Rainer Kurmayer Rubén Morón Asensio Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i> Toxins microcystin anabaenopeptin time-lapse experiments pulse-feeding chemo-selective labeling high-resolution microscopy |
| title | Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i> |
| title_full | Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i> |
| title_fullStr | Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i> |
| title_full_unstemmed | Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i> |
| title_short | Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria <i>Microcystis aeruginosa</i> and <i>Planktothrix agardhii</i> |
| title_sort | time resolved visualization of cyanotoxin synthesis via labeling by the click reaction in the bloom forming cyanobacteria i microcystis aeruginosa i and i planktothrix agardhii i |
| topic | microcystin anabaenopeptin time-lapse experiments pulse-feeding chemo-selective labeling high-resolution microscopy |
| url | https://www.mdpi.com/2072-6651/17/6/278 |
| work_keys_str_mv | AT rainerkurmayer timeresolvedvisualizationofcyanotoxinsynthesisvialabelingbytheclickreactioninthebloomformingcyanobacteriaimicrocystisaeruginosaiandiplanktothrixagardhiii AT rubenmoronasensio timeresolvedvisualizationofcyanotoxinsynthesisvialabelingbytheclickreactioninthebloomformingcyanobacteriaimicrocystisaeruginosaiandiplanktothrixagardhiii |