# Time-Resolved Visualization of Cyanotoxin Synthesis via Labeling by the Click Reaction in the Bloom-Forming Cyanobacteria Microcystis aeruginosa and Planktothrix agardhii

## TL;DR

Scientists used a chemical labeling method to track the production of toxic peptides in two types of cyanobacteria over time.

## Contribution

A novel method using click chemistry and fluorescent labeling to visualize cyanotoxin synthesis dynamics in live cyanobacteria.

## Key findings

- Fluorescent labeling with Alexa Fluor 488 effectively tracked cyanopeptide synthesis in Microcystis aeruginosa and Planktothrix agardhii.
- Labeling intensity correlated with chemical analysis of clickable peptides in individual cells and populations.
- Intracellular distribution of the label showed heterogeneous patterns during toxin synthesis.

## Abstract

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), N-propargyloxy-carbonyl-L-lysine (Prop-Lys), or O-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 Microcystis aeruginosa and AP biosynthesis in Planktothrix agardhii. 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.