Summary of my work to date

Cyanobacterial strains
In particular, the cyanobacterial harmful algal blooms of the genera Microcystis
aeruginosa and Planktothrix agardhii are being investigated in more detail. These
genera are known to produce high intracellular levels of the toxic peptides
microcystins (MCs) and anabaenopeptides (APs) [Morón-Asensio, R. et al., 2021]. It
is assumed that intracellular toxic peptides can be released in two main ways: by cell
lysis or by active transport mechanisms across the cell membrane into the
environment [Kurmayer, R., SLOT, 2018]. While cell lysis enables the direct exit of
peptides from the cell by dissolving the cell wall, active transport across the
membrane is probably carried out by specific transport proteins or transport
mechanisms that selectively guide the peptides out of the cell. Another important
aspect of this study concerns the structural variants of microcystins (MCs) and
anabaenopeptides (APs). These variants can vary both in terms of the composition of
the amino acids and the spatial arrangement of the peptide chains [Kurmayer, R. et
al., Proposal, 2013-2018].

Subcellular localisation of toxin production in cyanobacteria (SLOT)

Prof Rainer Kurmayer and Rubén Morón-Asensio are responsible for the supervision
of these methods. In the course of this study, a detailed analysis of the subcellular
localisation of chemically modified microcystins (MCs) and anabaenopeptins (APs) in
the cyanobacteria Microcystis and Planktothrix was carried out. For this purpose,
copper-catalysed alkyne azide cycloaddition (CuACC), also known as click chemistry
methodology, was applied to investigate the peptide storage and transport
mechanisms at the subcellular level. Using this molecular imaging technique with
bioorthogonal labelling, the modified peptides could be localised. A large number of
isolates, which differ significantly in their intra- and extracellular toxin content under
physiological stress conditions, were compared. To analyse the structural variants of
microcystins (MCs) and anabaenopeptins (APs), different non-natural amino acids
with alkyl and azide side chains were introduced into individual Microcystis strains
and Planktothrix strains. The labelled alkyne-modified peptides were then coupled
with two azide-modified fluorophores, Alexa 405 (blue fluorescence) and Alexa 488
(green fluorescence). Labelling was performed during non-ribosomal peptide
synthesis (NRPS). Confocal laser scanning microscopy with a resolution of 30 nm
was used to qualitatively and quantitatively analyse the cyano and peptide
distribution at the inter- and intracellular level.

Image processing analysis in Huygens

The subsequent image processing analysis was carried out using the Huygens
software, whereby 432 images (gas vesicle disruption) have already been processed.
During image processing, the cells were categorised into three compartments: intact
cell, cytoplasm and periplasmic membrane. By precisely measuring the spherical
intact cells (Maer) and the cylindrical cells (CYA126 and No371), the volume of the
cytoplasm and the periplasmic membrane could be calculated. The values were
entered into an Excel list with stored formulae. The statistical values of the cropped
images in Huygens, in particular the sum of the signal intensities for channels 0 and
1, were also transferred to the Excel list. The Pearson correlation coefficients for the
intact cell, the cytoplasm and the periplasmic membrane were calculated using
certain determining factors in Huygens. Finally, the results of the individual
calculations were documented.

Results to date

Examination of the percentage values of the periplasmic membrane shows a

variation between 5 and 23.5% of the total cell in the Microcystis strains (Maer). In
comparison, these values are somewhat lower for the Planktothrix strains (CYA126
and No371), ranging between 1.5 and 20% for CYA126 and between 4 and 22.5%
for No371. One limitation of this method is that the values are sometimes subject to
large fluctuations due to the manual measurement in Huygens using the Ortho Slicer
and therefore different results could be obtained. The analysis of the statistical values
shows that the signal intensities within a cell decrease from the intact cell to the
periplasmic membrane. Interestingly, when comparing the signal intensities between
the Microcystis and Planktothrix strains, no significant difference is recognisable, as
all of them decrease within the cell from the intact cell to the periplasmic membrane.
The Pearson correlation coefficients of the modified samples (intact cell, cytoplasm
and periplasmic membrane) show average values between +0.2 and +0.9. These
positive r-values indicate a positive correlation in which the values of both variables
(channel 0 and 1) tend to increase together. This indicates that there is a systematic
relationship between the analysed variables.

 There are another 2880 images (signal build-up) in Huygens in progress

(Maer and No371)