Unique Properties of Eukaryote-Type Actin and Profilin Horizontally Transferred to Cyanobacteria

A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 µm in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 5-10 µm and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity

Characterization of two eukaryotic cytoskeletal proteins horizontally transferred to a cyanobacterium

Das Cyanobakterium Microcystis aeruginosa PCC 7806 enthält zwei Proteine unbekannter Funktion, welche eine hohe Sequenzähnlichkeit mit Bausteinen des eukaryotischen Aktinzytoskeletts haben. Eines dieser Proteine ist Aktin selbst, das andere ist das Aktinbindeprotein Profilin. Die vorliegende Arbeit enthält eine detaillierte Charakterisierung beider Proteine sowie Vergleiche mit ihren eukaryotischen Verwandten. So inhibiert, im Gegensatz zu Eukaryoten, cyanobakterielles Aktin nicht das Enzym DNaseI. Es bildet jedoch Polymere, die hier mit Phalloidin visualisiert wurden. Konfokale Mikroskopie offenbart klare Unterschiede in den Polymeren, da die cyanobakteriellen eine Länge von 10 µm nicht überschreiten und breiter sind als die zylindrischen, ca. 100 µm langen Filamente eukaryotischen Aktins. Röntgen-Kleinwinkelstreuungsdaten zeigen, dass cyanobakterielle Aktinpolymere in ihrer Form am ehesten einem Band ähneln. Es bestehen auch Unterschiede hinsichtlich des Profilins: während es in Eukaryoten ausschließlich Aktinmonomere bindet, assoziiert cyanobakterielles Profilin mit Aktinfilamenten und vermittelt die Entstehung flächiger Heteropolymere. GFP-Fusionsstudien zeigen, dass die Koexpression von Aktin und Profilin die Bildung eines Hohlraumkompartiments in E.coli nach sich zieht. Ähnliche Gebilde wurden bereits in Microcystis gezeigt und könnten auf die beobachteten Heteropolymere zurückzuführen sein. Diese Arbeit verdeutlicht, dass beide Proteine in einer natürlichen Bakterienpopulation etabliert sind und dort Merkmale tragen, die ihre eukaryotischen Vorläufer nicht zeigen. Folglich könnte die Anpassung an die räumlichen Begrenzungen einer Bakterienzelle, welcher die für die Regulierung der Polymerisation notwendigen Aktinbindeproteine fehlen, die Triebkraft für eine Koevolution von cyanobakteriellem Aktin und Profilin gewesen sein. Dieser Prozess gipfelte möglicherweise in der Entstehung eines neuartigen intrazellulären Gebildes von potentiell struktureller Bedeutung.The cyanobacterium Microcystis aeruginosa PCC 7806 harbors two proteins with unknown functions that were transferred horizontally from eukaryotes and show a high degree of sequence identity with key components of the eukaryotic actin cytoskeleton. One is actin itself; the other is profilin, an actin binding protein. This work presents the detailed characterization of both proteins and comparisons with the eukaryotic archetype. In contrast to bona fide actin, its cyanobacterial counterpart does not inhibit DNaseI. It forms polymers that can be visualized with labeled phalloidin, resembling eukaryotic actin in that respect. However, confocal microscopy reveals key differences between polymers of eukaryotic and cyanobacterial actin. Whereas the former appear as cylindrical filaments about 100 µm in length, the latter are shorter and wider arresting polymerization at 5-10 µm. Structural elucidation by Small-angle X-ray scattering shows that cyanobacterial actin polymers are ribbon-shaped. This work also shows fundamental differences between cyanobacterial and eukaryotic profilin. Most importantly, cyanobacterial profilin binds actin filaments and mediates their assembly into heteropolymeric sheets. GFP labeling experiments show that the co-expression of cyanobacterial profilin and actin results in the formation of large hollow enclosures in E.coli. These structures resemble the shell-like distribution of actin in Microcystis aeruginosa and may be based on the actin/profilin heteropolymers observed in vitro. This work shows that both cyanobacterial proteins are established in a natural bacterial community where they have gained properties unknown from their eukaryotic ancestors. Consequently, the adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes may have driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity of potential structural relevance

Diel Variations of Extracellular Microcystin Influence the Subcellular Dynamics of RubisCO in Microcystis aeruginosa PCC 7806

The cyanobacterium Microcystis is widely known for the production of the hepatotoxin microcystin. While the aspects regarding its toxicity have been studied extensively, only little is known about the natural function of this compound. Here we show our latest findings on how microcystin interferes with the inorganic carbon metabolism in the model strain M. aeruginosa PCC 7806. Both intra- and extracellular functions as a signaling molecule are discussed, as microcystin can interact with proteins of the photosynthetic apparatus, especially with RubisCO. Diel experiments showed a direct link between microcystin and its intracellular targets, as fluctuations in the extracellular microcystin content correlated with changes in the microcystin binding pattern to intracellular proteins. Concomitantly, alterations in the accumulation of RubisCO products are occurring. Interestingly, we also observed changes in the subcellular localization of RubisCO associated with high levels of extracellular microcystin. Microcystin addition experiments demonstrated that effects of externally added microcystin appear strongest at high cell densities and high light intensities. This gives further insight into how microcystin could be part of a possible fast response mechanism to environmental changes like high light and high cell density and thus contribute to the incomparable success of Microcystis in the field

Transmission electron micrographs of ActM and rabbit actin with PfnM.

<p>Top row shows ActM filaments without PfnM (left, “-”) and with 4-fold molar excess of PfnM. Bottom row shows rabbit actin controls. Scale bars: 200 nm.</p

PfnM-GFP expression in <i>E.coli.</i>

<p>In <i>E.coli</i>, PfnM-GFP distributes evenly in the cytoplasm (A1-3) or is localized to the cell poles (B1-D). Scale bars: 2 µm.</p

Fluorescence microscopy of PfnM binding to ActM polymers.

<p>FITC stained PfnM (green) co-localizes with phalloidin-TRITC stained ActM polymers (red). Single ActM bundles are covered with PfnM along their length. Fluorescence signals increase with increasing PfnM concentration, molar ratios of ActM:PfnM are indicated. Images on the left hand side show an overview (scale bars: 5 µm), magnifications of corresponding single aggregates are on the right (scale bars: 2 µm).</p

High-density cultivation of terrestrial Nostoc strains leads to reprogramming of secondary metabolome

Terrestrial symbiotic cyanobacteria of the genus Nostoc exhibit a large potential for the production of bioactive natural products of the nonribosomal peptide, polyketide, and ribosomal peptide classes, and yet most of the biosynthetic gene clusters are silent under conventional cultivation conditions. In the present study, we utilized a high-density cultivation approach recently developed for phototrophic bacteria to rapidly generate biomass of the filamentous bacteria up to a density of 400 g (wet weight)/liter. Unexpectedly, integrated transcriptional and metabolomics studies uncovered a major reprogramming of the secondary metabolome of two Nostoc strains at high culture density and a governing effect of extracellular signals in this process. The holistic approach enabled capturing and structural elucidation of novel variants of anabaenopeptin, including one congener with potent allelopathic activity against a strain isolated from the same habitat. The study provides a snapshot on the role of cell-type-specific expression for the formation of natural products in cyanobacteria

ActM-GFP expression in <i>E.coli.</i>

<p>ActM-GFP adopts a variety of shapes and apparently is not freely diffusible in <i>E.coli</i>. All images show the GFP-channel, except for A2 and A3 which display the transmission channel and an overlay of transmission and GFP, respectively. Image F shows anti-actin/TRITC immunofluorescence of untagged ActM expressed in <i>E.coli</i>. Scale bars: 2 µm.</p

A hollow enclosure in cells co-expressing PfnM-GFP and ActM.

<p>Co-expression of PfnM-GFP and ActM gives rise to a hollow compartment. This enclosure is not dynamically rearranged, as fluorescence does not recover 30 minutes after bleaching (D4, E4, the bleached region is indicate by a red rectangle). Z-sectioning and 3D reconstructions of the cell shown in F. Stepwise rotations along the x-axis of the total enclosure (G1-4) or its “top” half (H1-5) is shown. Immunodetection of ActM in the enclosures of <i>E.coli</i> expressing both ActM and PfnM-GFP reveals a co-localization (J1-3). Images show either GFP-channel (“GFP”), transmission image (“trans”) or an overlay of both (“merge”). Scale bars in Z-sectioning and 3D reconstruction: 1 µm. All other scale bars: 2 µm.</p

Phalloidin staining of polymerized rabbit actin and ActM.

<p>Rabbit actin (left) polymerizes into long filaments forming an interwoven network. ActM polymers (right) appear as short filaments assembling in bundles and sheets. Scale bars: 5 µm.</p