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

Data_Sheet_1_Impact of temperature on the temporal dynamics of microcystin in Microcystis aeruginosa PCC7806.PDF

Cyanobacterial blooms pose a serious threat to water quality and human health due to the production of the potent hepatotoxin microcystin. In microcystin-producing strains of the widespread genus Microcystis, the toxin is largely constitutively produced, but there are fluctuations between the cellular and extracellular pool and between free microcystin and protein-bound microcystin. Here we addressed the question of how different temperatures affect the growth and temporal dynamics of secondary metabolite production in the strain Microcystis aeruginosa PCC7806 and its microcystin-deficient ΔmcyB mutant. While the wild-type strain showed pronounced growth advantages at 20°C, 30°C, and 35°C, respectively, the ΔmcyB mutant was superior at 25°C. We further show that short-term incubations at 25°C–35°C result in lower amounts of freely soluble microcystin than incubations at 20°C and that microcystin congener ratios differ at the different temperatures. Subsequent assessment of the protein-bound microcystin pool by dot blot analysis and subcellular localization of microcystin using immunofluorescence microscopy showed re-localization of microcystin into the protein-bound pool combined with an enhanced condensation at the cytoplasmic membrane at temperatures above 25°C. This temperature threshold also applies to the condensate formation of the carbon-fixing enzyme RubisCO thereby likely contributing to reciprocal growth advantages of wild type and ΔmcyB mutant at 20°C and 25°C. We discuss these findings in the context of the environmental success of Microcystis at higher temperatures.</p

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

The ubiquitous freshwater cyanobacterium Microcystis is remarkably successful, showing a high tolerance against fluctuations in environmental conditions. It frequently forms dense blooms which can accumulate significant amounts of the hepatotoxin microcystin, which plays an extracellular role as an infochemical but also acts intracellularly by interacting with proteins of the carbon metabolism, notably with the CO2 fixing enzyme RubisCO. Here we demonstrate a direct link between external microcystin and its intracellular targets. Monitoring liquid cultures of Microcystis in a diel experiment revealed fluctuations in the extracellular microcystin content that correlate with an increase in the binding of microcystin to intracellular proteins. Concomitantly, reversible relocation of RubisCO from the cytoplasm to the cell’s periphery was observed. These variations in RubisCO localization were especially pronounced with cultures grown at higher cell densities. We replicated these effects by adding microcystin externally to cultures grown under continuous light. Thus, we propose that microcystin may be part of a fast response to conditions of high light and low carbon that contribute to the metabolic flexibility and the success of Microcystis in the field.</jats:p

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

Enhancing photosynthesis at high light levels by adaptive laboratory evolution

Iterating mutagenesis and exposure to increasing light dramatically enhanced the light tolerance of a Synechocystis cyanobacterium strain. This involved over 100 mutations grouped around five haplotypes, as well as putative epistatic interactions. Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants

Nostopeptolide plays a governing role during cellular differentiation of the symbiotic cyanobacterium <i>Nostoc punctiforme</i>

Significance Nostoc symbioses with plants represent one of the most versatile and ancient types of symbioses. The infection process is a tug-of-war between the plant host and the cyanobacterial symbiont, with a reciprocal influence of both partners on the differentiation of the infectious motile filaments, hormogonia. In the present study, we have uncovered a major hormogonium-repressing factor of Nostoc punctiforme , nostopeptolide. To our knowledge, the nonribosomal peptide is the first complex secondary metabolite of cyanobacteria for which a governing role during cellular differentiation could be demonstrated. Different plant partners were shown to strictly downregulate the factor in symbiosis, thereby unveiling a complex cross-talk network between plants and Nostoc . </jats:p

Non-canonical localization of RubisCO under high light conditions in the toxic cyanobacterium<i>Microcystis aeruginosa</i>PCC7806

AbstractThe frequent production of the hepatotoxin microcystin and its impact on the life-style of bloom-forming cyanobacteria are poorly understood. Here we report that microcystin interferes with the assembly and the subcellular localization of RubisCO, inMicrocystis aeruginosaPCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes inM. aeruginosaand relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of microcystin to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2fixation product 3-phosphoglycerate are significantly higher in the microcystin-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction ofM. aeruginosawith its heterotrophic microbial community.</jats:p

Pair distance distribution function of PfnM-decorated ActM polymers.

<p>Cross section pair distribution functions of rabbit F-actin (dashed line), polymerized ActM (dotted) and polymerized ActM with PfnM in molar ratios of 2∶1, 1∶1 and 1∶2 (solid, dash-dotted, shot dotted line, respectively). Maximum dimensions at <i>p</i>_c(<i>r</i>)  = 0 are at 9 nm, 20 nm, 25 nm and 35 nm. Inset: Magnification of the region around the maxima of the <i>p</i>_c(<i>r</i>).</p