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

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

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

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

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

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

Protein refolding is required for assembly of the type three secretion needle

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from α-helix into β-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level