The AglRQS motor rotates the Nfs complex around the spore surface.

<p>(A) NfsD-mCherry moves in orbital trajectories around the surface of 4-h-old spores. Observation of dynamic NfsD-mCherry clusters at different focal planes, middle (i) and top (ii) sections of a spore. For each time lapse, trajectories were computed by summing the different time points in consecutive frames and shown in “fire” colors to the right. Scale bar = 1 µm. (B) Instantaneous speed histogram of tracked NfsD-mCherry clusters over time in WT spores and in the different mutants. (C) Distance from the origin of NfsD-mCherry clusters in WT spores and different mutants. (D) Mean square displacement (MSD) of NfsD-mCherry clusters over time in WT spores and in the different mutants. (E) NfsD-mCherry rotation is abolished by CCCP. (F) Rotation of NfsD-mCherry requires AglQ. (G) Movement of NfsD-mCherry in the <i>exoA</i> mutant.</p

The Agl interacts with Nfs to promote spore coat assembly.

<p>(A) Sporulation titers after heat and sonication counted by DAPI staining in various strains. Corresponding DAPI-staining images are shown. Note the 10² spores/ml detection limit of the assay. Scale bar = 5 µm. (B) Thin sections of myxospores observed by transmission electron microscopy. WT, <i>aglQ</i>, <i>nfsD</i>, and <i>exoA</i> strains were observed 24 h after the induction of sporulation. Arrows point to spore coat material that detaches from the surface of sporulating cells in <i>aglQ</i> and <i>nfsD</i> mutants. Scale bars = 0.1 µm. (C) GSLI-FITC staining of the spore coat material. WT, <i>aglQ</i>, <i>nfsD</i>, and <i>exoA</i> strains were observed 4 h after the induction of sporulation. Scale bar = 1 µm. (D) AglR interacts with GltG and NfsG in a bacterial two hybrid assay.</p

Phase specific interactions between Agl and Glt/Nfs promote motility or sporulation.

<p>The Agl motor (yellow), a three protein MotAB-like channel harvests the pmf and interacts either with Glt (orange) or Nfs (light blue) to transport slime (red) or the Exo polymer (blue) depending on the growth phase. Both Glt and Nfs are shown spanning the entire cell surface because they both contain predicted inner and outer membrane proteins. The connection between the Glt complex and the MreB cytoskeleton is shown in black. IM, inner membrane; OM, outer membrane; the PG is not represented because its connection with Glt is unresolved and spores apparently lack PG. In both cases, the transport mechanism remains to be elucidated. In motile cells, the current model proposes that Glt proteins and attached slime (orange-red dots circled in black) traffic along a closed loop helix (grey) and generate propulsion as they interact with the underlying substratum. At the lagging cell pole, the motility complexes become de-activated (orange circles), potentially by removing their connection to slime, which thus becomes deposited on the substrate. In spores, Nfs proteins may associate with Exo polymers following their secretion by the Exo export machinery (green). Distributed Agl motor units could move Exo-linked Nfs complexes from one motor to the next, guided by the Exo polymer.</p

A Versatile Class of Cell Surface Directional Motors Gives Rise to Gliding Motility and Sporulation in <i>Myxococcus xanthus</i>

<div><p>Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the <i>Myxococcus xanthus</i> Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.</p></div

The Nfs complex transports the spore coat polymer at the surface of the developing spores.

<p>(A) Snapshots of two beads moving on spore surface. Two beads are attached to a WT spore; another bead is stuck to the bottom of flow chamber providing a fixed reference. The bead in focus is tracked using the low-powered laser as in the Methods section. Both beads move independently at different times (arrows). Scale bar = 1 µm. (B) Speed histogram of tracked polystyrene beads at the spore surface: WT spores, <i>exoA</i> spores, <i>exoA</i> spores in the presence of CCCP (10 µM), and <i>exoA aglQ</i> spores. WT spores in the presence of CCCP and <i>aglQ</i> spores yielded similar results as the <i>exoA</i> CCCP and <i>exoA aqlQ</i> spores and are therefore not represented for improved clarity. (C) Dynamics of NfsD-mCherry and GSLI-FITC on a sporulating cell. A time lapse of a 4-h-old sporulating cell is shown with its corresponding kymograph. The white arrow points to the dissociation of the red and green signals. Scale bar = 1 µm.</p

An escherichia coli strain reference panel for genotype-to-penotype analysis

Poster presented at EESMicrobio and FEMS2017<br

The Agl-Nfs machinery is sporulation-specific.

<p>(A) <i>nfs</i> and <i>glt</i> genes are differentially regulated during sporulation. Expression of NfsD, GltD, and AglQ as inferred from the measurements of relative single cell mCherry/sfGFP fluorescence intensities over time after sporulation induction. Shown are the average fluorescence intensity ratios (measured intensity/maximum intensity) of 25 cells for each time points. (B) Time course of AglQ-sfGFP (left), GltD-mCherry (middle panel), and NfsD-mCherry (right panel) dynamics during sporulation.</p