FrzS regulates social motility in Myxococcus xanthus by controlling exopolysaccharide production

Abstract

Myxococcus xanthus Social (S) motility occurs at high cell densities and is powered by the extension and retraction of Type IV pili which bind ligands normally found in matrix exopolysaccharides (EPS). Previous studies showed that FrzS, a protein required for S-motility, is organized in polar clusters that show pole-to-pole translocation as cells reverse their direction of movement. Since the leading cell pole is the site of both the major FrzS cluster and type IV pilus extension/retraction, it was suggested that FrzS might regulate S-motility by activating pili at the leading cell pole. Here, we show that FrzS regulates EPS production, rather than type IV pilus function. We found that the frzS phenotype is distinct from that of Type IV pilus mutants such as pilA and pilT, but indistinguishable from EPS mutants, such as epsZ. Indeed, frzS mutants can be rescued by the addition of purified EPS, 1% methylcellulose, or co-culturing with wildtype cells. Our data also indicate that the cell density requirement in S-motility is likely a function of the ability of cells to construct functional multicellular clusters surrounding an EPS core.

Figure 1. Quantitative social motility assay.

Serial dilutions of mid exponential-phase cultures ranging from 2×10⁶ to 2×10⁸ cells/assay were prepared either in mono-cultures or co-cultures, then aliquoted onto 0.5% agar CYE plates. Swarm expansion was imaged and measured after 24 h incubation at 32°C. (A) Cell density-dependent social motility was assayed in wildtype (black ♦), frzS (green ▴) and a wildtype+frzS co-culture (green ▵), with the error bars representing one standard deviation. Corresponding images below show wildtype swarms, defective frzS swarms with small abortive flares, and full recovery of motility in the co-culture. (B) Analysis of wildtype (black ♦), S-motility defective pilA (orange ▪) and partial recovery of a wildtype+pilA co-culture (orange □) with corresponding images from the even number data points shown below. Scale bar is 1 mm.

Figure 2. Complementation of the frzS Social motility defect.

(A) Swarm expansion data for frzS-wildtype co-cultures at the ratios listed. (B,C) Transmission and fluorescence stereomicroscopy of 1∶1 co-cultures of wildtype with frzS^Y102A::gfp and (D,E) ΔfrzS with frzS^Y102A::gfp showing migration of the GFP-labeled cells corresponding to the colony edge. Scale bar is 1 mm.

Figure 3. Motility assay in methylcellulose.

Mid-exponential phase cells were resuspended in 1% methylcellulose on glass slides and S-motility observed by time-lapse microscopy. Images show 7 cell tracks for (A) frzS mutant; (B) wildtype strain DZ2: and (C) pilT mutant over 9 min (Top). Images at 0, 3, 6, and 9 min are shown below. Cells were tracked in three independent experiments each with frzS, wildtype and pilT, but movement was only observed with frzS and wildtype DZ2. Scale bar is 5 µm.

Figure 4. EPS analysis and agglutination assay.

(A) Congo red dye was added to mid-exponential phase cultures of the given strains, cells were centrifuged and the resulting supernatants analyzed in a spectrophotometer at 490 nm. Values were normalized to wildtype binding. An increase is indicative of more Congo red bound by the cells and increased EPS production, such as observed in pilT. pilA and frzS cells show reduced EPS production. Data is from three independent trials. (B) Cells were also analyzed with agglutination assays performed on wildtype (black ♦), frzS (green ▴), pilA (orange ▪), and pilT (red •), and a 1∶1 mixture of wildtype with the mutant (open symbols). In wildtype, absorbance at OD₆₀₀ at the top of a cuvette decreases over time as cells agglutinate and settle to the bottom of the cuvette. pilA and frzS inhibit agglutination by wildtype, whereas pilT was observed to behave similar to wildtype. Data shown is representative of three trials.

Figure 5. Complementation of S-motility with purified EPS.

Washed cells of wildtype (A–D), frzS (E–H), and pilA (I–L) were spotted onto 0.5% agar CYE plates adjacent to a thin line of either an aliquot of EPS purified from wildtype (left two columns) or a buffer control (right two columns). Images were captured at 0 and 48 h, showing stimulation of motility in wildtype and frzS cells, but not pilA cells. Stimulation of motility can be observed as downward outgrowth from the colony. Images show representative images from multiple trials.

Figure 6. Fluorescence microscopy analysis of EPS production.

DIC and fluorescence analysis of (A–D) wildtype M. xanthus cells from a liquid culture. (E–H) Wildtype M. xanthus cells after 4 h incubation with a solid surface in submerged culture, showing the formation of small and large rosette structures with centralized WGA-TR staining. (I–L) frzS cells after 4 h incubation with a solid surface, showing disorganized clustering and dispersed low levels of WGA-TR staining. Each data set depicts from left to right: DIC, red channel fluorescence, superimposed overlay, and surface plot of signal intensity.

Figure 7. EPS mutant analysis.

(A) Congo red analysis of EPS extracts from the given strains. (B) Quantitative social motility with wildtype (black ♦), S-motility defective epsZ (purple ▴), and a 1∶1 co-culture of the two strains (purple ▵) showing increased swarming relative to epsZ alone, with corresponding images shown below.

Figure 8. Pathway analysis.

(A) Quantitative S-motility assay examining wildtype (black ♦), frzS (green ▴), wildtype+frzS (green ▵), epsZ (purple ▪), wildtype+epsZ (purple □ ) and epsZ+frzS (gray ○). (B) Combinatorial quantitative social motility analysis of DZ2, difA, epsZ, epsU, frzS, pilA, and pilT strains. Mono-culture and co-culture combinations of these strains are shown, indicating that frzS, epsZ, and epsU are in the same functional pathway as they are both complemented by the presence of wildtype, but are not able to complement each other.