Localization of MglA, an essential gliding motility protein in Myxococcus xanthus

Abstract

MglA, a 22-kDa protein related to monomeric GTPases, is required for the normal operation of the A (Adventurous) and S (Social) motility and for multicellular development of Myxococcus xanthus. To determine how MglA controls A- and S-motility, MglA was assayed biochemically and its cellular location was determined. His-tagged MglA hydrolyzed GTP slowly in vitro at a rate nearly identical to that of Ras showing that MglA has GTPase activity. Immunofluorescence microscopy of fixed cells from liquid showed that MglA was associated with helical track similar to the MreB spiral that spanned the length of the cell. The distribution pattern of MglA depended on the type of surface from which cells were harvested. In cells gliding on 1.5% (w/v) agar, the helical pattern gave way to punctate clusters of MglA-Yfp at the poles and along the long axis (lateral clusters). The lateral clusters emerged near the leading pole as the cell advanced coincident with a decrease in the intensity of the MglA-Yfp cluster at the leading pole. Newly formed lateral clusters remained fixed with regard to the substratum as the cell moved forward, similar to focal adhesion complexes described for AglZ, a protein partner of MglA. Lateral clusters did not form in cells gliding in methylcellulose, a polymer that stimulates S-motility at low cell density; rather MglA-Yfp was diffuse in the cytoplasm and more concentrated at the poles. The results suggest that conditions that favor S-motility prevent the formation of lateral clusters of MglA, which are associated with A-motility functions.

Fig 1. Purification of MglA

(A) SDS-PAGE showing Ha-Ras and MglA purified from E. coli as described in Methods. Lane 1) Benchmark Protein Std 10–220 kDa (Invitrogen); 2) Ha-Ras-His fusion from pRJS2; 3) MglA-His from pAGS120. (B) Immunoblot analysis with antibodies directed against MglA. Lane 1) Magic Mark Western Std (Invitrogen) 20–120 kDa; 2) MglA-His from pAGS120.

Fig 2. MglA catalyzes hydrolysis of GTP

(A) Fluorescence decay was monitored at 435 nm and the rate of hydrolysis was determined from the single turnover rate constant using Prism Graphpad. The time-dependent change in fluorescence for mantGTP was monitored in the absence (mant GTP; solid grey line) and presence of 5 µM of MglA (dashed line) or Ha-Ras (solid black line) protein. The mean values for four independent assays are presented; the standard deviations for these measurements were <10%. (B) (C) Equilibrium binding of mantGTP and mantGDP to MglA-His. The K_d values of MglA for mantGTP (panel B) and mantGDP (panel C) were measured by plotting the amplitude of fluorescence intensity versus the concentration of mant nucleotide. MglA (1 µM) was mixed with concentrations of mantGTP varying from 0.5 µM to 10 µM. Results shown represent the average values from three independent series of measurements.

Fig 3. MglA associates with a helical structure in fixed M. xanthus cells

Epifluorescence was used to observe the pattern of MglA in fixed cells. (A) MxH2370 (no MglA control), (B) DK1622, (C) MxH2423 (DK1622 + mglBA), (D) MxH2424 (∆mglBA + mglBA-yfp), (E) MxH2265 (∆aglZ), and (F) DK10409 (∆pilA). IF was performed as described in Methods. Probe= anti-MglA antibody. Bars=10 µm

Fig 4. MglA-Yfp complements the ∆mglBA mutant

Colony morphology and microscopic motility assays show that MglA-Yfp can complement MxH2370, the ∆mglBA mutant. All images were taken at the same magnification after 5 d incubation on CTPM 1.5% (w/v) agar. (A) Colony morphology of DK1622 (WT); diameter 21 mm (B) ∆mglBA (MxH2370); diameter 4 mm, and (C) MxH2424 (MxH2370::pJP20 (mglBA-yfp)); diameter 12mm. Inset near (C) shows the colony edge of MxH2424. (D–F) Gliding patterns for 10 cells were generated from time-lapse video images of each strain on 1.5% (w/v) agarose pads using the Metamorph tracking software. (D)= DK1622, (E)= MxH2370, and (F)=MxH2424. (G–I) MglA-Yfp complements the development defect of the ∆mglBA mutant. (G)= DK1622, H= MxH2370, and I= MxH2424. Images were taken after 96 hr on TPM agar. Bar =50 µm.

Fig 5. Production of MglA-Yfp in M. xanthus

(A) Plasmid pJP20 contains a 2.34 kb mglB and mglA-yfp fragment under control of the mgl promoter. (B) Immunoblot analysis with antibodies directed against Gfp. Lane 1) MW standards; 2) DK1622; 3) MxH2423 (DK1622::pJP20); 4) MxH2424 (MxH2370 pJP20). The arrow indicates the position of MglA-Yfp (49 kDa). (C) Immunoblot analysis with antibodies directed against MglA. Lane 1) MW standards; 2) MxH2370 (∆mglBA); 3) MxH2424 (∆mglBA pJP20); 4) DK1622; 5) MxH2423 (DK1622 pJP20). Arrows indicate the position of MglA (22 kDa) and MglA-Yfp (49 kDa).

Fig 6. Clusters of MglA-Yfp form in actively gliding cells

Time lapse images of a cell gliding on 1.5% (w/v) agar show the formation of lateral clusters of MglA-Yfp. Images in column A (inverted images in column B) show the fluorescence distribution over the time (15 s intervals) of a typical MxH2424 cell moving right to left (arrow with asterisk (*) in t= 0 image). Two sets of arrows in (A) show the positions of lateral clusters, which remain fixed relative to the substratum. The large arrowhead in the center of t= 0 image does not move in subsequent images even though the cell has moved. The dashed line over the inverted image in panel aligns with this cluster. A second set of arrowheads (small arrowhead) shows the appearance of a new cluster at t= 45 s. Column C shows a pseudo 3D projection showing the relative intensity of fluorescence particles in the cell. The X and Y axes units are scaled in pixels and the Z axis represents the intensity pixel values (from 0 to 65535) for 24-bit images. The intensity of the cluster at the leading pole diminishes as new lateral clusters appear. Arrowheads in (C) correspond to the same clusters indicated in (A). Additional examples are shown in Supplemental material (SupFigtracking.pdf)

Fig 7. Image reconstruction of MglA-Yfp generated from sections taken through live cells

(A–C) Confocal microscopy was used to acquire a Z-series of fluorescence images of M xanthus cells producing MglA-Yfp (MxH2424); (D) Images from the Z-series were reconstituted to produce a composite image; (E) Cartoon depicting the arrangement of Z-section images to form a helix; (F–G) Left and right-handed helices were detected in some cells.

Fig 8. The distribution pattern of MglA-Yfp depends on the gliding matrix

The location of MglA-Yfp clusters was determined by analysis of fluorescence microscopy time-lapse images. (A) Seven distribution patterns for MglA-Yfp are shown in the left column. The percentage of cells that displayed a particular pattern is shown for WT cells on 1.5% CTPM (w/v) agar and CTPM plus 0.5% (v/v) methylcellulose. Only motile cells were included in the analysis; n=100 cells for 1.5% (w/v) agar and n=60 cells for MC. (B) Confocal image of MxH2424 cells on solid surface, bar = 10 µm; (C) Fluorescence image of MxH2424 cells in methylcellulose (taken from video file), bar = 10 µm.