Stationary bFACs drive M. xanthus gliding.

Motors carrying incomplete gliding complexes either diffuse or move rapidly along helical paths but do not generate propulsion. Motors stall and become nearly static relative to the substrate when they assemble into complete bFACs with other motor-associated proteins at the ventral side of the cell. Stalled motors push MreB and bFACs in opposite directions and thus exert force against outer membrane adhesins. Overall, as motors transport bFACs toward lagging cell poles, cells move forward but bFACs remain static relative to the substrate. IM, inner membrane; OM, outer membrane.

AgmT, a putative lytic transglycosylase, is required for M. xanthus gliding.

A) AgmT shows significant similarity to a widely conserved PG transglycosylase in YceG/MltG family. The conserved glutamine residue is marked by an asterisk. M_xant, M. xanthus; E_coli, E. coli; M_tube, Mycobacterium tuberculosis; S_coel, Streptomyces coelicolor; A_calc, Acinetobacter calcoaceticus; X_albi, Xanthomonas albilineans; N_meni, Neisseria menningitidis; R_prow, Rickettsia prowazekii; T_aqua, Thermus aquaticus. B) AgmT is required for M. xanthus gliding. Colony edges were imaged after incubating cells on 1.5% agar surface for 24 h. To eliminate S-motility, we further knocked out the pilA gene that encodes pilin for type IV pilus. Cells that move by gliding are able to move away from colony edges. Deleting agmT or disabling the active site of AgmT abolish gliding but fusing an PAmCherry (PAmCh) to its C-terminus does not. Heterologous Expression of E. coli MltG (MltGEc) restores gliding of agmT cells but not the cells that express AgmTEAEA. C, D) While cells lacking AgmT moved slower (C) and less persistently (D, measured by the distances cells traveled before pauses and reversals), the expression of MltGEc restores both the velocity and persistence of gliding in the agmT cells. Data were pooled from three independent experiments and p values were calculated using a one-way ANOVA test between two unweighted, independent samples. Boxes indicate the 25th - 75th percentiles and bars the median. The total number of cells analyzed is shown on top of each plot. *, p <0.001.

AgmT regulates cell morphology and integrity under antibiotic stress.

A) Purified AgmT solubilizes dye-labeled PG sacculi, but AgmTEAEA does not. Lysozyme serves as a positive control. Absorption at 595 nm was measured after 18 h incubation at 25 C. Data are presented as mean values ± SD from three replicates. B) AgmT regulates cell morphology. Compared to wild-type cells, cells that lack AgmT and express AgmTEAEA are significantly shorter and wider. A previously reported mutant that lacks all three class A penicillin-binding proteins (Δ3) displays similarly shortened and widened morphology but is still motile by gliding (Fig. S1). Heterologous expression of E. coli MltG partially restores cell length but not cell width in agmT and agmTEAEA backgrounds. Asterisks, 200 M sodium vanillate added. p values were calculated using a one-way ANOVA test between two unweighted, independent samples. Whiskers indicate the 25th - 75th percentiles and red dots the median. The total number of cells analyzed is shown on top of each plot. C) AgmT regulates cell morphology and integrity under mecillinam stress (100 μg/ml). Expressing E. coli MltG by a vanillate-inducible promoter (Pvan) restores resistance against mecillinam in agmT cells but not in the cells that express AgmTEAEA. van, 200 M sodium vanillate. Arrows point to newly lysed cells. Scale bars, 5 µm.

AgmT and its LTG activity are essential for proper bFAC assembly.

A) Overall distribution of AglR-PAmCherry particles on 1.5% agar surface is displayed using the composite of 100 consecutive frames taken at 100-ms intervals. Single-particle trajectories of AglR-PAmCherry (AglR-PAmCh) were generated from the same frames. Individual trajectories are distinguished by colors. Deleting agmT or disabling the transglycosylase activity of AgmT (AgmTEAEA) decrease the stationary population of AglR particles. B) The stationary population of AglR-PAmCherry particles, which reflects the AglR molecules in bFACs, changes in response to substrate hardness (controlled by agar concentration) and the presence and function of AgmT. The total number of particles analyzed is shown on top of each plot. C) AglR fails to assemble into bFACs in the absence of active AgmT, even on 5.0% agar surfaces. D) AgmT and its LTG activity also support the assembly of AglZ into bFACs. White arrows point to bFACs. Scale bars, 5 µm.

AgmT does not assemble into bFACs but connects bFACs to PG.

A) Immunoblotting using M. xanthus cell lysates and an anti-mCherry antibody shows that PAmCherry (PAmCh)-labeled AgmT and AgmTEAEA (592 amino acids) accumulate as full-length proteins. AgmT is significantly more abundant than the PAmCherry-labeled motor protein AglR (498 amino acids). The bacterial actin homolog MreB visualized using an MreB antibody is shown as a loading control. B) AgmT does not aggregate into bFACs. C) The LTG activity of AgmT is required for connecting bFACs to PG and expressing the E. coli LTG MltG (MltGEc) restores PG binding by bFACs in cells that lack AgmT but not in the ones that express an inactive AgmT variant (AgmTEAEA). AglR-PAmCherry was detected using an anti-mCherry antibody to mark the presence of bFACs that co-precipitate with PG-containing (lysozyme-) pellets. Lysates from the cells that express AglR-PAmCherry in different genetic backgrounds were pelleted by centrifugation in the presence and absence of lysozyme. The loading control of AglR-PAmCherry in the whole cell is shown as “total”.

A possible mechanism by which AgmT connect bFACs to PG.

AgmT could generate short glycan strands through its LTG activity and thus uniquely modify the overall structure of M. xanthus PG, such as producing small pores that retard and retain the inner subcomplexes of bFACs. Likewise, the M. xanthus mutants that lack active AgmT could produce PG with increased strain length, which precludes bFACs from binding to the cell wall.