Article Figures & Data
Figures
- FIG 1
Images demonstrating the flexibility of P. mirabilis and V. parahaemolyticus swarmer cells. (A) Time series of P. mirabilis swarmer cells in a colony actively moving across the surface of a 1.5% agarose gel. A representative cell, false-colored green, had a generally straight shape at t = 0 s and was bent in half at t = 0.98 s. Most of the cells in this frame were bending during this imaging sequence. (B) A time series of V. parahaemolyticus swarmer cells in a colony actively moving across the surface of a 1.4% agarose gel. A representative cell (false-colored purple) had a generally straight shape at t = 0 s and was bent in half at t = 4.02 s.
- FIG 2
Using a reloadable microfluidics-based assay to determine bacterial cell stiffness. (A) Schematic of the microfluidic channel used to apply a user-defined shear force to bend filamentous or swarmer cells. Single-sided green arrows depict the flow of fluid through the central channel; the parabolic flow profile of the fluid is shown. Double-sided green arrows indicate the vacuum chamber used to load cells into side channels and to empty the device. (B) Cartoon of a flexible bacterium (left) and a stiff bacterium (right) under conditions of flow force (Vflow). “xmax” indicates the deflection of cells in the flow. 2r = cell diameter; L = cell length in contact with the flow force. (C) Representative images of filamentous cells of P. mirabilis under no-flow (left) and flow (right) conditions (top) and P. mirabilis swarmer cells (bottom). Purple dashed lines indicate the position of a cell tip under no-flow conditions, and black dashed lines illustrate the position after flow is applied using a gravity-fed mechanism. The arrow indicates the direction of fluid flow in the channel.
- FIG 3
P. mirabilis and V. parahaemolyticus swarmer cells have a lower bending rigidity than vegetative cells. We measured the bending rigidity of P. mirabilis and V. parahaemolyticus swarmer cells and filamentous vegetative cells in a microfluidic flow assay and included measurements of vegetative E. coli cells. P. mirabilis swarmers exhibited 26-fold-lower bending rigidity than vegetative cells; V. parahaemolyticus swarmers were 2-fold less rigid than vegetative cells. Overexpression of FlhDC (from the plasmid-carried pflhDC genes) had little effect on the stiffness of P. mirabilis vegetative and swarmer cells. The values for each cell type represent an average of the data from two fitting models (see the supplemental material), and the bracket bars represent the upper and lower limits of the two models. More than 100 cells were used for each cell type from at least 3 independent experiments. The 95% confidence intervals associated with the fits are shown in Fig. S19. The plot has a logarithmic y-axis scale.
- FIG 4
Swarmer cells increase in cell extension during osmotic shock. We calculated ΔL as (cell length in water – cell length in 1 M NaCl) and performed a similar calculation for ΔW, substituting cell width. Cell length indicates length prior to osmotic shock. We filamented all vegetative cells using aztreonam to grow them to lengths that were comparable with the lengths of P. mirabilis and V. parahaemolyticus swarmers. Lines indicate linear fits to single-cell measurements (circles) of more than 100 cells from at least 3 independent experiments. (A) P. mirabilis swarmer cells showed an increase in extension (ΔL) under conditions of osmotic shock compared to E. coli and P. mirabilis vegetative cells. (B) V. parahaemolyticus vegetative and swarmer cells showed an increase in extension (ΔL) under conditions of osmotic shock compared to E. coli. (C) P. mirabilis swarmer cells showed an increase in ΔW compared to E. coli; P. mirabilis vegetative cells displayed a slight decrease in width and increased cell length. (D) There was no observable change in ΔW of V. parahaemolyticus swarmer and vegetative cells.
- FIG 5
Alterations in the PG muropeptide composition of P. mirabilis and V. parahaemolyticus swarmer cells. (A) UPLC-MS data reveal that the muropeptide compositions of P. mirabilis and V. parahaemolyticus vegetative and swarmer cells differed slightly in the abundances of monomers, dimers, and anhydrous-substance-terminated saccharides. (B) We observed a relative increase in the amount of anhydrous-substance-containing saccharides in swarmers consistent with a decrease in polysaccharide strand length. (C) There was no change in PG cross-linking of P. mirabilis and V. parahaemolyticus vegetative and swarmer cells, although V. parahaemolyticus did display a lower level of cross-linking. n = 3 biological replicates. Error bars represent the standard deviations of the means. For (panels A to C, significance was determined via two-way analysis of variance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant (P > 0.05).
- FIG 6
AFM revealed that the PG layer of P. mirabilis and V. parahaemolyticus swarmer cells is thinner than in vegetative cells, and ECT demonstrated a reduced membrane-to-membrane distance. (A) Sacculi were isolated from cells, dried, and imaged by AFM. The thickness of swarmer cell PG was reduced compared to that seen with vegetative cells. We analyzed >65 vegetative cells of E. coli, P. mirabilis, and V. parahaemolyticus; >65 P. mirabilis swarmer cells; and 7 V. parahaemolyticus swarmer cells. Error bars represent the standard deviations of the means. (B) Bending rigidity and cell wall thickness display an approximately exponential relationship (R2 = 0.9874). (C) Subtomograph-averaged ECT volume images of the P. mirabilis vegetative (left) and swarmer (right) cell wall. Two central slices of subtomogram average volume images with normalized image densities are shown. Yellow dashed lines indicate the orientation used for gray-value measurements. ES, extracellular space; OM, outer membrane; PG, peptidoglycan; IM, inner membrane; CP, cytoplasm. (D) The density profile of subtomogram-averaged ECT volume images reveals reduced membrane-to-membrane distance in swarmer cells. The vertical-axis data represent normalized values corresponding to the shades of gray shown in panel C, with the darkest value equal to 0 and the lightest value equal to 1. The red dashed lines in the left and right panels denote the average value of the extracellular space represented by the shades of gray and serves as a reference for the background; the blue-shaded area indicates the thickness of the putative PG layer.
- FIG 7
Swarmer cells are more susceptible to antibiotics that target the cell wall than vegetative cells. (A) Survival of cells treated with 1× MIC of cephalexin after 3 h of incubation. We define percent survival as follows: (cell countno lysis/cell counttotal) × 100. P. mirabilis and V. parahaemolyticus swarmer cells exhibited levels of survival that were decreased (∼30%) compared to those seen with vegetative cells (n = ≥90 cells from at least two independent experiments). A similar decrease occurred when P. mirabilis was treated with penicillin G (n = ≥77 cells from at least two independent experiments). Error bars represent the standard deviations of the means. (B) After exposure to cephalexin or penicillin G, the survival time of P. mirabilis and V. parahaemolyticus swarmers was ∼2-fold to ∼3-fold lower than that of vegetative cells. Survival time was determined for ≥49 cells that lysed from at least 2 independent experiments. The statistical significance of the data presented in panels A and B was determined using a two-tailed t test. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001.
