Altered motility of Caulobacter Crescentus in viscous and viscoelastic media

Abstract

Background: Motility of flagellated bacteria depends crucially on their organelles such as flagella and pili, as well as physical properties of the external medium, such as viscosity and matrix elasticity. We studied the motility of wild-type and two mutant strains of Caulobacter crescentus swarmer cells in two different types of media: a viscous and hyperosmotic glycerol-growth medium mixture and a viscoelastic growth medium, containing polyethylene glycol or polyethylene oxide of different defined sizes.

Results: For all three strains in the medium containing glycerol, we found linear drops in percentage of motile cells and decreases in speed of those that remained motile to be inversely proportional to viscosity. The majority of immobilized cells lost viability, evidenced by their membrane leakage. In the viscoelastic media, we found less loss of motility and attenuated decrease of swimming speed at shear viscosity values comparable to the viscous medium. In both types of media, we found more severe loss in percentage of motile cells of wild-type than the mutants without pili, indicating that the interference of pili with flagellated motility is aggravated by increased viscosity. However, we found no difference in swimming speed among all three strains under all test conditions for the cells that remained motile. Finally, the viscoelastic medium caused no significant change in intervals between flagellar motor switches unless the motor stalled.

Conclusion: Hyperosmotic effect causes loss of motility and cell death. Addition of polymers into the cell medium also causes loss of motility due to increased shear viscosity, but the majority of immobilized bacteria remain viable. Both viscous and viscoelastic media alter the motility of flagellated bacteria without affecting the internal regulation of their motor switching behavior.

Figure 1

Schematics of three strains of Caulobacter crescentus used: CB15 wild-type, CB15 Δpilin, and SB3860. The CB15 wild-type swarmer cell expresses pili (thin hairs) and the flagellar motor switches rotation directions to cause alternating forward and backward motion. The Δpilin cell expresses no pili, but the motor operates as the wild-type. The SB3860 (cheR138::Tn5 in YB375) is a double mutant, expressing no pili and moving exclusively forward.

Figure 2

Loss in motility with polymer added into the medium of synchronized SB3860 swarmer cells. A. Fraction of motile cells plotted versus PEG 4000, PEG 35000, and PEO 400000 in weight percentage. B. The same data collapsed to a single line when re-plotted as a function of viscosity. The errors bars, some smaller than the symbols, represent the standard errors of the mean. The numbers of cells counted were 531 in PEG 4000, 5884 in PEG 35000, and 1191 in PEO 400000.

Figure 3

Motility of swarmer cells as a function of viscosity in viscous and viscoelastic media. The three types of symbols represent wild-type (wt), Δpilin (deltaP), and SB3860 (3860) strains, in glycerol (solid symbols, with corresponding weight percentages listed on Table 1) and PEG 35000 (empty symbols, with corresponding weight percentages noted on the upper axis). The swarmer cells were counted using videos captured a few minutes after mixing with either pure glycerol or a concentrated PEG solution. The numbers of cells counted were, in PEG 35000, 4639 for wild-type, 4035 for Δpilin, and 5884 for SB3860; and in glycerol, 7174 for wild-type, 3509 for Δpilin, and 3603 for SB3860. The error bars, some smaller than the symbols, are standard errors of three separate sets of measurements.

Figure 4

Fractions of dead, non-motile and motile cells under three medium conditions. Cells whose membrane became permeable to DiBAC were considered dead. The non-motile cells were those whose membrane was still intact, so that the cells were not labeled by DiBAC. Observations were made with SB3860 cells within minutes following the preparation as described under Materials and Methods. The fractions of motile cells were determined prior to the labeling experiments, with the results taken from Figure 3. The numbers of non-motile cells counted in the labeling experiments were 467 in PYE, 1207 in 4% PEG 35000, and 565 in 44% glycerol.

Figure 5

Average swimming speed of cells as a function of viscosity, varied by addition of glycerol or PEG 35000. The speed data were generated by MATLAB analysis of videos taken at 5 minutes after mixing bacteria with the viscous solution and within 15 minutes of synchronizing the swarmer cells. The solid lines are power law fits to collective data of all three strains, yielding exponents of −1.06 in viscous (glycerol) and −0.45 in viscoelastic (PEG 35000) solutions, respectively. The numbers of cells with their speeds measured were, for wild-type, 80 in PYE (control), 2084 in glycerol, and 773 in PEG 35000; for Δpilin, 23 in PYE, 270 in glycerol, and 170 in PEG 35000; and for SB3860, 181 in PYE, 1033 in glycerol, and 868 in PEG 35000. The error bars represent standard deviations, to convey the fact that there was a natural spread of swimming speed under each condition.

Figure 6

Overlays of selected frames at 0.17 s intervals out of a 3-second movie, to illustrate switches during the motion of Δpilin (left panels) and wild-type (right panels) cells in PYE (top panels) and 4% PEG 35000 (bottom panels). The faint grey lines following the swimming trajectories were produced with darkened overlay of all frames over the 3-second movies at 125 frames/second. Arrows next to the trajectories were drawn to point out the directions of motion, with switches indicated by grey circles. The scale bar applies to all 4 images.

Figure 7

Histograms of intervals between motor switches. The intervals were measured for wild-type (wt, circles) and Δpilin (deltaP, triangles) cells in the standard PYE medium (solid symbols) and in the PYE medium containing 4% PEG 35000 (hollow symbols). The data show slight shift of switch intervals to lower values due to addition of PEG 35000. More details of these data are tabulated in Table 2.