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Three-Dimensional Observations of an Aperiodic Oscillatory Gliding Behavior in Myxococcus Xanthus Using Confocal Interference Reflection Microscopy

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Three-Dimensional Observations of an Aperiodic Oscillatory Gliding Behavior in Myxococcus Xanthus Using Confocal Interference Reflection Microscopy

Liam M Rooney et al. mSphere.

Abstract

The deltaproteobacterium Myxococcus xanthus is a model for bacterial motility and has provided unprecedented insights into bacterial swarming behaviors. Fluorescence microscopy techniques have been invaluable in defining the mechanisms that are involved in gliding motility, but these have almost entirely been limited to two-dimensional (2D) studies, and there is currently no understanding of gliding motility in a three-dimensional (3D) context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, revealing aperiodic oscillatory behavior with changes in the position of the basal membrane relative to the substrate on the order of 90 nm in vitro First, we use a model planoconvex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus bacteria and show that cells undergo previously unobserved changes in their adhesion profile as they glide. We compare the wild type with mutants that have reduced motility, which also exhibit the same changes in the adhesion profile during gliding. We find that the general gliding behavior is independent of the proton motive force-generating complex AglRQS and suggest that the novel behavior that we present here may be a result of recoil and force transmission along the length of the cell body following firing of the type IV pili.IMPORTANCE 3D imaging of live bacteria with optical microscopy techniques is a challenge due to the small size of bacterial cells, meaning that previous studies have been limited to observing motility behavior in 2D. We introduce the application of confocal multiwavelength interference reflection microscopy to bacteria, which enables visualization of 3D motility behaviors in a single 2D image. Using the model organism Myxococcus xanthus, we identified novel motility behaviors that are not explained by current motility models, where gliding bacteria exhibit aperiodic changes in their adhesion to an underlying solid surface. We concluded that the 3D behavior was not linked to canonical motility mechanisms and that IRM could be applied to study a range of microbiological specimens with minimal adaptation to a commercial microscope.

Keywords: 3D imaging; bacterial motility; gliding motility; label free; live-cell imaging; optical microscopy.

Figures

FIG 1
FIG 1
IRM image and schematic diagram of a planoconvex lens specimen. (a) Composite IRM image acquired using wavelengths at 488 nm, 514 nm, and 543 nm, which are false colored as indicated. As the concentric fringes propagate away from the cover glass, we observed spectral separation of the fringes. Bar = 200 μm. (b) Cross-sectional schematic of the lens specimen showing the color ordering of each acquisition wavelength as the fringes propagate axially from the cover glass.
FIG 2
FIG 2
3D reconstruction of a planoconvex lens specimen. (a) Radial intensity profile of the interference fringe pattern shown in Fig. 1a. We observed the fringe periodicity decrease as observed in the RGB IRM image. (b) Using a priori knowledge of the lens specimen, the axial height was calculated and used to plot the intensity of each pixel as a function of height. arb.u., arbitrary units. (c) 3D reconstruction of the lens specimen using the known x, y, and z values and intensity extracted from the 2D IRM image.
FIG 3
FIG 3
IRM reveals axial movements along the cell body during gliding motility. (a) A single frame from a wild-type DK1622 gliding specimen with 4 magnified regions of interest (ROI) of a single representative cell over the course of the time-lapse (from t = 1 min 27 s to t = 5 min 24 s). Images were acquired using a multiwavelength approach, with the reflected 488-nm signal false colored in cyan and the reflected 635-nm signal shown in magenta. As the cell glides across the solid substrate, the interference fringe pattern changes as the relative position of the cell to the cover glass fluctuates. (b) A single frame of DK1622 ΔaglQ with magnified ROI from a single representative gliding cell over the course of the time-lapse (from t = 3 min 1 s to t = 5 min 18 s). Images were acquired using incident light at 488 nm (cyan) and 635 nm (magenta). DK1622 ΔaglQ exhibits the same axial movements as the wild type, demonstrated by the presence of interference fringes which fluctuate as the cell glides. Full time series data for DK1622 and DK1622 ΔaglQ are presented in Movies S1 and S2 in the supplemental material, respectively. Bars = 20 μm (single frame) and 5 μm (ROI).
FIG 4
FIG 4
The interference fringe patterns of a gliding cell reveal the axial profile of the cell. (a) A representative DK1622 cell from a time series data set with the location where the intensity profile in panel b was measured. Interference fringes along the cell body can be observed, with the reflected 488-nm signal shown in cyan and the 635-nm signal in magenta. Bar = 5 μm. (b) Intensity plot profile from the line through the cell presented in panel a. The plot shows the maxima and minima of the interference fringes acquired using both 488-nm (cyan) and 635-nm (magenta) light. The spectral separation of the two interference patterns can also be observed. Axial directionality of the cell can be determined by interpreting the color ordering of the fringes, where fringes arising from the longer wavelength appear after those from the shorter wavelength when the cell is inclined, and the opposite is observed for declining slopes. The plot was acquired by averaging the signal over a line width of 3 pixels (0.3 μm). (c) Schematic of the x, z profile of the cell shown in panel a according to the color ordering and intensity profile in panel b. The axial positions of the colored fringes from each acquisition wavelength are also shown. The cell has not adhered to the solid surface during gliding and does not maintain a linear cylindrical profile along the length of the cell body. According to theory, regions that intersect the axial position of the first-order 488-nm maxima are located 91.7 nm above the substratum, and those for the first-order 635-nm maxima are located 119.3 nm above the substratum. The schematic is not drawn to scale.
FIG 5
FIG 5
IRM as a method for measuring the velocity of adherent cells. (a) Path lengths of DK1622 and DK1622 ΔaglQ over the course of a time series. Overall, DK1622 has an increased mean path length compared to DK1622 ΔaglQ, which shows that DK1622 ΔaglQ has a lower adhesion profile than the wild type. (b) Deletion of aglQ results in a decrease in the mean velocity of 41.2%, from 15.76 ± 0.89 μm/min to 9.26 ± 0.72 μm/min, compared with the wild type (nDK1622 = 21; nDK1622 ΔaglQ = 22 [****, P < 0.0001]).

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