Bacterial motility measured by a miniature chamber for high-pressure microscopy

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Abstract

Hydrostatic pressure is one of the physical stimuli that characterize the environment of living matter. Many microorganisms thrive under high pressure and may even physically or geochemically require this extreme environmental condition. In contrast, application of pressure is detrimental to most life on Earth; especially to living organisms under ambient pressure conditions. To study the mechanism of how living things adapt to high-pressure conditions, it is necessary to monitor directly the organism of interest under various pressure conditions. Here, we report a miniature chamber for high-pressure microscopy. The chamber was equipped with a built-in separator, in which water pressure was properly transduced to that of the sample solution. The apparatus developed could apply pressure up to 150 MPa, and enabled us to acquire bright-field and epifluorescence images at various pressures and temperatures. We demonstrated that the application of pressure acted directly and reversibly on the swimming motility of Escherichia coli cells. The present technique should be applicable to a wide range of dynamic biological processes that depend on applied pressures. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

Figures

  • Figure 1. High-pressure microscope. (A) Photograph of the high-pressure chamber (HPC) mounted on an upright microscope without any modifications. (B) Cross section of HPC. MB, main body; FP; U-shaped flow path; WS, window support; OW, observation window; MW, medium window; RW, rear window; O1, O2 and O3, O-rings. The orange and green areas were filled with assay buffer and distilled water, respectively.
  • Figure 2. Motility of smooth-swimming cells. The motility assay was performed by two different systems. The current high-pressure chamber was equipped with a “built-in” separator, in which water pressure was properly transduced to that of the sample solution (See Section 3.1). On the other hand, the previous one was equipped with an “external” separator [29]. (A and B) Swimming fraction and speed during the pressurization (closed circles) and depressurization processes (open squares). Swimming fractions, Fbuilt-in, were based on the number of cells that swam with a speed of > 2 µm s−1 at each pressure. The speed, Sbuilt-in, was the average value of the swimming cells in A. Error bars are the SD. (C and D) Correlations between the results measured by “built-in” and “external” separator systems. The swimming fraction (C) and speed (D) at 0.1 (blue), 20 (green), 40 (yellow), 60 (pink) and 80 MPa (red). The plots in C and D were fitted to lines with slopes of 1.02 ± 0.01 and 1.07 ± 0.02 (± fitting error), respectively.
  • Figure 3. Rotational speed of single flagellar motors. (A) Schematic drawing of the experimental system (not to scale). (B and C) Sequential bright-field images of the same rotating tethered cell at 33 ms intervals at 0.1 MPa (B) and 80 MPa (C). The images are displayed after processing contrast enhancement and brightness offset. Blue arrowheads indicate completion of a turn. Scale bar, 2 µm. (D) The plots are mean values (n = 52) of the rotational speed in the pressurization (circles) and depressurization processes (diamonds). Each speed was obtained from the rotation number during 10 s. Data for the motors that were in the stop state were excluded from calculations of the speed.
  • Figure 4. Purification of the E. coli flagellin. Protein samples in each purification step are resolved on a Coomassie-stained 12% SDS-PAGE gel. (A) Lane 1, whole cell lysate; lane 2, supernatant of low speed centrifugation after shearing flagella by a blender; lane 3 and 4, supernatant and pellet of the ultracentrifugation of the flagella-containing suspension; lane 5 and 6, supernatant and pellet of the ultracentrifugation after heat treatment; lane 7 to 9, peak fractions of the HiTrap Q column. (B) Immunoblot detection of flagellin by using the antibody raised against the purified E. coli flagellin. A strong flagellin band can be seen for the whole cell sample of the wild-type E. coli strain RP437, but not for that of the strain RP3098, which does not produce any flagellar proteins. An arrowhead indicates E. coli flagellin (51 kDa).

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APA

Nishiyama, M., & Kojima, S. (2012). Bacterial motility measured by a miniature chamber for high-pressure microscopy. International Journal of Molecular Sciences, 13(7), 9225–9239. https://doi.org/10.3390/ijms13079225

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