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Measurements of Free-swimming Speed of Motile Salmonella Cells in Liquid Media
液体培养基中活性沙门氏菌细胞自由游动速度的测定   

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Abstract

Bacteria such as Escherichia coli and Salmonella enterica swim in liquid media using the bacterial flagella. The flagellum consists of the basal body (rotary motor), the hook (universal joint) and the filament (helical screw). Since mutants with a defect in flagellar assembly and function cannot swim smoothly, motility assay is an easy way to characterize flagellar mutants. Here, we describe how to measure free-swimming speeds of Salmonella motile cells in liquid media. Free-swimming behavior under a microscope shows a significant variation among bacterial cells.

Keywords: Bacterial flagella(细菌鞭毛), Motility(能动性), Motor(马达), Optical microscopy(光学显微镜), Proton motive force(质子动力), Salmonella(沙门氏菌)

Background

The flagellar motor of E. coli and Salmonella is powered by downhill proton translocation along proton motive force (PMF) across the cytoplasmic membrane (Morimoto and Minamino, 2014; Minamino and Imada, 2015). The rotational speed of the proton-driven flagellar motor is proportional to total PMF (Gabel and Berg, 2003). Therefore, measurements of free-swimming speeds of motile cells allow us not only to analyze motor performance of various mutants but also to examine whether there is a significant difference in total PMF under experimental conditions (Minamino et al., 2016).

Materials and Reagents

  1. 1.5 ml Eppendorf tubes
  2. Double-sided tape (NICHIBAN, catalog number: NW-5 )
  3. Glass slide (Matsunami Glass, catalog number: S1126 )
  4. 18 x 18 mm coverslip (thickness: 0.12-0.17 mm) (Matsunami Glass, catalog number: C018181 )
  5. Pipette tips  
  6. Filter paper
  7. Salmonella SJW1103 strain (wild type for motility and chemotaxis) (Yamaguchi et al., 1984)
  8. Salmonella MMHI0117 strain [∆fliH-fliI flhB(P28T)] (Minamino and Namba, 2008)
  9. Bacto tryptone (BD, catalog number: 211705 )
  10. Potassium dihydrogenphosphate (Wako Pure Chemical Industries, catalog number: 164-22635 )
  11. Dipotassium hydrogenphosphate (Wako Pure Chemical Industries, catalog number: 164-04295
  12. Bacto yeast extract (BD, catalog number: 212750 )
  13. Bacto agar (BD, catalog number: 214010 )
  14. Sodium chloride (Wako Pure Chemical Industries, catalog number: 192-13925 )
  15. T-broth (TB) (see Recipes)
  16. L-broth agar plate (see Recipes)

Equipment

  1. Selection of single channel pipettes (1,000 µl, 100 µl) (Gilson, model: P-1000 , P-100 )
  2. Shaking incubator (30 °C, at 200 rpm) (TAITEC, model: BR-40LF )
  3. Centrifuge (able to hold 1.5 ml tube, spin at 6,000 x g) (TOMY SEIKO, model: MX-305 )
  4. Spectrophotometer (able to measure OD600) (GE Healthcare, model: GeneQuant 1300 )
    Note: This product has been discontinued by the manufacturer. 
  5. Phase contrast microscope (Olympus, model: CH40 )
    1. 40x objective lens
    2. CCD camera (Hamamatsu Photonics, model: C5405 )
  6. Objective micrometer (10 µm/pitch)
  7. Hard-disk video recorder (Panasonic, model: DMR-XP25V )

Software

  1. Move-tr/2D (Library Co., Tokyo)
  2. Microsoft Excel (Microsoft)

Procedure

Note: Carry out procedures at ca. 23 °C unless otherwise specified.

  1. Pick a single colony from L-broth agar plate and inoculate it into 5 ml of fresh TB.
  2. Incubate overnight at 30 °C with shaking at 150 rpm.
  3. Inoculate 50 µl of overnight culture of Salmonella SJW1103 and MMHI0117 strains into 5 ml of fresh TB and incubate at 30 °C for 5 h with shaking at 150 rpm. (The cell density reaches an OD600 of ca. 1.0-1.2.)
  4. Transfer 100 µl of the culture into a 1.5 ml Eppendorf tube.
  5. Collect the cells by centrifugation (6,000 x g, 2 min).
  6. Suspend the cell pellet in 1.0 ml of fresh TB.
  7. Centrifuge at 6,000 x g for 2 min.
  8. Discard supernatant.
  9. Repeat steps 4-6.
  10. Resuspend the cells in 1.0 ml of fresh TB.
  11. Make a tunnel slide by sandwiching double-sided tape between glass slide (bottom side) and 18 x 18 mm coverslip (top side) (see Figure 1 and Video 1).


    Figure 1. Tunnel slide for observation of bacteria cells under optical microscopy. Double-sided tapes are sandwiched between a glass slide (bottom side) and an 18 x 18 mm coverslip (top side). Cells are added to the space between the glass slide and the coverslip. Observation area is magnified in a circle.

    Video 1. Preparation for a tunnel slide

  12. Add the cell suspension to the tunnel slide and absorb excess medium with a piece of filter paper.
  13. Set the tunnel slide on the stage of a phase contrast microscope.
  14. Select a 40x objective lens.
  15. Observe motile cells under the phase contrast microscope.

    Video 2. Free-swimming of Salmonella cells

  16. Record a movie of motile cells for 10-30 sec with a hard-disk video recorder.
  17. Move a field of view under the microscope.
  18. Repeat steps 14 and 15 more than 5 times.
  19. Capture an image of an objective micrometer in the same setting.
  20. Tansfer the movies recorded on the hard-disk video recorder to a DVD.

Data analysis

  1. Calibrate a scale of a movie using the image of the objective micrometer.
  2. Track motile cells in the movie with the length of 1 sec (30 sequential images recorded at 30 frames per second) using a motion analysis software Move-tr/2D (Library Co., Tokyo).
  3. Measure the velocity (µm/sec) of each cell by the Move-tr/2D.
  4. Calculate the average velocity and standard deviation from the data of more than 30 cells using Microsoft Excel (Microsoft).


    Figure 2. Free-swimming speed of Salmonella cells. SJW1103 (WT) and MMHI0117 (∆fliHI flhB*) were grown at 30 °C for 4 h in T-broth and observed by a phase contrast microscope. Vertical bars indicate standard deviations. (Modified from Minamino et al., 2016)

Notes

  1. When free swimming of motile cells is observed by phase contrast microscopy, a proper cell concentration is required. If the cell concentration is low, only a small number of cells are observed in a single movie. If the cell concentration is quite high, traces of each motile cell are overlapped, thereby making precise cell tracking difficult. The free-swimming motility is not affected at all by cell-cell interactions.
  2. Cells nonspecifically attached on a glass surface are often observed and so cannot show any free-swimming motility. So these cells must not be judged as non-motile cells.

Recipes

  1. T-broth (TB)
    1% Bacto tryptone, 10 mM potassium phosphate, pH 7.5
  2. L-broth agar plate
    1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl, 1.5% Bacto agar

Acknowledgments

This protocol was modified from a previous work (Minamino et al., 2003). This research has been supported in part by JSPS KAKENHI Grant Numbers JP15K14498 and JP15H05593 to YVM, JP21227006 and JP25000013 to KN and JP26293097 to TM and MEXT KAKENHI Grant Numbers JP26115720 and JP15H01335 to YVM and JP23115008, JP24117004, JP25121718 and JP15H01640 to TM.

References

  1. Gabel, C. V. and Berg, H. C. (2003). The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force. Proc Natl Acad Sci USA 100(15): 8748-8751.
  2. Minamino, T. and Imada, K. (2015). The bacterial flagellar motor and its structural diversity. Trends Microbiol 23(5): 267-274.
  3. Minamino, T., Imae, Y., Oosawa, F., Kobayashi, Y. and Oosawa, K. (2003) Effect of intracellular pH on rotational speed of bacterial flagellar motors. J Bacteriol 185(4):1190-1194.
  4. Minamino, T., Morimoto, Y. V., Hara, N., Aldridge, P.D. and Namba, K. (2016). The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na+ for flagellar protein export. PLoS Pathog 12(3): e1005495.
  5. Minamino, T. and Namba, K. (2008). Distinct role of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature 451(7177): 485-488.
  6. Morimoto, Y. V. and Minamino, T. (2014). Structure and function of the bi-directional bacterial flagellar motor. Biomolecules 4(1): 217-234.
  7. Yamaguchi, S., Fujita, H., Sugata, K., Taira, T. and Iino, T. (1984). Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella. J Gen Microbiol 130(2): 255-265.

简介

细菌如大肠杆菌和肠炎沙门氏菌在液体培养基中使用细菌鞭毛游泳。鞭毛由基体(旋转马达),钩(万向接头)和丝(螺旋螺钉)组成。由于鞭毛组装和功能缺陷的突变体不能顺利游动,所以运动测定是描绘鞭毛突变体的简单方法。在这里,我们描述如何测量液体介质中沙门氏菌活力细胞的游泳速度。显微镜下的游泳行为显示出细菌细胞之间的显着变化。

背景 fl ar。。。大肠杆菌和沙门氏菌由跨越细胞质膜的质子动力(PMF)的下坡质子易位(Morimoto和Minamino,2014; Minamino和Imada,2015)提供动力。质子驱动的鞭毛马达的旋转速度与总PMF成比例(Gabel和Berg,2003)。因此,运动细胞的游泳速度的测量使得我们不仅可以分析各种突变体的运动性能,还可以检查在实验条件下总PMF是否存在显着差异(Minamino等人)。 ,2016)。

关键字:细菌鞭毛, 能动性, 马达, 光学显微镜, 质子动力, 沙门氏菌

材料和试剂

  1. 1.5 ml Eppendorf管
  2. 双面胶带(NICHIBAN,目录号:NW-5)
  3. 玻璃滑梯(Matsunami Glass,目录号:S1126)
  4. 18 x 18毫米盖玻片(厚度:0.12-0.17毫米)(松本玻璃,目录号:C018181)
  5. 移液器提示
  6. 滤纸
  7. 沙门氏菌 SJW1103菌株(运动性和趋化性的野生型)(Yamaguchi等人,1984)
  8. MMHI0117菌株[Δfli-fliI flhB (P28T)](Minamino和Namba,2008)
  9. Bacto胰蛋白胨(BD,目录号:211705)
  10. 磷酸二氢钾(Wako Pure Chemical Industries,目录号:164-22635)
  11. 磷酸氢二钾(Wako Pure Chemical Industries,目录号:164-04295) 
  12. 细菌酵母提取物(BD,目录号:212750)
  13. Bacto琼脂(BD,目录号:214010)
  14. 氯化钠(和光纯药,目录号:192-13925)
  15. T型肉汤(TB)(见食谱)
  16. L-肉汤琼脂平板(参见食谱)

设备

  1. 选择单通道移液管(1,000μl,100μl)(Gilson,型号:P-1000,P-100)
  2. 摇动培养箱(30℃,200rpm)(TAITEC,型号:BR-40LF)
  3. 离心机(能够容纳1.5ml管,以6,000×g旋转)(TOMY SEIKO,型号:MX-305)
  4. 分光光度计(能够测量OD 600)(GE Healthcare,型号:GeneQuant 1300)
    注意:本产品已被制造商停产。
  5. 相位显微镜(Olympus,型号:CH40)
    1. 40x物镜
    2. CCD相机(滨松光子,型号:C5405)
  6. 目标千分尺(10μm/间距)
  7. 硬盘录像机(松下,型号:DMR-XP25V)

软件

  1. Move-tr/2D(东京图书馆有限公司)
  2. Microsoft Excel(Microsoft)

程序

注意:执行程序在ca. 23°C,除非另有说明

  1. 从L-肉汤琼脂平板上挑取一个菌落并将其接种到5ml新鲜结核菌中
  2. 在30℃下以150rpm摇动孵育过夜。
  3. 将50μl沙门氏菌SJW1103和MMHI0117菌株的过夜培养物接种到5ml新鲜的TB中,并在30℃下以150rpm摇动孵育5小时。 (细胞密度达到约1.0-1.2的OD 600)
  4. 将100μl培养物转移到1.5ml Eppendorf管中
  5. 通过离心(6,000 x g,2分钟)收集细胞。
  6. 将细胞沉淀物悬浮于1.0ml新鲜结核菌中。
  7. 以6,000 x g离心2分钟。
  8. 弃去上清液。
  9. 重复步骤4-6。
  10. 将细胞重新悬浮于1.0ml新鲜结核菌中。
  11. 在玻璃滑板(底侧)和18 x 18 mm盖玻片(上侧)之间夹住双面胶带(见图1和视频1),制作隧道滑轨。


    图1.在光学显微镜下观察细菌细胞的隧道载玻片双面胶带夹在玻璃滑块(底侧)和18 x 18 mm盖玻片(上侧)之间。将细胞加入到玻片与盖玻片之间的空间中。观察区域被放大一圈。

    <! - flashid2093v152开始 - >
    视频1.准备隧道幻灯片
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    Get Adobe Flash Player

    - ! - [if!IE]> - >
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    <! - flashid2093v152结束 - >
  12. 将细胞悬浮液加入隧道载玻片,用一张滤纸吸收多余的培养基
  13. 在相位显微镜的舞台上设置隧道滑梯。
  14. 选择40x物镜。
  15. 观察相位显微镜下的运动细胞
    <! - flashid2093v153开始 - >
    视频2.沙门氏菌单元格的游泳
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    要播放视频,您需要安装较新版本的Adobe Flash Player。

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    - ! - [if!IE]> - >
    <! - <![endif] - >
    <! - flashid2093v153结束 - >
  16. 用硬盘录像机录制10-30秒的动作电池片。
  17. 在显微镜下移动视野。
  18. 重复步骤14和15超过5次。
  19. 在相同的设置中捕获目标测微计的图像。
  20. 将录制在硬盘录像机上的电影转换成DVD。

数据分析

  1. 使用目标测微计的图像校准电影的比例。
  2. 使用运动分析软件Move-tr/2D(Library Co.,Tokyo)跟踪长度为1秒的长度为1秒(30个连续图像以30帧/秒记录)的电影轨道中的运动细胞。
  3. 通过Move-tr/2D测量每个单元格的速度(μm/sec)。
  4. 使用Microsoft Excel(Microsoft)计算超过30个单元格的数据的平均速度和标准偏差。


    图2.沙门氏菌细胞的游泳速度在30℃下生长SJW1103(WT)和MMHI0117(ΔfliHIflhB *)在T-broth中培养4小时,并用相差显微镜观察。垂直条表示标准偏差。 (从Minamino等人修改,2016)

笔记

  1. 当通过相差显微镜观察运动细胞的游泳时,需要适当的细胞浓度。如果细胞浓度低,在单个影片中仅观察到少量的细胞。如果细胞浓度相当高,每个运动细胞的痕迹重叠,从而使精确的细胞追踪变得困难。游泳动力不受细胞间相互作用影响。
  2. 经常观察到非特异性附着在玻璃表面上的细胞,因此不能显示出任何游泳动力。所以这些细胞不能被判断为不能运动的细胞

食谱

  1. T-broth(TB)
    1%细菌胰蛋白胨,10mM磷酸钾,pH7.5
  2. L-肉汤琼脂板
    1%Bacto胰蛋白胨,0.5%Bacto酵母提取物,1%NaCl,1.5%Bacto琼脂

致谢

该协议是从以前的工作(Minamino等人,2003)修改的。该研究部分由JSS KAKENHI Grant Numbers JP15K14498和JP15H05593授予YVM,JP21227006和JP25000013至KN和JP26293097以及TMXT KAKENHI Grant Numbers JP26115720和JP15H01335授予YVM和JP23115008,JP24117004,JP25121718和JP15H01640。

参考文献

  1. Gabel,CV和Berg,HC(2003)。  大肠杆菌的鞭毛旋转电动机的速度随着质子动力线性变化。 100(15):8748-8751。 >
  2. Minamino,T.和Imada,K.(2015)。  细菌鞭毛运动及其结构多样性。 趋势微生物 23(5):267-274。
  3. Minamino,T.,Imae,Y.,Oosawa,F.,Kobayashi,Y.and Oosawa,K。(2003)  细菌鞭毛III型出口门复合物是双燃料发动机,可以同时使用H + 和Na + 进行鞭毛蛋白质输出。 > PLoS Pathog 12(3):e1005495。
  4. Minamino,T.and Namba,K。(2008)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/18216858"target ="_ blank" > FliI ATP酶和质子动力在细菌鞭毛蛋白质出口中的独特作用。自然 451(7177):485-488。
  5. Morimoto,YV和Minamino,T。(2014)。 双向细菌鞭毛马达的结构和功能。生物分子 4(1):217-234。
  6. Yamaguchi,S.,Fujita,H.,Sugata,K.,Taira,T。和Iino,T.(1984)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/6374019"target ="_ blank"> H2的遗传分析,沙门氏菌中2期鞭毛蛋白的结构基因。 J Gen Microbiol/em> 130(2):255-265。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Morimoto, Y. V., Namba, K. and Minamino, T. (2017). Measurements of Free-swimming Speed of Motile Salmonella Cells in Liquid Media. Bio-protocol 7(1): e2093. DOI: 10.21769/BioProtoc.2093.
  2. Minamino, T., Morimoto, Y. V., Hara, N., Aldridge, P.D. and Namba, K. (2016). The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na+ for flagellar protein export. PLoS Pathog 12(3): e1005495.
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