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Jun 2019
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Preparation of Actinoplanes missouriensis Zoospores and Assay for Their Adherence to Solid Surfaces
密苏里游动放线菌孢子的制备及其固体表面黏附力检测   

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Abstract

Spherical zoospores of a rare actinomycete, Actinoplanes missouriensis, adhere to various hydrophobic solid surfaces by means of type IV pili. The purpose of this protocol is to provide detailed descriptions of the preparation of A. missouriensis zoospores and an assay for the adhesion of the zoospores to solid surfaces. This adhesion assay, which measures numbers of zoospores that adhered to the dish surface and swimming zoospores in a tunnel chamber by using a phase-contrast microscope, can also be used for swimming cells of other microorganisms.

Keywords: Adhesion (黏附), Tunnel chamber (隧道硐室), Rare actinomycete (稀有放线菌), Zoospore (游动孢子), Solid surface (固体表面), Type IV pili (IV型纤毛)

Background

Zoospores are motile asexual cells for reproduction that swim in aquatic environments by means of flagella. Although zoospores are often described as a kind of spore because of their function in the life cycle of producing organisms, attention must be paid to the fact that they are not dormant cells when they are swimming. Both eukaryotic and prokaryotic organisms produce zoospores, but eukaryotic zoospores produced by protists and fungi are more well-known compared with prokaryotic zoospores. Members of a fungal phylum of chytridiomycota, as well as oomycetes (which are pseudomycetes), are known to develop zoospores (Sharma et al., 2015; Letcher and Powell, 2017). Zoospores of these microorganisms swim in aquatic environments and adhere to the surface of organic substances, including parasitism hosts and dead bodies of animals and plants. In bacteria, several rare actinomycetes are known to produce zoospores, which are developed in a sporangium or by the fragmentation of aerial hyphae. A wide variety of bacterial zoospores have been isolated from natural environments by taking advantage of their chemotactic property (Hayakawa et al., 1991). Importantly, bacterial zoospores arise from dormant sporangiospores or arthrospores. A rare actinomycete Actinoplanes missouriensis produces terminal sporangia that contain a few hundred flagellated spores. The spores are dormant in a sporangium and, are activated and released to external environments when the sporangium is immersed in water. Although the presence of pili had not been reported in bacterial zoospores, we recently found a biosynthetic gene cluster for functional type IV pili in the A. missouriensis genome sequence, genetically analyzed the gene cluster, and successfully observed the unprecedented zoospore pili in A. missouriensis (Kimura et al., 2019). Furthermore, we developed an adhesion assay for A. missouriensis zoospores to characterize the function of the zoospore pili to attach the zoospores to solid surfaces. A similar adhesion assay for Mycoplasma has already been published by Kasai and Miyata (2013). The adhesion assay described in this protocol can be used not only for zoospores of other species but also for swimming cells of other microorganisms.

Materials and Reagents

  1. Coverslips (18 x 18 mm, Matsunami Glass Industry, catalog number: C218181)
  2. Glass dish (f 35 mm, AGC Techno Glass, catalog number: 3970-035)
  3. Polystyrene dish (f 35 mm, AGC Techno Glass, catalog number: 1000-035)
  4. Polystyrene dish (f 90 mm, Sansei Medical, catalog number: 01-013)
  5. 1.5-ml centrifuge tube (Greiner Bio-One, catalog number: J618201)
  6. 50-ml centrifuge tube (Corning, catalog number: 430829)
  7. Shaking (Sakaguchi) flask (Sansyo, catalog number: 82-0317)
  8. Double-sided tape (width 15 mm, thickness 0.086 mm, NICETACK, Nichiban)
  9. WhatmanTM Filter paper (GE Healthcare Life Sciences, catalog number: 3030-917)
  10. Toothpick
  11. A. missouriensis 431T (NBRC 102363T)
  12. Yeast extract (Becton, Dickinson and Company, catalog number: 212750)
  13. Meat extract (Kyokuto, catalog number: 551-01240-8)
  14. N-Z-Amine® (FUJIFILM Wako Pure Chemical, catalog number: 146-08675)
  15. D (+)-Maltose monohydrate (FUJIFILM Wako Pure Chemical, catalog number: 130-00615)
  16. Agar powder (Kokusan Chemical, catalog number: 2111136)
  17. Peptone (Becton, Dickinson and Company, catalog number: 211677)
  18. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Kokusan Chemical, catalog number: 2114992)
  19. Saccharose (Kokusan Chemical, catalog number: 2111624)
  20. Casamino acids, technical (Becton, Dickinson and Company, catalog number: 223120)
  21. Dipotassium hydrogen phosphate (K2HPO4) (Kokusan Chemical, catalog number: 2115140)
  22. Nitrohumic acid (Tokyo Chemical Industry, catalog number: H0161)
  23. Sodium hydroxide (NaOH) (Kokusan Chemical, catalog number: 2112744)
  24. Zinc chloride (ZnCl2) (FUJIFILM Wako Pure Chemical, catalog number: 263-00271)
  25. Iron (III) chloride hexahydrate (FeCl3·6H2O) (FUJIFILM Wako Pure Chemical, catalog number: 091-00872)
  26. Cupric chloride, dihydrate (CuCl2·2H2O) (Kokusan Chemical, catalog number: 2150417)
  27. Manganese (II) chloride tetrahydrate (MnCl2·4H2O) (FUJIFILM Wako Pure Chemical, catalog number: 139-00722)
  28. Sodium tetraborate (Na2B4O7·10H2O) (Kokusan Chemical, catalog number: 2114089)
  29. Ammonium molybdate ((NH4)6Mo7O24·4H2O) (Kokusan Chemical, catalog number: 2152738)
  30. Sodium chloride (NaCl) (Kokusan Chemical, catalog number: 2110733)
  31. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647)
  32. Ammonium hydrogencarbonate (NH4HCO3) (FUJIFILM Wako Pure Chemical, catalog number: 017-02875)
  33. YBNM agar medium (see Recipes)
  34. PYM broth (see Recipes)
  35. HAT agar medium (see Recipes)
  36. Nitrohumic acid solution (see Recipes)
  37. Trace element solution (see Recipes)

Equipment

  1. Mortar and pestle
  2. Laminar flow cabinet
  3. Flask shaker
  4. Centrifuge (Kubota Corp., model: 5200)
  5. Incubator (PHC Holdings, model: MIR-154)
  6. Micropipette
  7. Phase-contrast microscope (Olympus, model: IX73)
  8. Objective lens (Olympus, model: UPLFLN20×PH)
  9. Optical table (JVI, model: HAX-0605)
  10. High speed recorder system (Digimo, model: LRH1540) with complementary metal-oxide semiconductor (CMOS) camera

Software

  1. ImageJ (https://imagej.nih.gov/ij/)

Procedure

  1. Preparation of A. missouriensis zoospores
    1. Inoculate A. missouriensis cells from a glycerol stock on YBNM agar medium and cultivate them at 30 °C in an incubator for 2 or 3 days (Figure 1).


      Figure 1. A YBNM agar plate (diameter, 9 cm) on which A. missouriensis mycelia are grown for 4 days at 30 °C

    2. Using a sterilized toothpick, cut the agar medium to prepare an agar piece (approximately 1 cm square), on the surface of which A. missouriensis mycelium has proliferated (Figure 2). Put the agar piece into PYM broth (100 ml) in a shaking flask to inoculate the mycelium, and cultivate the A. missouriensis cells by shaking at 120 rpm at 30 °C for 2 days (Figure 3).


      Figure 2. Preparation of an agar piece for the inoculation of A. missouriensis


      Figure 3. Liquid culture of A. missouriensis in a shaking flask (at 30 °C for 2 days)

    3. Collect the cells by centrifugation at 2,330 x g for 10 min at RT.
    4. Suspend the cells in 0.75% NaCl solution (50 ml) and centrifuge them at 2,330 x g for 10 min at RT.
    5. Resuspend the cells in 0.75% NaCl solution (10 ml; Figure 4) and inoculate a portion (0.1 ml) of the cell suspension on one HAT agar plate.


      Figure 4. A. missouriensis mycelia suspended in 0.75% NaCl solution

    6. Spread the cell suspension and completely dry the surface of the HAT agar medium.
    7. Incubate the plate at 30 °C for at least 7 days (Figure 5).


      Figure 5. A HAT agar plate (diameter, 9 cm) on which A. missouriensis are grown for 7 days at 30 °C. Many sporangia are produced on substrate mycelium.

    8. Pour 25 mM NH4HCO3 solution (10 ml) onto the HAT agar plate (Figure 6) and incubate it at 30 °C for 1 h.


      Figure 6. Pouring of NH4HCO3 solution onto HAT agar

    9. Collect the poured solution, which contains swimming zoospores (approximately 104-105 cells/μl; Figure 7). Keep the solution at RT until the use.


      Figure 7. A. missouriensis zoospore-containing solution

  2. Zoospore adhesion test
    1. Attach a coverslip to a hydrophobic polystyrene dish or a hydrophilic glass dish by using two pieces of double-sided tape. Make a tunnel chamber by arranging the tape as parallel lines and making open slits on both sides of the coverslip (Figure 8). This arrangement enables a reproducible distance between the coverslip and dish, ensuring the fixed volume of the tunnel chamber, and also enables a replacement of the solution inside the chamber using a filter paper. The protocol using a similar tunnel chamber has already been published by Kasai and Miyata (2013).


      Figure 8. Illustration of a tunnel chamber

    2. (Optional; BSA-coating on the surface of a hydrophobic polystyrene dish) Using a micropipette, pour 1% BSA solution into the space between the dish and coverslip. Keep it at RT for 1 min. Absorb 1% BSA solution from a side of the chamber using a filter paper. By a similar procedure, wash the chamber with 25 mM NH4HCO3 solution. The BSA-coating treatment renders the dish surface hydrophilic. Through the treatment, the average proportion of the zoospores that adhered to the dish surface decreased from 41% (non-treated dish surface) to 0.2% (BSA-treated dish surface; Kimura et al., 2019).
    3. Using a micropipette, put the zoospore-containing solution (12 µl) carefully into the space between the dish and coverslip without introducing air bubbles. Incubate the chamber at RT for 10 min.
    4. Record the zoospores that adhered to the dish surface and the swimming zoospores in the tunnel by using a phase-contrast microscope equipped with a 20x objective lens. A lab recorder system and a CMOS camera enable the high-speed (200 frames per second) imaging. Scan the microscopic fields along the vertical direction for the analyzing region of the tunnel chamber in 3 s (600 images in total).
    5. Using a micropipette, put the 25 mM NH4HCO3 solution (150 μl) on one side of the tunnel and absorb the zoospore-containing solution from the other side using a filter paper to completely exchange the solution for 25 mM NH4HCO3 solution for the removal of the zoospores that did not adhere to the solid surface.
    6. Photograph the zoospores that adhered to the dish surface by using the microscope.

  3. Data analysis
    1. Convert the movie files recorded in Step B4 into AVI files. Convert the 8-bit images recorded in Step B6 into TIF files without compression using ImageJ, an image analysis software.
    2. Count the total number of swimming zoospores and those adhered to the dish surface in the analyzing region of the tunnel chamber by ImageJ using the microscopic images recorded in Step B4. The Color_FootPrint macro for ImageJ (http://www.jaist.ac.jp/ms/labs/hiratsuka/images/0/09/Color_FootPrint.txt) enables the visualization of the zoospore swimming trajectories (Hiratsuka et al., 2006).
      1. Open a movie file by using the command “File > Open”. Check off the boxes for “Use Virtual Stack” and “Convert to Grayscale”.
      2. Start the Color_FootPrint macro by using the command “Plugins > Macros > Color Footprint Rainbow”. The macro visualizes zoospore swimming trajectories (Figure 9B).
      3. Count the number of the stationary cells on the dish surface (Figure 9A) and the number of the colored trajectories, which represents the number of swimming zoospores, in the analyzing region of the tunnel chamber scanned in Step B4, by visual inspection.


        Figure 9. Counting the number of zoospores in the tunnel chamber by ImageJ. A. Input image; the first image of the total 600 scanning images is shown as a representative. B. Output image; colored trajectories are shown. Scale bar = 50 μm.

    3. Count the number of zoospores that adhered to the dish surface using the microscopic images recorded in Step B6 by ImageJ.
      1. Open an image by using the command “File > Open”.
      2. Select the analyzed region of the microscopic field scanned in Step B4 by using the “Rectangle” tool and clip the field by using the command “Image > Crop”.
      3. Set a threshold level by using the command “Image > Adjust > Threshold”.
      4. Count the number of zoospores by using the command “Analyze > Analyze Particles”. Set appropriate values of the parameters “Size” and “Circularity” (Figure 10).


        Figure 10. Counting the number of zoospores on the dish surface by ImageJ. A. Input image. Scale bar = 20 μm. B. Output image. A part of the original image is enlargedly shown.

    4. Calculate the adhesion ratio, i.e., the proportion of adhesive zoospores to whole zoospores: C3/C2. Correction values per unit area should be used for the calculation.

Notes

In the adhesion test, zoospores must be freshly prepared. Use zoospores within 1 h after collection. The collected zoospores should not be diluted because the dilution with a buffer may affect the motility of zoospores.

Recipes

  1. YBNM agar medium
    0.1% yeast extract
    0.2% meat extract
    0.2% N-Z-Amine®
    1% D (+)-maltose monohydrate
    Adjust pH to 7.0
    Add agar to 2% prior to autoclaving
  2. PYM broth
    0.5% peptone
    0.3% yeast extract
    0.1% MgSO4·7H2O
    Adjust pH to 7.0
    Autoclave the broth
  3. HAT agar medium
    0.1% saccharose
    0.01% casamino acids, technical
    0.05% K2HPO4
    2% nitrohumic acid solution
    1% trace element solution
    Adjust pH to 7.5
    Add agar to 2% prior to autoclaving
  4. Nitrohumic acid solution
    1. Grind 10 g of nitrohumic acid with a mortar and pestle
    2. Add 100 ml of 0.8% NaOH solution little by little and suspend the nitrohumic acid powder
    3. Autoclave the suspension at 105 °C for 15 min
    4. Stir the suspension until cooling down to RT
    5. Autoclave again the suspension at 105 °C for 15 min
    6. Stir the suspension until cooling down to RT
    7. Centrifuge the suspension at 1,500 x g for 10 min at 4 °C
    8. Transfer the supernatant to a sterilized glass bottle and store at 4 °C
  5. Trace element solution
    0.004% ZnCl2
    0.02% FeCl3·6H2O
    0.001% CuCl2·2H2O
    0.001% MnCl2·4H2O
    0.001% Na2B4O7·10H2O
    0.001% (NH4)6Mo7O24·4H2O
    Autoclave the solution

Acknowledgments

This protocol is adapted from Kimura et al. (2019). The above work was supported in part by Grants-in-Aid for Scientific Research no. 19H05685 (to Y.O.), 18H02122 (to Y.O.), 26252010 (to Y.O.), and 17K07711 (to T.T.), Grants-in-Aid for Young Scientists no. 16H06230 (to D.N.) and 15K18669 (to T.T.), and a Grant-in-Aid for JSPS Research Fellow no. 15J07768 (to T.K.) from the Japan Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).

Competing interests

The authors declare no conflicts of interest associated with this manuscript.

References

  1. Hayakawa, M., Tamura, T. and Nonomura, H. (1991). Selective isolation of Actinoplanes and Dactylosporangium from soil by using γ-collidine as the chemoattractant. J Ferment Bioeng 72(6): 426-432.
  2. Hiratsuka, Y., Miyata, M., Tada, T. and Uyeda TQ. (2006). A microrotary motor powered by bacteria. Proc Natl Acad Sci USA 103(37): 13618-13623.
  3. Kasai, T. and Miyata, M. (2013). Analyzing inhibitory effects of reagents on Mycoplasma gliding and adhesion. Bio-protocol 3(14): e829.
  4. Kimura, T., Tezuka, T., Nakane, D., Nishizaka, T., Aizawa, S. and Ohnishi, Y. (2019). Characterization of zoospore type IV pili in Actinoplanes missouriensis. J Bacteriol 201(14): e00746-18.
  5. Letcher, P. M. and Powell, M. J. (2017). Hypothesized evolutionary trends in zoospore ultrastructural characters in Chytridiales (Chytridiomycota). Mycologia 106(3): 379-396.
  6. Sharma, M., Ghosh, R., Tarafdar, A. and Telangre, R. (2015). An efficient method for zoospore production, infection and real-time quantification of Phytophthora cajani causing Phytophthora blight disease in pigeonpea under elevated atmospheric CO2. BMC Plant Biol 15: 90.

简介

稀有的放线菌 Actinoplanes missouriensis 的球形游动孢子通过IV型菌毛附着在各种疏水性固体表面上。该协议的目的是提供有关制备 A的详细说明。密苏里州游动孢子和游动孢子粘附到固体表面的测定。该粘附测定法通过使用相衬显微镜来测量粘附在培养皿表面的游动孢子的数量以及在隧道室内游动游动孢子的数量,也可用于游动其他微生物的细胞。

【背景】游动孢子是能繁殖的运动性无性细胞,通过鞭毛在水生环境中游动。尽管游动孢子因其在生产生物的生命周期中的功能而经常被描述为一种孢子,但必须注意这样一个事实,即游动时它们不是休眠细胞。真核生物和原核生物都产生游动孢子,但是与原核游动孢子相比,由原生生物和真菌产生的真核游动孢子更为人所共知。壶菌属真菌门以及卵菌(假真菌)的成员已知会形成游动孢子(Sharma et al。,2015; Letcher and Powell,2017)。这些微生物的游动孢子在水生环境中游动并粘附在有机物质的表面,包括寄生动物宿主和动植物的尸体。在细菌中,已知几种稀有的放线菌会产生游动孢子,游动孢子在孢子囊中或通过气生菌丝的分裂而发育。利用其趋化特性,已从自然环境中分离出各种各样的细菌游动孢子(Hayakawa et al。,1991)。重要的是,细菌游动孢子产生于休眠的孢子孢子或节孢子。稀有的放线菌 Actinoplanes missouriensis 产生的末端孢子囊含有数百个鞭毛孢子。孢子在孢子囊中处于休眠状态,并在孢子囊浸入水中后被激活并释放到外部环境中。尽管尚未在细菌游动孢子中报道菌毛的存在,但我们最近在 A中发现了功能性IV型菌毛的生物合成基因簇。missouriensis 基因组序列,对基因簇进行遗传分析,并成功观察到 A中前所未有的游动孢子菌毛。密苏里州(Kimura et al。,2019)。此外,我们开发了 A的粘附力测定。Missouriensis 游动孢子表征了游动孢子菌毛将游动孢子附着在固体表面上的功能。Kasai和Miyata(2013)已发表了一种针对支原体的相似粘附试验。该协议中描述的粘附测定不仅可以用于其他物种的游动孢子,还可以用于其他微生物的游动细胞。

关键字:黏附, 隧道硐室, 稀有放线菌, 游动孢子, 固体表面, IV型纤毛

材料和试剂

  1. 盖玻片(18 x 18 mm,松浪玻璃工业公司,目录号:C218181)
  2. 玻璃皿(f 35 mm,AGC Techno Glass,货号:3970-035)
  3. 聚苯乙烯盘(f 35 mm,AGC Techno玻璃,目录号:1000-035)
  4. 聚苯乙烯盘(f 90 mm,Sansei Medical,目录号:01-013)
  5. 1.5 ml离心管(Greiner Bio-One,目录号:J618201)
  6. 50 ml离心管(Corning,目录号:430829)
  7. 摇晃(阪口)烧瓶(Sansyo,目录号:82-0317)
  8. 双面胶带(宽度15毫米,厚度0.086毫米,NICETACK,Nichiban)
  9. Whatman TM 滤纸(GE Healthcare Life Sciences,目录号:3030-917)
  10. 牙签
  11. A。密苏里州 431 T (NBRC 102363 T )
  12. 酵母提取物(Becton,Dickinson and Company,目录号:212750)
  13. 肉提取物(京都,目录号:551-01240-8)
  14. NZ-Amine ®(FUJIFILM Wako Pure Chemical,目录号:146-08675)
  15. D(+)-麦芽糖一水合物(FUJIFILM Wako Pure Chemical,目录号:130-00615)
  16. 琼脂粉(Kokusan Chemical,目录号:2111136)
  17. 蛋白ept(Becton,Dickinson and Company,目录号:211677)
  18. 七水合硫酸镁(MgSO 4 ·7H 2 O)(Kokusan Chemical,目录号:2114992)
  19. 蔗糖(Kokusan Chemical,目录号:2111624)
  20. 工业级氨基酸(Becton,Dickinson and Company,目录号:223120)
  21. 磷酸氢二钾(K 2 HPO 4 )(Kokusan Chemical,目录号:2115140)
  22. 腐殖酸(东京化学工业,目录号:H0161)
  23. 氢氧化钠(Kokusan Chemical,目录号2112744)
  24. 氯化锌(ZnCl 2 )(FUJIFILM Wako Pure Chemical,目录号263-00271)
  25. 六水合氯化铁(III)(FeCl 3 ·6H 2 O)(FUJIFILM Wako Pure Chemical,目录号:091-00872)
  26. 二水合氯化铜(CuCl 2 ·2H 2 O)(Kokusan Chemical,目录号:2150417)
  27. 四水合氯化锰(MnCl 2 ·4H 2 O)(FUJIFILM Wako Pure Chemical,目录号139-00722)
  28. 四硼酸钠(Na 2 B 4 O 7 ·10H 2 O)(Kokusan Chemical,目录号:2114089 )
  29. 钼酸铵((NH 4 ) 6 Mo 7 O 24 ·4H 2 O)(国山化学,目录号:2152738)
  30. 氯化钠(NaCl)(Kokusan Chemical,目录号:2110733)
  31. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9647)
  32. 碳酸氢铵(NH 4 HCO 3 )(FUJIFILM Wako Pure Chemical,目录号:017-02875)
  33. YBNM琼脂培养基(请参阅食谱)
  34. PYM汤(请参阅食谱)
  35. HAT琼脂培养基(请参见食谱)
  36. 硝酸腐殖酸溶液(请参阅食谱)
  37. 痕量元素解决方案(请参见食谱)

设备

  1. 研钵和研杵
  2. 层流柜
  3. 烧瓶
  4. 离心机(久保田公司,型号:5200)
  5. 孵化器(PHC Holdings,型号:MIR-154)
  6. 微量移液器
  7. 相衬显微镜(奥林巴斯,型号:IX73)
  8. 物镜(奥林巴斯,型号:UPLFLN20×PH)
  9. 光学平台(JVI,型号:HAX-0605)
  10. 高速记录器系统(Digimo,型号:LRH1540),带有互补金属氧化物半导体(CMOS)摄像机

软件

  1. ImageJ( https://imagej.nih.gov/ij/ )

程序

  1. 制备 A。密苏里州游动孢子
    1. 接种 A。从YBNM琼脂培养基上的甘油储备液中提取出misouriensis 细胞,并将其在30°C的培养箱中培养2或3天(图1)。


      图1. YBNM琼脂平板(直径9厘米),在其上 A。missouriensis 菌丝体在30°C下生长4天

    2. 用消毒的牙签切开琼脂培养基,制备琼脂片(约1平方厘米),其表面为A。Missouriensis 菌丝体已增殖(图2)。将琼脂片放入摇瓶中的PYM肉汤(100 ml)中以接种菌丝体,并培养 A。通过在30°C下以120 rpm的频率摇动2天,从而获得了密苏里州的密苏里州细胞(图3)。


      图2.准备用于接种 A的琼脂片。密苏里州


      图3. A的液体培养。密苏里州 放在摇瓶中(在30°C下放置2天)

    3. 通过在RT下以2,330 x g 离心10分钟收集细胞。
    4. 将细胞悬浮在0.75%NaCl溶液(50毫升)中,在室温下以2,330 x g 的速度离心10分钟。
    5. 将细胞重悬在0.75%NaCl溶液(10 ml;图4)中,并在一个HAT琼脂平板上接种一部分细胞悬浮液(0.1 ml)。


      图4. A。密苏里州 菌丝体悬浮在0.75%NaCl溶液中

    6. 铺展细胞悬浮液,并完全干燥HAT琼脂培养基的表面。
    7. 将板在30°C下孵育至少7天(图5)。


      图5.上有 A的HAT琼脂平板(直径9厘米)。missouriensis 在30°C下生长7天。许多孢子囊在底质菌丝体上产生。

    8. 将25 mM NH 4 HCO 3 溶液(10 ml)倒入HAT琼脂平板上(图6),并在30°C下孵育1 h。


      图6.倒入NH 4 HCO 3 在HAT琼脂上的溶液

    9. 收集倒入的溶液,其中包含游泳游动孢子(大约10 4 -10 5 细胞/μl;图7)。保持溶液在室温下直至使用。


      图7. A。密苏里州 含游动孢子的溶液

  2. 游动孢子附着力测试
    1. 使用两片双面胶带将盖玻片附着到疏水性聚苯乙烯盘或亲水性玻璃盘上。通过将胶带布置成平行线并在盖玻片的两侧都开缝(图8)来制作隧道室。这种布置使得盖玻片和培养皿之间具有可再现的距离,从而确保了通道腔室的固定体积,并且还能够使用滤纸替换腔室内部的溶液。Kasai和Miyata(2013)已发布了使用类似隧道室的协议。


      图8.隧道室的图示

    2. (可选;在疏水性聚苯乙烯皿的表面上进行BSA涂层)使用微量移液器,将1%BSA溶液倒入皿和盖玻片之间的空间中。保持室温1分钟。使用滤纸从腔室的一侧吸收1%BSA溶液。通过类似的程序,用25 mM NH 4 HCO 3 溶液清洗反应室。BSA涂层处理使碟子表面具有亲水性。通过处理,附着在碟子表面上的游动孢子的平均比例从41%(未经处理的碟子表面)下降到0.2%(BSA处理的碟子表面;木村等人,2019年) )。
    3. 用微量移液器小心地将含游动孢子的溶液(12 µl)放入培养皿和盖玻片之间的空间,而不会引入气泡。在室温下孵育室10分钟。
    4. 使用配备有20倍物镜的相差显微镜记录粘附在培养皿表面的游动孢子和隧道中游动的游动孢子。实验室记录仪系统和CMOS相机可实现高速(每秒200帧)成像。沿垂直方向扫描显微区域,在3 s内对隧道室的分析区域进行扫描(总共600张图像)。
    5. 使用微量移液器将25 mM NH 4 HCO 3 溶液(150μl)置于隧道的一侧,并使用另一端从另一侧吸收含游动孢子的溶液滤纸将溶液完全替换为25 mM NH 4 HCO 3 溶液,以除去不粘附在固体表面上的游动孢子。
    6. 用显微镜拍摄粘附在培养皿表面的游动孢子。

  3. 数据分析
    1. 将步骤B4中录制的电影文件转换为AVI文件。使用图像分析软件ImageJ无需压缩即可将步骤B6中记录的8位图像转换为TIF文件。
    2. 使用步骤B4中记录的显微图像,通过ImageJ计算游泳游动孢子的总数以及附着在隧道室分析区域中的碟子表面的总数。用于ImageJ的Color_FootPrint宏( http:// www .jaist.ac.jp / ms / labs / hiratsuka / images / 0/09 / Color_FootPrint.txt )可以使游动的游动轨迹可视化(Hiratsuka et al。,2006年) 。
      1. 使用命令“文件>打开电影文件”。打开”。选中“使用虚拟堆栈”和“转换为灰度”复选框。
      2. 使用命令“插件>启动Color_FootPrint宏”。宏> 彩色足迹彩虹”。该宏将游动孢子的游泳轨迹可视化(图9B)。
      3. 通过目视检查,计算在步骤B4中扫描的隧道室的分析区域中,碟形表面上的静止细胞的数量(图9A)和彩色轨迹的数量(代表游泳游动孢子的数量)。


        图9.通过ImageJ计算隧道室内游动孢子的数量。 以600张扫描图像中的第一张图像为代表。B.输出图像;显示了彩色的轨迹。比例尺= 50μm。

    3. 使用ImageJ在步骤B6中记录的显微图像,计算粘附在碟子表面上的游动孢子的数量。
      1. 使用命令“文件>打开图像”。打开”。
      2. 使用“矩形”工具选择在步骤B4中扫描的显微视野的分析区域,并使用命令“图像>剪切”修剪视野。作物”。
      3. 使用命令“ Image> 调整> 阈”。
      4. 使用命令“ Analyze> 分析粒子”。设置参数“大小”和“圆度”的适当值(图10)。


        图10.通过ImageJ计数培养皿表面上的游动孢子数量。 A.输入图像。比例尺= 20μm。B.输出图像。原始图像的一部分被放大显示。

    4. 计算粘附率即,即粘附游动孢子与整个游动孢子的比例:C3 / C2。计算中应使用每单位面积的校正值。

笔记

在附着力测试中,游动孢子必须新鲜制备。收集后1小时内使用游动孢子。不应稀释收集的游动孢子,因为用缓冲液稀释可能会影响游动孢子的运动性。

菜谱

  1. YBNM琼脂培养基
    0.1%酵母提取物
    0.2%肉提取物
    0.2%NZ-胺®
    1%D(+)-麦芽糖一水合物
    将pH调整至7.0
    在高压灭菌之前将琼脂添加至2%
  2. PYM汤
    0.5%蛋白ept
    0.3%酵母提取物
    0.1%MgSO 4 ·7H 2 O
    将pH调整至7.0
    高压灭菌肉汤
  3. HAT琼脂培养基
    0.1%蔗糖
    0.01%酪蛋白氨基酸,工业
    0.05%K 2 HPO 4
    2%硝酸腐殖酸溶液
    1%微量元素溶液
    调节pH至7.5
    在高压灭菌之前将琼脂添加至2%
  4. 腐殖酸溶液
    1. 用研钵和研杵研磨10克硝酸腐植酸
    2. 一点一点地添加100 ml的0.8%NaOH溶液并悬浮硝化腐殖酸粉末
    3. 将悬浮液在105°C高压灭菌15分钟
    4. 搅拌悬浮液直至冷却至室温
    5. 再次将悬浮液在105°C高压灭菌15分钟
    6. 搅拌悬浮液直至冷却至室温
    7. 将悬浮液在4°C下以1,500 x g 离心10分钟
    8. 将上清液转移到灭菌的玻璃瓶中并在4°C下保存
  5. 微量元素解决方案
    0.004%ZnCl 2
    0.02%FeCl 3 ·6H 2 O
    0.001%CuCl 2 ·2H 2 O
    0.001%MnCl 2 ·4H 2 O
    0.001%Na 2 B 4 O 7 ·10H 2 O
    0.001%(NH 4 ) 6 Mo 7 O 24 ·4H 2 O
    高压灭菌解决方案

致谢

该协议改编自Kimura et al。(2019)。以上工作得到了No. 19H05685(至YO),18H02122(至YO),26252010(至YO)和17K07711(至TT),青年科学家资助计划。16H06230(至DN)和15K18669(至TT),以及JSPS研究研究员的No. 日本科学促进会(JSPS)和日本教育,文化,体育,科学与技术部(MEXT)的15J07768(致TK)。

利益争夺

作者声明与此手稿无关的利益冲突。

参考文献

  1. Hayakawa,M.,Tamura,T.和Nonomura,H.(1991)。选择性隔离 Actinoplanes 和 Dactylosporangium J发酵生物工程 72(6):426-432。
  2. Y.平冢,M。宫田,Tada和T. Uyeda TQ。(2006)。由细菌提供动力的微旋转马达。 美国国家自然科学中心(Proc Natl Acad Sci USA) 103(37):13618-13623。
  3. Kasai,T.和Miyata,M.(2013)。分析试剂对支原体滑动和粘附的抑制作用。 Bio-协议 3(14):e829。
  4. T.木村(T.Kimura),T. 密苏里放线猕猴中IV型游动孢子菌毛的特征。 J Bacteriol 201(14):e00746-18。
  5. 总理莱彻(Lettercher)和新泽西州鲍威尔(2017)。 Ch虫(Chytridiomycota)中游动孢子超微结构特征的假想进化趋势。 Mycologia 106(3):379-396。
  6. Sharma,M.,Ghosh,R.,Tarafdar,A.和Telangre,R.(2015年)。一种有效的游动孢子生产,感染和实时定量< Phytophthora cajani 的定量方法在大气CO 2 升高下引起木豆疫霉病。 BMC植物生物学报 15:90.
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引用:Tezuka, T., Nakane, D., Kimura, T. and Ohnishi, Y. (2019). Preparation of Actinoplanes missouriensis Zoospores and Assay for Their Adherence to Solid Surfaces. Bio-protocol 9(24): e3458. DOI: 10.21769/BioProtoc.3458.
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