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Phototaxis Assays of Synechocystis sp. PCC 6803 at Macroscopic and Microscopic Scales

宏观和微观尺度上集胞藻PCC 6803的趋光性分析

AJ Annik Jakob
NS Nils Schuergers
AW Annegret Wilde

发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2328 浏览次数: 10490

评审: Maria SinetovaDennis NürnbergAnonymous reviewer(s)

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Abstract

Phototaxis is a mechanism that allows cyanobacteria to respond to fluctuations in the quality and quantity of illumination by moving either towards or away from a light source. Phototactic movement on low concentration agar or agarose plates can be analyzed at macroscopic and microscopic scales representing group behavior and single cell motility, respectively. Here, we describe a detailed procedure for phototaxis assays on both scales using the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Keywords: Cyanobacteria (蓝藻细菌) search Synechocystis (集胞藻属) search Motility (动力性) search Phototaxis (趋光性) search Photoreceptors (光受体) search

Background

The model organism Synechocystis sp. PCC 6803 uses retractile type IV pili (T4P) to move across moist surfaces in a jerky motion referred to as twitching motility. Two secretion ATPases (PilB and PilT) are responsible for the extension and retraction of the pilus apparatus, thus pulling the cells forward. Synechocystis sp. PCC 6803 harbors a variety of photoreceptors covering the entire visible spectrum. Absorption of light can stimulate either positive or negative phototaxis depending on wavelength and intensity. Recently, it was demonstrated that single cells of Synechocystis sp. PCC 6803 are able to directly detect unidirectional illumination by focusing the light in a sharp focal point on the distal side (Schuergers et al., 2016). Moreover, it was shown that the direction of twitching motility correlates with a specific proximal localization of the motor ATPase PilB (Schuergers et al., 2015). A model was proposed that the focusing leads to a local inhibition of the motility apparatus, thus determining the direction of movement of single cells as a photophobic response away from the focal light spot (Schuergers et al., 2016).

Materials and Reagents

  1. Sterile square (120 x 120 x 17 mm) Petri dishes (Greiner Bio One International, catalog number: 688161 ; supplied by VWR)
  2. Sterile 1 µl inoculation loops (SARSTEDT, catalog number: 86.1567.010 )
  3. Sterile 1.5 ml Eppendorf tubes 3810X (Eppendorf, catalog number: 0030125150 )
  4. Sterile µ-dishes 35 mm high glass bottom (ibidi, catalog number: 81158 )
  5. Microscope coverslips 20 x 20 mm (Carl Roth, catalog number: H873 )
  6. Sterile syringes 50 ml (SARSTEDT, catalog number: 94.6077.137 )
  7. Sterile syringe filters Filtropur S 0.2 (SARSTEDT, catalog number: 83.1826.001 )
  8. Sterile 50 ml Greiner centrifuge tubes (Greiner Bio One International, catalog number: 227261 )
  9. Serological pipettes 25 ml (SARSTEDT, catalog number: 86.1685.020 )
  10. Serological pipettes 5 ml (SARSTEDT, catalog number: 86.1253.025 )
  11. Synechocystis sp. PCC 6803 strain (motile wild type obtained from S. Shestakov, Moscow State University, Russia), resequenced by Trautmann et al. (2012)
  12. Liquid paraffin, viscous (Carl Roth, catalog number: 8904 )
  13. Ultrapure water
  14. Ethylenediamine tetraacetic acid disodium salt dehydrate (Na2EDTA·2H2O) (Carl Roth, catalog number: 8043 )
  15. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771 )
  16. di-Potassium hydrogen phosphate trihydrate (K2HPO4·3H2O) (EMD Millipore, catalog number: 105099 )
  17. Sodium carbonate (Na2CO3) (Carl Roth, catalog number: P028 )
  18. Boric acid (H3BO3) (Carl Roth, catalog number: 6943 )
  19. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Carl Roth, catalog number: T881 )
  20. Zinc sulphate heptahydrate (ZnSO4·7H2O) (Carl Roth, catalog number: T884 )
  21. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Carl Roth, catalog number: 0274 )
  22. Copper(II) sulphate pentahydrate (CuSO4·5H2O) (Carl Roth, catalog number: P024 )
  23. Cobalt(II) nitrate hexahydrate, Co(NO3)2·6H2O (Carl Roth, catalog number: HN16 )
  24. Ammonium ferric citrate (Carl Roth, catalog number: CN77 )
  25. 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES) (Carl Roth, catalog number: 9137 )
  26. Sodium thiosulphate (Na2S2O3) (Carl Roth, catalog number: HN25 )
  27. D(+)-glucose (Carl Roth, catalog number: X997 )
  28. Sodium nitrate (NaNO3) (Carl Roth, catalog number: A136 )
  29. Magnesium sulphate heptahydrate (MgSO4·7H2O) (Carl Roth, catalog number: P027 )
  30. Calcium chloride dihydrate (CaCl2·2H2O) (Carl Roth, catalog number: 5239 )
  31. Citric acid (Carl Roth, catalog number: X863 )
  32. Agar-agar, Kobe I (Carl Roth, catalog number: 5210 )
  33. UltraPureTM agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  34. 0.5 M Na2EDTA pH 8.0 (see Recipes)
  35. 3% w/v K2HPO4·3H2O solution (see Recipes)
  36. 2% w/v Na2CO3 solution (see Recipes)
  37. Trace metal mix solution (see Recipes)
  38. 0.6% w/v ammonium ferric citrate solution (see Recipes)
  39. 1 M TES buffer pH 8.0 (see Recipes)
  40. 30% w/v Na2S2O3 solution (see Recipes)
  41. 40% w/v D(+)-glucose solution (see Recipes)
  42. 100x BG11 medium (see Recipes)
  43. 2x BG11 medium (see Recipes)
  44. 1x BG11 medium (see Recipes)
  45. 1% w/v agar solution (see Recipes)
  46. 0.6% w/v agarose solution (see Recipes)
  47. Macroscopic phototaxis plate medium (see Recipes)
  48. Microscopic phototaxis plate medium (see Recipes)

Equipment

  1. Laboratory bottles 1,000 ml (Duran Group, catalog number: 21 801 54 5 )
  2. Laboratory bottles 500 ml (Duran Group, catalog number: 21 801 44 5 )
  3. Laboratory bottles 250 ml (Duran Group, catalog number: 21 801 36 5 )
  4. Laboratory bottles 100 ml (Duran Group, catalog number: 21 801 24 5 )
  5. Non-transparent square box (127 x 127 x 19 mm) with a one-sided opening (custom-made from polyvinyl chloride)
  6. Silicon ring (øo: 30 mm, øi: 16 mm) custom-made
  7. Non-transparent hollow cylinder (ø: 40 mm, h: 30 mm) with 4 holes (ø: 5 mm, h: 7 mm) positioned in increments of 90° for the insertion of LEDs (custom-made from polyvinyl chloride)
  8. Balance (Denver Instrument)
  9. pH meter (Mettler-Toledo)
  10. UV/Vis spectrophotometer (Shimadzu, model: UV-2401PC )
  11. Light source (Philips Lighting Holding, model: MASTER TL-D Super 80 18W/840 1SL/25 )
  12. Pipetting aid PIPETBOY acu 2 (INTEGRA Biosciences, model: PIPETBOY acu 2, catalog number: 155 017 )
  13. Quantum Sensor LI-190R (LI-COR, model: LI-190R )
  14. Transmitted light scanner (Epson, model: Epson Perfection V700 Photo , with adjustable cover)
  15. Fluorescence microscope (Nikon Instruments, model: Eclipse Ni-U , with CFI Plan Fluor 40X/0.75)
  16. Digital CCD camera (Hamamatsu Photonics, model: ORCA®-05G )
  17. Microcontroller board (Arduino, model: Arduino UNO R3 )
  18. RGB-LEDs (625 nm, 525 nm, 470 nm) 5 mm (World Trading Net)
  19. Biological safety cabinet (NuAire, model: Class II Type A2 )

Software

  1. NIS-Elements Basic Research 4.20.01
  2. Arduino 1.0.6
  3. MATLAB Runtime 8.3
  4. BacteriaMobilityQuant (https://web.fe.up.pt/~dee11017/software/BacterialMobilityQuant.zip)
  5. R

Procedure

English
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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Jakob, A., Schuergers, N. and Wilde, A. (2017). Phototaxis Assays of Synechocystis sp. PCC 6803 at Macroscopic and Microscopic Scales. Bio-protocol 7(11): e2328. DOI: 10.21769/BioProtoc.2328.

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分类

微生物学 > 微生物细胞生物学 > 细胞染色

微生物学 > 微生物生理学 > 趋光性

细胞生物学 > 细胞成像 > 活细胞成像

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