铜绿​​假单胞菌中细胞内钙浓度测定

Manita  Guragain; Anthony K. Campbell; Marianna A. Patrauchan

doi:10.21769/BioProtoc.2041

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Peer-reviewed

Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa

铜绿假单胞菌中细胞内钙浓度测定

Manita Guragain

AC Anthony K. Campbell

Marianna A. Patrauchan email

发布: 2016年12月05日第6卷第23期 DOI: 10.21769/BioProtoc.2041 浏览次数: 8767

评审: Valentine V TrotterAmit DeyAnonymous reviewer(s)

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Abstract

Characterization of the molecular mechanisms of calcium (Ca²⁺) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca²⁺_in). Here, we describe a protocol optimized to use a recombinantly expressed Ca²⁺-binding protein, aequorin, for detecting Ca²⁺_in in Pseudomonas aeruginosa. Upon binding to free Ca²⁺, aequorin undergoes chromophore oxidation and emits light, the log of which intensity linearly correlates with the amount of bound Ca²⁺, and therefore, can be used to measure the concentration of free Ca²⁺ available for binding. This protocol involves the introduction of the aequorin gene into P. aeruginosa, induction of apoaequorin production, reconstitution of the holoenzyme with its chromophore, and monitoring its luminescence. This protocol allows continuous measuring of Ca²⁺_in concentration in vivo in response to various stimuli.

Keywords: Intracellular calcium (细胞内钙离子)

Pseudomonas aeruginosa (铜绿假单胞菌)

Background

Ca²⁺ regulates physiology and virulence of P. aeruginosa (Guragain et al., 2013; Patrauchan et al., 2005; Sarkisova et al., 2014), however, the molecular mechanisms of Ca²⁺ regulation are not well understood. To characterize these mechanisms, it is critically important to not only measure the concentration of Ca²⁺_in ([Ca²⁺_in]), but to monitor its changes in response to various stimuli. Considering that [Ca²⁺_in] may change in response to even minute alterations in cell physiology (reviewed in [Dominguez et al., 2015]), measuring [Ca²⁺_in] requires a tool specifically recognizing Ca²⁺ without significantly disturbing cells. One such tool is aequorin, a Ca²⁺-binding protein, which upon binding to free Ca²⁺, undergoes chromophore oxidation and emits light. The emitted light can be recorded as a measure of free Ca²⁺. Aequorin has been successfully used to monitor Ca²⁺_in in eukaryotes (Bonora et al., 2013), as well as several bacterial species (Herbaud et al., 1998; Naseem et al., 2007; Rosch et al., 2008). Sufficient level of aequorin production and its stability within a cell enables continuous monitoring of Ca²⁺_in (Naseem et al., 2007). Use of aequorin offers additional advantages such as targeted intracellular distribution (cytoplasm or periplasm), high dynamic range, high signal-to-noise ratio, and low Ca²⁺ buffering effect (Bonora et al., 2013). Alternative approaches include application of chemical indicators, such as Fura. However, due to reduced cell membrane permeability in P. aeruginosa, loading cells of this bacterium even with membrane permeable Fura acetoxymethyl (AM, ester form) is challenging and requires additional treatments, which limits physiological relevance of the measurements (not published observations). Therefore, our group pioneered the use of aequorin for measuring [Ca²⁺_in] in P. aeruginosa (Guragain et al., 2013). The original protocol was developed for Escherichia coli (Knight et al., 1991) and further developed in (Jones et al., 1999). Here we present a modified adaptation of the protocol, successfully used to study Ca²⁺ homeostasis in P. aeruginosa, clinically and environmentally important organism (Guragain et al., 2013).

Materials and Reagents

General supplies
1. Centrifuge bottles (No specific brand is required)
2. Microfuge tubes (No specific brand is required)
3. Lumitrac 96 well white microplates (Greiner Bio One, catalog number: 655075 )
4. Aluminum foil
5. Plastic cuvettes (BrandTech Scientific, catalog number: 759086D )
Strains and plasmids
1. Pseudomonas aeruginosa strain PAO1 carrying pMBB66EH containing aequorin gene
2. Plasmid pMBB66EH encoding aequorin gene from Aequoria victoria (courtesy: Drs. Delfina Dominguez)
Culture medium
1. Luria bertani (LB) agar (see Recipes)
2. Biofilm minimal medium (BMM) (see Recipes)
  1. 10x basal salt solution
  2. Vitamin solution
  3. Trace metal
  4. 1 M MgSO₄
Chemicals and buffers
1. Carbenicillin, disodium (Gold Bio, catalog number: C-103-5 )
2. IPTG (Gold Bio, catalog number: I2481C )
3. Calcium chloride dehydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C5080 )
4. Live-Dead staining (Molecular Probes)
5. Yeast extract (BD, Bacto, catalog number: 212750 )
6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 or VWR, catalog number: E529-500ML )
7. Tryptone (BD, Bacto, catalog number: 211705 )
8. Agar (BD, Bacto, catalog number: 214010 )
9. Nanopure water
10. Monosodium glutamate (Sigma-Aldrich, catalogue number: 1446600 )
11. Glycerol (Thermo Fisher Scientific, Fisher Scientific, catalog number: G33 )
12. Sodium phosphate monobasic dihydrate (NaH₂PO₄) (Sigma-Aldrich, catalog number: 71500 )
13. Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: 17835 )
14. Biotin (Gold Bio, catalog number: B-950-1 )
15. Thiamine HCl (Gold Bio, catalog number: T-260-25 )
16. HCl (Pharmco-Aaper, catalog number: 284000ACS )
17. Copper(II) sulfate pentahydrate (CuSO₄·5H₂O) (Sigma-Aldrich, catalog number: 209198 ).
18. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Sigma-Aldrich, catalog number: Z0251 )
19. Iron(II) sulfate heptahydrate (FeSO₄·7H₂O) (Sigma-Aldrich, catalog number: 215422 )
20. Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Avantor Performance Materials, J.T. Baker, catalog number: 2540-04 )
21. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: 230391 )
22. HEPES (Sigma-Aldrich, catalog number: H3375 )
23. MgCl₂ (Sigma-Aldrich, catalog number: 230391 )
24. Tergitol Type NP-40, 70% in H₂O (Sigma-Aldrich, catalog number: T1135 )
25. Coelenterazine (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: C2944 )
26. Ethanol (Pharmco-Aaper, catalog number: AAP-111000190CSGL )
27. HEPES buffer (see Recipes)
28. Discharge buffer (see Recipes)
29. 1 M CaCl₂ solution (see Recipes)
30. 6 mM CaCl₂ solution for injection (see Recipes)
31. Coelenterazine (see Recipes)

Equipment

Multichannel pipette (Finnipipette F1, Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 4661030 )
Pipetman classic pipettes (Gilson, catalog numbers: F123600 , F123615 , F123601 , F123602 )
500 ml glass flasks (No specific brand is required)
Synergy^TM Mx multimode microplate reader (Biotek Instruments) with Gen5^TM 2.05 PC software (BioTek Instruments)
37 °C incubator (Bench top incubator) (VWR, catalog number: 89409-314 )
37 °C shaking incubator (MaxQ 4000 table top shake incubator) (Thermo Fisher Scientific, Thermo Scientific^TM, model: SHKA4000 )
Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Sorvall^TM RC 6 Plus Centrifuge ) with rotor type (Thermo Fisher Scientific, Thermo Scientific^TM, model: F13-14x50 cy )
Centrifuge (Eppendorf, model: 5424 )

Procedure

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如何引用

Guragain, M., Campbell, A. K. and Patrauchan, M. A. (2016). Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa. Bio-protocol 6(23): e2041. DOI: 10.21769/BioProtoc.2041.