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Identification and Characterization of Bacterial Chemoreceptors Using Quantitative Capillary and Gradient Plate Chemotaxis Assays
使用量化毛细管和梯度平板趋化性试验法进行细菌化学受体的识别和特性描述   

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Abstract

Bacterial chemotaxis is a motility-based response that biases cell movement toward beneficial molecules, called attractants, and away from harmful molecules, also known as repellents. Since the species of the genus Pseudomonas are characterized by a metabolic versatility, these bacteria have developed chemotactic behaviors towards a wide range of different compounds. The specificity of a chemotactic response is determined by the chemoreceptor, which is at the beginning of the signaling cascade and which receives the signal input. The basic elements of a typical chemoreceptor are the periplasmic ligand binding domain (LBD), responsible for sensing environmental stimuli, and the cytosolic methyl-accepting (MA) domain, that interacts with other components of the cellular signaling cascade. Escherichia coli (E. coli), the traditional model in chemotaxis research, has 5 well-characterized chemoreceptors. However, genome sequence analyses have revealed that many other bacteria possess many more chemoreceptors, some of which with partially overlapping signal profiles. This high number of chemoreceptors complicates their study by the analysis of single chemoreceptor mutants. We have pursued an alternative strategy for chemoreceptor characterization which corresponds to the generation of chimeric receptors composed of the LBD of the chemoreceptor under investigation and the MA domain of an E. coli receptor (Tar). The chimer is then introduced into a chemoreceptor free mutant of E. coli and the chemotaxis of the resulting strain is entirely due to the action of this chimeric receptor. In this publication we describe the use of quantitative capillary and gradient plate assays to study Pseudomonas chemotaxis as well as E. coli strains harboring chimeric receptors.

Keywords: Chemotaxis capillary assays(趋化性毛细管法), Chemoreceptors(化学受体), Receptor chimeras(受体嵌合体), Chemotaxis(趋化性), Gradient plate assays(梯度平板法)

Materials and Reagents

  1. Materials
    1. SterilinTM Standard 90 mm Petri Dishes (Thermo Fisher Scientific, catalog number: 101/IRR )
    2. Microtest plate 96-well, F (SARSTEDT AG & Co, catalog number: 82.1581.501 )
    3. 1.5 ml Eppendorf tubes
    4. Square petri dishes (120 mm x 120 mm) with grid (Greiner Bio-One GmbH, catalog number: 688102 )
    5. Capillaries (Sigma-Aldrich, Drummond Microcaps®, catalog number: P1424 )
    6. Bulb for Pasteur pipette
    7. Erlenmeyer flasks 100 ml

  2. Strains
    1. P. aeruginosa PAO1 (Stover et al., 2000)
    2. E. coli HD49 (Reyes-Darias et al., 2015a), chemoreceptor free strain E. coli UU1250 (Ames et al., 2002) harboring a plasmid encoding a chimeric receptor comprising the LBD of the PctC chemoreceptor of P. aeruginosa PAO1 (Taguchi et al., 1997; Rico-Jimenez et al., 2013) and the MA domain of the E. coli Tar receptor.

  3. Reagents
    1. HEPES sodium salt (Sigma-Aldrich, catalog number: H7006 )
    2. Potassium phosphate dibasic (HK2PO4) (Sigma-Aldrich, catalog number: P3786 )
    3. Sodium salicylate (Sigma-Aldrich, catalog number: S3007 )
    4. Chloramphenicol (Sigma-Aldrich, catalog number: C-0378 )
    5. Potassium phosphate monobasic (H2KPO4) (Sigma-Aldrich, catalog number: 60220 )
    6. Ammonium sulfate [(NH4)2SO4] (Merck Millipore Corporation, catalog number: 1.01217 )
    7. Sodium citrate tribasic dihydrate (Sigma-Aldrich, catalog number: C7254 )
    8. Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: 63140 )
    9. Thiamine hydrochloride (Thiamine HCl) (Sigma-Aldrich, catalog number: 47858 )
    10. Glycerol (Scharlab, S.L., catalog number: GL0027005P )
    11. L-threonine (Sigma-Aldrich, Fluka, catalog number: 89179 )
    12. L-methionine (Sigma-Aldrich, catalog number: M9625 )
    13. L-leucine (Merck Millipore Corporation, catalog number: 5360 )
      Note: Currently, it is “Merck Millipore Corporation, catalog number: 105360 .
    14. L-histidine (Sigma-Aldrich, catalog number: 153688 )
    15. γ-Aminobutyric acid (GABA) (Sigma-Aldrich, catalog number: A2129 )
    16. Bacto-Agar (BD, DifcoTM, catalog number: 281230 )
    17. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
    18. Tryptone Broth (TB) medium (see Recipes)
    19. 0.9% NaCl solution (see Recipes)
    20. LB medium (see Recipes)
    21. 5x minimal A salts (see Recipes)
    22. 5 mg/ml Aminoacid-mix (see Recipes)
    23. Minimal A gradient plate medium (see Recipes)

Equipment

  1. Spectrophotometer (Perkin Elmer, model: uv/vis lambda 20 )
  2. Incubators (30 °C and 37 °C) (Thermo Fisher Scientific, Heraeus, model: B6060 incubator)
  3. Orbital shaker incubator SH maxi (Controltécnica Instruments)
  4. Centrifuge Allegra X-22R (Beckman Coulter)
  5. pH meter GLP22 (HACH LANGE SPAIN, Crison)
  6. Bunsen burner
  7. Two pairs of tweezers
  8. Bulb dispenser (Drummond Scientific Company, catalog number: 1-000-9000 )

Procedure

In the first part of this publication we will describe the quantitative capillary assay using as an example the chemotaxis towards g-aminobutyrate (GABA). These experiments have shown that P. aeruginosa PAO1 shows GABA chemotaxis and that this response is mediated by the PctC chemoreceptor (Reyes-Darias et al., 2015a). To characterize in more detail the PctC chemoreceptor, we have produced a chimeric receptor (Figure 1), which is reported in further detail (Reyes-Darias et al., 2015a). There is now a significant body of information showing that different receptor chimera constructs are functional (Feng et al., 1997; Krikos et al., 1985; Kristich et al., 2003; Repik et al., 2000; Reyes-Darias et al., 2015a; Reyes-Darias et al., 2015b; Weerasuriya et al., 1998).
In the second part of this publication we will describe the gradient plate chemotaxis assay on the example of the chemoreceptor free strain E. coli UU1250 into which a construct encoding the PctC-Tar chimera had been introduced. This approach is a convenient means to determine the chemoeffector profile by in vivo experimentation.

Figure 1. Construction of the PctC-Tar chimeric receptor. Tm: Transmembrane region; LBD: ligand binding domain; The HAMP linker domain (present in Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases) is an approximately 50-amino acid alpha-helical region; MA: Methylaccepting domain; Modified version of Figure taken from (Reyes-Darias et al., 2015a).

Part I. Quantitative capillary chemotaxis assay

The principle of this assay consists of immersing chemoeffector filled capillaries into a bacterial suspension. In the case of chemotaxis, cells will preferentially swim into the capillary whereas in the case of chemorepellation, fewer cells will swim into the capillary as compared to the buffer filled control capillary. Capillaries are then emptied and the number of colony forming units is determined. We detail here a modified version of the original capillary assay developed by Adler (1973).

  1. P. aeruginosa PAO1 is grown overnight (12-18 h) in LB medium (pH=7.4) on a rotator shaker at 200 rpm and 37 °C.
  2. Subsequently 0.1-0.2 ml overnight culture is transferred to 20 ml of fresh LB medium (pH=7.4) in 100 ml Erlenmeyer flasks. The initial OD600 should be between 0.05-0.07.
  3. The culture is grown to early stationary phase (OD600 0.25-0.35) at 37 °C and 200 rpm.
  4. The cells then have to be changed to an appropriate medium for mobility and chemotaxis. Wash twice 4 ml of the culture with four ml of chemotaxis buffer (HEPES, pH=7.0) by centrifuging at 1,667 x g (4 °C) for 5 min, followed by resuspension of cell pellet to a density of OD600= 0.04-0.05 in 4-8 ml of chemotaxis buffer. Harsh cell treatment may result in the loss of the flagella.
  5. Subsequently, 230 μl aliquots of bacterial suspension are placed into the wells of a 96-well plate.
  6. Capillaries are heat-sealed at one end over the flame of a burner (Figure 2A). The open end is then inserted into the chemoattractant solution (Figure 2B), of which the pH had been adjusted to that measured for the bacterial suspension (pH=7.0). As negative controls, capillaries are filled with chemotaxis buffer and for positive controls, capillaries are filled with a known, strong chemoattractant such, in the case of Pseudomonas strains, with 0.1 % of casamino acids. The closed end of the capillary is inserted into the rubber adaptor, which is then placed onto the wells in a way that the open end of the capillary is immersed into the bacterial suspension (Figure 2C).
  7. After incubation for 30 min, the capillaries are removed from the plate and the section of the capillary that was in contact with bacteria is rinsed with water. The sealed end of the capillary is broken and its contents emptied into a microfuge tube containing 1 ml of 0.9% (w/v) NaCl solution (Figure 2D) using a bulb dispenser.


    Figure 2. Experimental setup of capillary assays.
    A. One end of the capillary is sealed on open fire. B. Sealed and warmed capillary is inserted into the chemoattractant solution for filling. C. The rubber adaptor with the capillary is placed onto the well, which submerges the open end of capillary into the bacterial suspension that had been placed previously into the well. D. The capillary content is emptied into an Eppendorf tube containing 0.9% (w/v) NaCl.

  1. After a short centrifugation (5-10 sec), serial 10-fold dilutions of the resulting cell suspension are prepared in 0.9% (w/v) NaCl. For cell counting, a modified version of the protocol described in (Hoben and Somasegaran, 1982) is used. Agar plates containing LB medium (pH=7.4) supplemented with 17.5 µg/ml chloramphenicol are divided into six sectors, and three individual 20 µl aliquots are placed onto a plate sector as shown in Figure 3.
  2. Plates are incubated at 37 °C for 20-24 h prior to cell counting.


    Figure 3. LB agar plates containing chloramphenicol used for cell counting in the quantitative capillary chemotaxis assay.
    P. aeruginosa PAO1 colonies from the buffer control (left) and from chemotaxis towards 1 mM GABA (right). Shown are 10-fold dilutions and further dilutions are necessary to precisely quantify colonies of the GABA chemotaxis experiment.

Part II. Gradient plate assays

In this assay aliquots of chemoeffectors are deposited on an agar plate. After incubation permitting chemoeffector gradient formation, aliquots of the bacterial suspension are placed at a defined distance from the deposited chemoeffector. After approx. 1 day, plates are inspected. In the case of chemoattraction, the bacterial halo is acentric towards the chemoeffector, in the case of chemorepellation the halo is acentric away from the chemoeffector.

  1. Square petri dishes (120 mm x 120 mm) with vents are filled with 80 ml of Minimal A gradient plate medium (pH=7) (see Note 1). Plates are cooled at room temperature for at least 3 h. Care must be taken when moving the plates since the agar is semi-solid and can be damaged easily.
  2. Along the vertical central line of the plate, 10 μl aliquots of a concentrated chemoeffector solution (10 mM GABA in the case shown, see Note 2), dissolved in sterile water, are placed at regular distances (Figure 4). Plates are incubated for 12-16 h at 4 °C for gradient formation.
  3. Bacteria, in the case shown E. coli HD49, are grown overnight in TB medium supplemented with 17.5 µg/ml chloramphenicol at 30 °C.
  4. 1 ml of the cell culture is then washed twice with 1 ml of 0.9 % (w/v) NaCl by consecutive centrifugation at 1,667 x g at 4 °C for 5 min and resuspension. The cell suspension is then diluted with 0.9% (w/v) NaCl to an OD600 of 0.4-0.6.
  5. Initially, 2 microliter aliquots of bacterial suspension are placed horizontally to each of the chemoattractant spots but with varying distances of 0.5-3.5 cm to the chemoeffector deposit (Figure 4). Plates are incubated at 30 °C for 16-30 h. The distance at which chemotaxis is seen best is then used for further analyses. Typically, cells can be deposited on either side of the attractant, which is a way to generate duplicate measurements or to assess taxis of different strains (wt and mutant strain) towards the same chemoeffector deposit.
  6. From these plates the magnitude of chemotaxis can be determined in a semi-quantitative manner by calculating the response index as described by (Pham and Parkinson, 2011). Briefly, the distances from the site of inoculation to the colony edges closest to (D1) and furthest from (D2) the chemoattractant source are determined to calculate the response index (RI) using RI = D1/(D1 + D2). RIs superior to 0.52 indicate chemotaxis, whereas RIs inferior to 0.48 represent chemorepellation. RI values between 0.48 and 0.52 indicate neutral behavior.


    Figure 4. The experimental set-up of the gradient plate assay

Representative data

Figure 5 shows results from quantitative capillary assays of P. aeruginosa PAO1, its mutant deficient in the PctC chemoreceptor and the mutant complemented with a plasmid harboring the mcpG gene towards GABA. Data were corrected with the number of cells that swam into buffer containing capillaries and are expressed as bacterial cells that migrate into the GABA containing capillary.

Figure 5. Quantitative capillary chemotaxis assays of wild type, mutant and complemented mutant strains of P. aeruginosa PAO1 to GABA. This figure was taken from Rico-Jimenez et al. (2013).

Figure 6 shows gradient plate assays towards GABA of E. coli strains containing either the Tar receptor or the PctC-Tar chimera as a sole chemoreceptor. In the case of Tar, the bacterial halo is circular and consequently an RI of 0.50 was determined indicating the absence of chemotaxis. In the PctC-Tar experiment, the halo is acentric towards deposited GABA and the derived RI of 0.88 indicates strong chemotaxis.

Figure 6. Gradient plate assay of E. coli expressing either Tar (top) or PctC-Tar (bottom) as the only chemoreceptor. Shown are duplicate samples. The response indices were 0.5 (± 0.05) for Tar and 0.88 (± 0.02) for PctC-Tar. Modified version of a figure is taken from Reyes-Darias et al. (2015a).

Notes

  1. The plate contained 1 μM sodium salicylate to induce expression of the chimeric receptors and 17.5 μg/ml chloramphenicol to select for plasmid maintenance.
  2. The optimal attractant concentration in the gradient plate assay varies. Initial experiments with different concentrations for chemoeffector deposit may be carried out (1 to 100 mM). Frequently, good responses are observed with 10 mM solutions and a distance of 2.5 cm between bacteria and the chemoeffector.
  3. Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25 °C) and analytical grade reagents.

Recipes

Note: Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25 °C) and analytical grade reagents.

  1. Tryptone Broth (TB) medium
    10 g tryptone
    5 g NaCl
    Add dH2O to 1,000 ml
    Autoclaved and stored at room temperature
  2. 0.9% NaCl solution
    9 g of NaCl
    Add dH2O to 1,000 ml
    Autoclaved and stored at room temperature
  3. Luria-Bertani (LB) medium
    5 g yeast extract
    10 g tryptone broth medium
    5 g NaCl
    Add dH2O to 1,000 ml
    Adjust pH to 7.4
    Autoclaved and stored at room temperature
  4. 5x minimal A salts
    26.25 g K2HPO4
    11.25 g KH2PO4
    2.5 g (NH4)2SO4
    1.25 g Na citrate
    Add dH2O to 500 ml
    Autoclaved and stored at room temperature
  5. 5 mg/ml Aminoacid-mix
    250 mg of L-threonine
    250 mg of L-histidine
    250 mg L-methionine
    250 mg L-leucine
    dH2O to 50 ml
    Filtered and stored at 4 °C
  6. Minimal A gradient plate medium
    1x minimal A salts
    0.2% (v/v) glycerol
    1 mM MgSO4.7H2O
    0.04 mg/ml aminoacid-mix
    0.1 mg/ml thiamine
    0.25% (w/v) agar
    dH2O to 100 ml
    Adjust pH to 7
    Gradient plates are allowed to solidify between 2-4 h at room temperature

Acknowledgments

This work was supported by FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grants P09-RNM-4509 and CVI-7335 to T. K.), the Spanish Ministry for Economy and Competitiveness (grant Bio2010-16937 to T. K.) and EMBO short term fellowship grant ASTF 479 -2012 to JA-R.

References

  1. Adler, J. (1973). A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J Gen Microbiol 74(1): 77-91.
  2. Ames, P., Studdert, C. A., Reiser, R. H. and Parkinson, J. S. (2002). Collaborative signaling by mixed chemoreceptor teams in Escherichia coli. Proc Natl Acad Sci U S A 99(10): 7060-7065.
  3. Feng, X., Baumgartner, J. W. and Hazelbauer, G. L. (1997). High- and low-abundance chemoreceptors in Escherichia coli: differential activities associated with closely related cytoplasmic domains. J Bacteriol 179(21): 6714-6720.
  4. Hoben, H. J. and Somasegaran, P. (1982). Comparison of the pour, spread, and drop plate methods for enumeration of rhizobium spp. in inoculants made from presterilized peat. Appl Environ Microbiol 44(5): 1246-1247.
  5. Krikos, A., Conley, M. P., Boyd, A., Berg, H. C. and Simon, M. I. (1985). Chimeric chemosensory transducers of Escherichia coli. Proc Natl Acad Sci U S A 82(5): 1326-1330.
  6. Kristich, C. J., Glekas, G. D. and Ordal, G. W. (2003). The conserved cytoplasmic module of the transmembrane chemoreceptor McpC mediates carbohydrate chemotaxis in Bacillus subtilis. Mol Microbiol 47(5): 1353-1366.
  7. Pham, H. T. and Parkinson, J. S. (2011). Phenol sensing by Escherichia coli chemoreceptors: a nonclassical mechanism. J Bacteriol 193(23): 6597-6604.
  8. Repik, A., Rebbapragada, A., Johnson, M. S., Haznedar, J. O., Zhulin, I. B. and Taylor, B. L. (2000). PAS domain residues involved in signal transduction by the Aer redox sensor of Escherichia coli. Mol Microbiol 36(4): 806-816.
  9. Reyes-Darias, J. A., Garcia, V., Rico-Jimenez, M., Corral-Lugo, A., Lesouhaitier, O., Juarez-Hernandez, D., Yang, Y., Bi, S., Feuilloley, M., Munoz-Rojas, J., Sourjik, V. and Krell, T. (2015a). Specific gamma-aminobutyrate chemotaxis in pseudomonads with different lifestyle. Mol Microbiol 97(3): 488-501.
  10. Reyes-Darias, J. A., Yang, Y., Sourjik, V. and Krell, T. (2015b). Correlation between signal input and output in PctA and PctB amino acid chemoreceptor of Pseudomonas aeruginosa. Mol Microbiol 96(3): 513-525.
  11. Rico-Jimenez, M., Munoz-Martinez, F., Garcia-Fontana, C., Fernandez, M., Morel, B., Ortega, A., Ramos, J. L. and Krell, T. (2013). Paralogous chemoreceptors mediate chemotaxis towards protein amino acids and the non-protein amino acid gamma-aminobutyrate (GABA). Mol Microbiol 88(6): 1230-1243.
  12. Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J., Lagrou, M., Garber, R. L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L. L., Coulter, S. N., Folger, K. R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G. K., Wu, Z., Paulsen, I. T., Reizer, J., Saier, M. H., Hancock, R. E., Lory, S. and Olson, M. V. (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406(6799): 959-964.
  13. Taguchi, K., Fukutomi, H., Kuroda, A., Kato, J. and Ohtake, H. (1997). Genetic identification of chemotactic transducers for amino acids in Pseudomonas aeruginosa. Microbiology 143 ( Pt 10): 3223-3229.
  14. Weerasuriya, S., Schneider, B. M. and Manson, M. D. (1998). Chimeric chemoreceptors in Escherichia coli: signaling properties of Tar-Tap and Tap-Tar hybrids. J Bacteriol 180(4): 914-920.

简介

细菌趋化性是基于运动性的反应,其将细胞运动偏向有益分子,称为引诱剂,并远离有害分子,也称为驱避剂。由于假单胞菌属的种属的特征在于代谢多样性,这些细菌已经对宽范围的不同化合物产生趋化行为。趋化反应的特异性由化学感受器确定,化学感受器处于信号级联的开始并且接受信号输入。典型的化学感受器的基本元件是负责感测环境刺激的周质配体结合结构域(LBD)和与细胞信号传导级联的其它组分相互作用的细胞溶质甲基接受(MA)结构域。大肠杆菌(大肠杆菌)是趋化性研究中的传统模型,具有5个良好表征的化学感受器。然而,基因组序列分析已经揭示许多其他细菌具有更多的化学感受器,其中一些具有部分重叠的信号谱。这种高数目的化学感受器通过分析单个化学感受器突变体使他们的研究变得复杂。我们已经寻求化学受体表征的替代策略,其对应于由所研究的化学受体的LBD和E的MA结构域组成的嵌合受体的产生。大肠杆菌受体(Tar)。然后将嵌合体引入E的无化学感受器的突变体。并且所得菌株的趋化性完全是由于该嵌合受体的作用。在本公开中,我们描述了使用定量毛细管和梯度板测定来研究假单胞菌趋化性以及E。含有嵌合受体的大肠杆菌菌株。

关键字:趋化性毛细管法, 化学受体, 受体嵌合体, 趋化性, 梯度平板法

材料和试剂

  1. 材料
    1. Sterilin TM 标准90mm培养皿(Thermo Fisher Scientific,目录号:101/IRR)
    2. 微测试板96孔,F(SARSTEDT AG& Co,目录号:82.1581.501)
    3. 1.5 ml Eppendorf管
    4. 带有格栅的方形培养皿(120mm×120mm)(Greiner Bio-One GmbH,目录号:688102)
    5. 毛细管(Sigma-Aldrich,Drummond Microcaps ,目录号:P1424)
    6. 巴斯德吸管灯泡
    7. 锥形瓶100ml
  2. 菌株
    1. p。铜绿假单胞菌PAO1(Stover等人,2000)
    2. E。大肠杆菌HD49(Reyes-Darias等人,2015a),无化学感受器菌株E。大肠杆菌UU1250(Amers等人,2002),其携带编码包含Pct的PctC化学感受器的LBD的嵌合受体的质粒。铜绿假单胞菌PAO1(Taguchi等人,1997; Rico-Jimenez等人,2013)和E的MA结构域。大肠杆菌 Tar受体
  3. 试剂
    1. HEPES钠盐(Sigma-Aldrich,目录号:H7006)
    2. 磷酸氢二钾(HK2 PO 4)(Sigma-Aldrich,目录号:P3786)
    3. 水杨酸钠(Sigma-Aldrich,目录号:S3007)
    4. 氯霉素(Sigma-Aldrich,目录号:C-0378)
    5. 磷酸二氢钾(H 2 KPO 4)(Sigma-Aldrich,目录号:60220)
    6. 硫酸铵[(NH 4)2 SO 4](Merck Millipore Corporation,目录号:1.01217)
    7. 柠檬酸三钠二水合物(Sigma-Aldrich,目录号:C7254)
    8. 硫酸镁七水合物(MgSO 4·7H 2 O 7H 2 O)(Sigma-Aldrich,目录号:63140)
    9. 盐酸硫胺素(盐酸硫胺素)(Sigma-Aldrich,目录号:47858)
    10. 甘油(Scharlab,S.L.,目录号:GL0027005P)
    11. L-苏氨酸(Sigma-Aldrich,Fluka,目录号:89179)
    12. L-甲硫氨酸(Sigma-Aldrich,目录号:M9625)
    13. L-亮氨酸(Merck Millipore Corporation,目录号:5360)
      注意:目前,它是"默克密理博公司,目录号: 105360 "。
    14. L-组氨酸(Sigma-Aldrich,目录号:153688)
    15. γ-氨基丁酸(GABA)(Sigma-Aldrich,目录号:A2129)
    16. Bacto-Agar(BD,Difco TM ,目录号:281230)
    17. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
    18. 胰蛋白胨肉汤(TB)培养基(见配方)
    19. 0.9%NaCl溶液(见配方)
    20. LB介质(见配方)
    21. 5x最小A盐(见配方)
    22. 5 mg/ml氨基酸混合物(见配方)
    23. 最小梯度板介质(见配方)

设备

  1. 分光光度计(Perkin Elmer,型号:uv /visλ20)
  2. 孵育器(30℃和37℃)(Thermo Fisher Scientific,Heraeus,型号:B6060培养箱)
  3. 轨道摇床孵化器SH maxi(ControltécnicaInstruments)
  4. 离心机Allegra X-22R(Beckman Coulter)
  5. pH计GLP22(HACH LANGE SPAIN,Crison)
  6. 本生灶
  7. 两对镊子
  8. 灯泡分配器(Drummond Scientific Company,目录号:1-000-9000)

程序

在本出版物的第一部分中,我们将描述定量毛细管测定,其中使用对 g span> -aminobutyrate(GABA)。这些实验已经显示出P.铜绿假单胞菌PAO1显示GABA趋化性,并且该反应由PctC化学感受器介导(Reyes-Darias等人,2015a)。为了更详细地表征PctC化学受体,我们产生了嵌合受体(图1),其进一步详细报道(Reyes-Darias等人,2015a)。现在有大量的信息显示不同的受体嵌合构建体是功能性的(Feng等人,1997; Krikos等人,1985; Kristich等人2003; Repik等人,2000; Reyes-Darias等人,2015a; Reyes-Darias等人,2003; ,2015b; Weerasuriya等人,1998)。
在该出版物的第二部分中,我们将描述对无化学感受器应变的实例的梯度板趋化性测定。其中导入了编码PctC-Tar嵌合体的构建体的大肠杆菌UU1250。这种方法是通过体内实验确定化学效应子谱的方便手段。

图1.PctC-Tar嵌合受体的构建。 Tm:跨膜区; LBD:配体结合结构域; HAMP接头结构域(存在于组氨酸激酶,腺苷酸环化酶,甲基接受蛋白和磷酸酶中)是约50个氨基酸的α-螺旋区; MA:甲基受体结构域;图的修改版本(Reyes-Darias等人,2015a)。

定量毛细管趋化性测定

该测定法的原理包括将化学感受器填充的毛细血管浸入细菌悬浮液中。在趋化性的情况下,细胞将优先游进毛细管,而在化学打印的情况下,与缓冲液填充的对照毛细管相比,更少的细胞将游入毛细管。然后清空毛细管,并确定菌落形成单位的数量。我们在这里详述由Adler(1973)开发的原始毛细管测定法的修改版本。

  1. p。铜绿假单胞菌PAO1在旋转振荡器上在200rpm和37℃下在LB培养基(pH = 7.4)中生长过夜(12-18小时)。
  2. 随后将0.1-0.2ml过夜培养物转移到100ml的Erlenmeyer烧瓶中的20ml新鲜LB培养基(pH = 7.4)中。初始OD <600> 应在0.05-0.07之间
  3. 在37℃和200rpm下培养物生长至早期的固定相(OD <600)0.25-0.35。
  4. 然后必须将细胞改变为用于迁移和趋化性的适当培养基。通过在1,667×g(4℃)离心5分钟用4ml趋化性缓冲液(HEPES,pH = 7.0)洗涤4ml培养物两次,然后将细胞沉淀重悬浮至密度OD 600 = 0.04-0.05在4-8ml趋化缓冲液中。严酷的细胞治疗可能导致鞭毛的损失。
  5. 随后,将230μl等分的细菌悬浮液置于96孔板的孔中。
  6. 毛细管在一端热密封在燃烧器的火焰上(图2A)。然后将开放端插入化学引诱物溶液(图2B),其pH已经调节至对于细菌悬浮液(pH = 7.0)测量的。作为阴性对照,用趋化缓冲液填充毛细血管,对于阳性对照,用已知的强化学引诱物填充毛细血管,在假单胞菌菌株的情况下,用0.1%酪蛋白氨基酸填充。将毛细管的封闭端插入橡胶适配器中,然后将其放置在孔上,使得毛细管的开口端浸入细菌悬浮液中(图2C)。
  7. 孵育30分钟后,从平板上除去毛细管,并用水冲洗与细菌接触的毛细管部分。毛细管的密封端破碎,并使用球形分配器将其内容物倒入含有1ml 0.9%(w/v)NaCl溶液的微量离心管中(图2D)。


    图2.毛细管测定的实验设置 A.毛细管的一端在明火密封。 B.将密封并加温的毛细管插入化学引诱物溶液中用于填充。 C.将具有毛细管的橡胶适配器放置在孔上,其将毛细管的开口端浸入先前已置于孔中的细菌悬浮液中。 D.将毛细管内容物倒入含有0.9%(w/v)NaCl的Eppendorf管中
  1. 在短暂离心(5-10秒)后,在0.9%(w/v)NaCl中制备所得细胞悬浮液的系列10倍稀释液。对于细胞计数,使用(Hoben和Somasegaran,1982)中描述的方案的修改版本。将含有补充有17.5μg/ml氯霉素的LB培养基(pH = 7.4)的琼脂平板分成六个扇区,将三个单独的20μl等分试样放置在如图3所示的平板扇区上。
  2. 将板在37℃下孵育20-24小时,然后进行细胞计数

    图3.含有氯霉素的LB琼脂平板,用于定量毛细管趋化性测定中的细胞计数。铜绿色PAO1菌落从缓冲液对照(左)和趋化性趋向于1mM GABA(右)。显示10倍稀释,并且进一步稀释是精确定量GABA趋化性实验的菌落所必需的。

第二部分。梯度板测定

在该测定中,将化学效应子的等分试样沉积在琼脂板上。在温育允许化学效应物梯度形成之后,将细菌悬浮液的等分试样放置在距沉积的化学效应器的限定距离处。约后。 1天,检查板。在化学吸引的情况下,细菌卤素朝向化学效应器是非中心的,在化学打印的情况下,卤素远离化学效应子。

  1. 用80ml Minimal A梯度板培养基(pH = 7)填充带有通气孔的方形培养皿(120mm×120mm)(参见注释1)。将板在室温下冷却至少3小时。移动板时必须小心,因为琼脂是半固体的并且容易损坏
  2. 沿着板的垂直中心线,将10μl等分试样的溶解于无菌水中的浓缩化学作用物溶液(在所示的情况下,10mM GABA,参见注释2)以规则的距离放置(图4)。将平板在4℃孵育12-16小时以形成梯度
  3. 细菌,在所示的情况下。大肠杆菌HD49在补充有17.5μg/ml氯霉素的TB培养基中于30℃生长过夜。
  4. 然后通过在4℃下以1,667×g连续离心5分钟并重悬浮,用1ml的0.9%(w/v)NaCl洗涤1ml的细胞培养物两次。然后将细胞悬浮液用0.9%(w/v)NaCl稀释至OD 600为0.4-0.6。
  5. 最初,将2微升等分的细菌悬浮液水平放置到每个化学引诱剂点,但与化学引诱物沉积物的距离为0.5-3.5cm(图4)。将平板在30℃孵育16-30小时。然后将趋化性被最佳地看到的距离用于进一步分析。通常,细胞可以沉积在引诱剂的任一侧,这是产生重复测量或评估针对相同化学效应物沉积物的不同菌株(wt和突变株)的出血的方式。
  6. 从这些平板中,通过计算如(Pham和Parkinson,2011)所述的反应指数,可以半定量的方式测定趋化性的大小。简言之,使用RI = D1 /(D1 + D2),确定从接种位点到最接近(D1)和距离(D2)化学引诱源最远的菌落边缘的距离,以计算响应指数(RI)。 RI高于0.52表示趋化性,而低于0.48的RI表示化学打印。 0.48和0.52之间的RI值表示中性行为。


    图4.梯度板测定的实验装置

代表数据

图5显示了来自P的定量毛细管测定的结果。铜绿假单胞菌PAO1,其突变体缺乏在pctC 化学感受器中,突变体用携带针对GABA的mcpG 基因的质粒补充。数据用游泳到含有毛细管的缓冲液中的细胞数量校正,并表示为迁移到含有GABA的毛细管中的细菌细胞。

图5.野生型,突变体和互补的突变株p的定量毛细管趋化性测定。铜绿假单胞菌 PAO1到GABA。这个数字取自Rico-Jimenez等人(2013)。

图6显示了针对E的GABA的梯度板测定。含有Tar受体或PctC-Tar嵌合体作为唯一化学感受器的大肠杆菌菌株。在Tar??的情况下,细菌晕是圆形的,因此确定RI为0.50,表明不存在趋化性。在PctC-Tar实验中,光晕对于沉积的GABA是偏心的,并且衍生的RI为0.88表示强的趋化性。

图6. 的梯度板测定 E。大肠杆菌 表示Tar(顶部)或PctC-Tar(下)作为化学感受器。 显示重复样本。 Tar的响应指数为0.5(±0.05),PctC-Tar的响应指数为0.88(±0.02)。一个数字的修改版本取自Reyes-Darias等人。(2015a)。

笔记

  1. 该板含有1μM水杨酸钠诱导嵌合受体的表达和17.5μg/ml氯霉素以选择质粒维持。
  2. 梯度板测定中的最佳引诱剂浓度变化。可以进行具有不同浓度的化学效应沉积物的初始实验(1至100mM)。通常,用10mM溶液观察到良好的反应,并且细菌和化学作用物之间的距离为2.5cm
  3. 使用超纯水(通过纯化去离子水以在25℃下获得18MΩcm的灵敏度)和分析级试剂制备所有溶液。

食谱

注意:使用超纯水(通过纯化去离子水以在25℃下获得18MΩcm的灵敏度)和分析级试剂制备所有溶液。

  1. 胰蛋白胨肉汤(TB)培养基
    10g胰蛋白胨
    5克NaCl
    将dH <2> O添加至1,000 ml
    高压灭菌并在室温下贮存
  2. 0.9%NaCl溶液
    9克NaCl 将dH <2> O添加至1,000 ml
    高压灭菌并在室温下贮存
  3. Luria-Bertani(LB)培养基
    5g酵母提取物
    10克胰蛋白胨肉汤培养基
    5克NaCl
    将dH <2> O添加至1,000 ml
    将pH调节至7.4
    高压灭菌并在室温下贮存
  4. 5x最小A盐
    26.25克K sub 2 HPO 4
    11.25g KH 2 PO 4 sub/
    2.5g(NH 4)2 SO 4>/
    1.25克柠檬酸钠 将dH <2> O添加到500ml
    高压灭菌并在室温下贮存
  5. 5mg/ml氨基酸混合物
    250毫克L-苏氨酸 250mg L-组氨酸 250毫克L-甲硫氨酸 250毫克L-亮氨酸 dH 2 O至50ml ml 过滤并储存在4℃下
  6. 最小梯度平板培养基
    1×最小A盐
    0.2%(v/v)甘油 1mM MgSO 4。7H 2 O 3 0.04mg/ml氨基酸混合物
    0.1mg/ml硫胺素 0.25%(w/v)琼脂 dH 2 O至100毫升
    将pH调节至7
    将梯度板在室温下固化2-4小时

致谢

这项工作得到了FEDER基金和Fondo社会Europeo通过拨款从安达卢西亚Junta(拨款P09-RNM-4509和CVI-7335到TK),西班牙经济和竞争力部(授予Bio2010-16937 TK)和EMBO短期奖学金资助ASTF 479 -2012 to JA-R。

参考文献

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  2. Ames,P.,Studdert,C.A.,Reiser,R.H。和Parkinson,J.S。(2002)。 混合化学感受器团队在大肠杆菌中的协同信号。。 Proc Natl Acad Sci USA 99(10):7060-7065。
  3. Feng,X.,Baumgartner,J.W.and Hazelbauer,G.L。(1997)。 大肠杆菌中的高丰度和低丰度化学感受器:差异活动与密切相关的细胞质结构域。细菌细胞179(21):6714-6720。
  4. Hoben,H.J。和Somasegaran,P。(1982)。 倾倒,展开和滴板方法的比较根据根瘤菌的计数。在由预灭菌的泥炭制成的接种物中。 Appl Environ Microbiol 44(5):1246-1247。
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  6. Kristich,C.J.,Glekas,G.D.and Ordal,G.W。(2003)。 跨膜化学感受器McpC的保守细胞质模块在枯草芽孢杆菌中介导碳水化合物趋化作用。 Mol Microbiol 47(5):1353-1366。
  7. Pham,HT和Parkinson,JS(2011)。由大肠杆菌感染的苯酚em> chemoreceptors:a nonclassical mechanism。 J Bacteriol 193(23):6597-6604。
  8. Repik,A.,Rebbapragada,A.,Johnson,M.S.,Haznedar,J.O.,Zhulin,I.B.and Taylor,B.L。(2000)。 参与大肠杆菌Aer氧化还原传感器信号转导的PAS结构域残基。 Mol Microbiol 36(4):806-816。
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引用:Reyes-Darias, J. A., García, V., Rico-Jiménez, M., Corral-Lugo, A. and Krell, T. (2016). Identification and Characterization of Bacterial Chemoreceptors Using Quantitative Capillary and Gradient Plate Chemotaxis Assays. Bio-protocol 6(8): e1789. DOI: 10.21769/BioProtoc.1789.
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