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Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC
HPLC法估测不同黄单胞菌菌株培养的无细胞上清液中黄单胞菌铁载体   

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Abstract

Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.

Keywords: Xanthomonas siderophores(黄单胞菌属铁载体), Xanthoferrin(黄单胞菌铁蛋白), Bacterial siderophore estimation(细菌铁载体估测), HPLC(HPLC), Amberlite XAD-16 resin columns(Amberlite XAD-16树脂柱)

Background

The Xanthomonas group of bacterial phytopathogens possess an xss (Xanthomonas siderophore synthesis) operon, which is required to produce xanthoferrin (an α-hydroxy carboxylate-type siderophore; similar to vibrioferrin) and to encode an outer membrane receptor involved in the siderophore-mediated iron uptake (Pandey and Sonti, 2010; Pandey et al., 2016a and 2016b). Siderophores are small iron-chelating compounds secreted by bacteria to utilize the insoluble form of iron (Neilands, 1995). For the past three decades, CAS (Chrome azurol S)-agar plate assay has been mostly employed to measure bacterial siderophores by monitoring the halo formation around bacterial colonies (Schwyn and Neilands, 1987; Pandey and Sonti, 2010). However, the above assay cannot reliably be used to quantify siderophores from bacteria which also secrete organic acids (e.g., oxalic acid, citric acid, and isocitric acids) along with siderophores, as the organic acids are capable of chelating iron from CAS dye to compromise the accuracy to quantify siderophore (Rai et al., 2015). Since the above mentioned assay only gives the idea about the bacterial siderophore production qualitatively, hence HPLC-mediated siderophore quantification is a better method than the CAS-agar plate assay for bacteria that produce both siderophores along with organic acids; such as Xanthomonas species (Rai et al., 2015; Pandey et al., 2016a and 2016b).

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog numbers: TF-300-R-S , TF-200-R-S , and TF-1000-R-S )
  2. Culture tubes (Borosil, catalog number: 9800U06 )
  3. Petri dishes (Tarson Products, catalog number: 460140-140MM )
  4. Stericup® filter units with 0.22 μm porosity (EMD Millipore, catalog number: SCGPU02RE )
  5. Centrifuge tubes: 500 ml, 50 ml, 15 ml, 2 ml, and 1.5 ml (Tarson Products, Kolkata, India)
  6. Cork borer, 8 ± 0.2 mm (HiMedia Laboratories, catalog number: LA737 )
  7. Syringe driven filter unit with 0.22 μm porosity (Millex®-GS) (EMD Millipore, catalog number: SLGS033SS )
  8. Xanthomonas campestris pv. campestris 8004, X. oryzae pv. oryzicola BXOR1, X. oryzae pv. oryzae (Indian isolate) and ∆xssA mutant in Xanthomonas siderophore synthesis gene A (Lab collections)
  9. Rifampicin
  10. 2,2’-dipyridyl (DP) (Sigma-Aldrich, catalog number: 14453 )
    Note: This product has been discontinued.
  11. Amberlite XAD16N, 20-60 mesh (Sigma-Aldrich, catalog number: XAD16-1KG )
  12. Methanol (Thermo Fisher Scientific, catalog number: Q32407 )
  13. Standard vibrioferrin (Fujita et al., 2011; see Notes 2 and 3)
  14. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: T6508-25ML )
  15. Acetonitrile (CH3CN) (Sigma-Aldrich, catalog number: 34888-1L )
    Note: This product has been discontinued.
  16. Peptone (HiMedia Laboratories, catalog number: CR001 )
  17. Sucrose (HiMedia Laboratories, catalog number: GRM601-500G )
  18. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  19. Chrome Azural S (CAS) (Merck, catalog number: 1.02477.0025 )
  20. Ferric chloride (FeCl3) (Standard Reagents, catalog number: SRCF005C )
  21. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: 29507 )
  22. Hexadecyltrimethylammonium bromide (HDTMA) (Sigma-Aldrich, catalog number: H6269-250G )
  23. BactoTM agar (BD, BactoTM, catalog number: 214010 )
  24. BactoTM peptone (BD, BactoTM, catalog number: 211677 )
  25. Agar (HiMedia Laboratories, catalog number: RM026-500G )
  26. Peptone-sucrose (PS) medium (Tsuchia et al., 1982; see Recipes)
  27. CAS solution (Schwyn and Neilands, 1987; see Recipes)
  28. Peptone-sucrose-agar (PSA) medium (see Recipes)
  29. PSA-CAS-DP plate (see Recipes)

Equipment

  1. Conical flasks: 500 ml (Vensil Glass Works, catalog number: 1161 )
  2. Conical flasks: 2,000 ml (Borosil, catalog number: 4980030 )
  3. Micro-pipettes (Eppendorf, catalog number: EPPR4396 )
  4. Shaking incubator (Eppendorf, New BrunswickTM, model: Innova® 43 )
  5. SORVALL RC-5B PLUS Superspeed centrifuge (Thermo Fisher Scientific, Waltham, MA, USA)
  6. pH meter (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EC-PH510/11S )
  7. Agilent 1100 series HPLC system (Agilent Technologies, model: Agilent 1100 Series )
  8. Agilent C-18 (4.6 mm x 250 mm x 5 μm) column (Agilent Technologies, Santa Clara, CA, USA)
  9. Burette (BOROSIL, catalog number: 2123016 ), burette support stand and clamps
  10. Vacuum concentrator plus (Eppendorf, model: Concentrator Plus , catalog number: 5305000304)
  11. Millipore vacuum, pressure pump (EMD Millipore, catalog number: XI04 220 50 )
  12. Autoclave

Software

  1. ChemStation Software; Rev. A.10.02 [1757] (Agilent, Santa Clara, CA, USA)
  2. Microsoft Office Excel 2013 (Microsoft Corporation, Redmond, USA)

Procedure

  1. Preparation of the cell-free culture supernatant
    1. Streak the bacterial strain from its glycerol stock on a PSA plate containing rifampicin (50 μg/ml) and incubate in a static incubator at 28 °C for 2-3 days. To start a primary culture, transfer a single colony from the above mentioned plate to an autoclaved culture tube containing 3 ml fresh PS medium (see Recipes) with rifampicin (50 μg/ml). The primary culture of Xanthomonas can be grown to late-exponential growth (OD600 ~1.0) phase at 28 °C and 200 rpm in shaking incubator for 16-20 h.
    2. Start a secondary culture by using 0.2% inoculum (2 ml) from the above primary culture into fresh PS medium (1 L) supplemented with the appropriate amount of 2,2’-dipyridyl (150 μM for X. campestris pv. campestris str. 8004, 100 μM for X. oryzae pv. oryzae, and 50 μM for X. oryzae pv. oryzicola). Incubate the secondary culture in a 28 °C incubator with shaking at 200 rpm for 48 h.
    3. Centrifuge the bacterial cultures at 17,000 x g for 1 h at 4 °C to pellet cells and exopolysaccharides.
    4. To remove the remaining cells from the supernatant, filter the supernatant with Stericups (pore size 0.22 μm) and a vacuum pressure pump.
    5. Adjust pH to 2.0 with concentrated HCl (6 N) to reduce the solubility of siderophore in water (Rey et al., 1996).

  2. Column chromatography using XAD-16 resin columns
    1. Prepare a 2.4 x 30 cm column by packing 220 g of Amberlite XAD-16 polymeric resin.
      Note: Prepare the Amberlite resin by soaking in water overnight on the day before the column is needed.
    2. Wash the packed column with two-times the bed-volume (440 ml) of methanol and equilibrate the column with three-times the bed-volume (660 ml) of sterile MilliQ water.
    3. Pass the cell-free bacterial supernatant (Procedure A; step A5) through the equilibrated XAD-16 resin column (see Note 7).
    4. Elute with 200 ml methanol and collect approximately 100 fractions (~2 ml each) in 2.0 ml centrifuge tubes (see Note 7; Figure 1).


      Figure 1. Xad-16 resin column (prepared in the burette) with burette support equipment for the collection of different fractions of the elute for xanthoferrin siderophores

  3. CAS-Plate assay
    1. Prepare a PSA-CAS-DP plate (see Recipes).
    2. Punch several holes in the plate with a sterile cork borer keeping approximately 2.5 cm distance between the holes.
    3. Put approximately 250 μl of each fraction (according to elution order, Procedure B; step B4) into separate holes, placed upright with the lid on top, and incubate at 28 °C in a static incubator.
    4. Observe the activity of the fractions after 24 h.
    5. The characteristic, yellow-colored haloes (haloes 13-24) indicate the presence of siderophores while blue- and red-colored haloes (haloes 1-12) indicate the presence of organic acids (Figure 2).


      Figure 2. A PSA-CAS-DP plate with multiple holes representing different colored haloes formation after 24 h incubation with different fractions of the eluate (separated by XAD-16 resin column) for siderophore isolation from X. oryzae pv. oryzicola. Blue- and red-colored haloes (haloes 1-12) indicate the fractions with organic acids while yellow-colored haloes (haloes 13-24) indicate the fractions with xanthoferrin siderophores.
      Notes:
      1. We have not taken the 21st-24th fractions of the siderophore elutes for HPLC run. Because we have already observed there was negligible amount of siderophores present in those fractions in comparison to the presence of other impurities which create more noise during the HPLC run. Addition of those fragments for the HPLC run along with the active fragments (13th-20th) would have increased the impurity level to more extent without significantly increasing the total amounts of the siderophores in the sample. Note that Student’s t-test (paired) was used to analyze extent of contribution for those fractions towards the impurity levels and siderophore amounts in the main active fractions (13th-20th) for HPLC run.
      2. In order to minimize the extent of interference (after heat and trial methods) due to other unwanted ingredients present in the fractions corresponding to the halos 21-24, only the active fractions of the siderophores corresponding to the haloes 13-20 should be collected from their respective remaining main stocks (~1.75 ml each) for HPLC quantification.

  4. HPLC quantification 
    1. Pool all the active fractions (corresponding to haloes 13-20) from Procedure B; step B4 into a 15 ml centrifuge tube that display characteristic yellow haloes on the PSA-CAS-DP plate (Procedure C; step C5).
    2. To remove the solvent, dry the sample completely using a vacuum concentrator (at 30 °C in alcohol mode).
    3. Reconstitute in 1 ml methanol and filter with a syringe driven filter unit with 0.22 μm pore size.
    4. Inject the recommended volume of filtered sample for each strain (25 μl for X. campestris pv. campestris, and 10 μl for X. oryzae pv. oryzicola and X. oryzae pv. oryzae) into the Agilent 1100 series HPLC system (Figures 3A-3B).
    5. Achieve the separation with Agilent C-18 column (4.6 mm x 250 mm x 5 μm) using gradient: A = H2O/0.1% TFA, B = CH3CN/0.1% TFA; 0-30% B in 10 min, 30-45% B in 15 min, 45-0% B in 20 min at a flow rate of 1 ml/min.
    6. Make the standard curve, draw trendline, and get equation (using Microsoft Excel) from either known amounts of vibrioferrin (Fujita et al., 2011) or xanthoferrin versus their respective peak areas obtained by HPLC chromatograms (see Notes 1, 2 and 3).
    7. Calculate the amount of xanthoferrin siderophores (present in the unknown sample) from the formula obtained either by standard vibrioferrin or xanthoferrin.


      Figure 3. HPLC program and instrument. A. HPLC program in ChemStation Software; Rev. A.10.02 [1757]. B. A part of HPLC machine indicating samples orientation and automatic injection needle. 

Data analysis

Load HPLC signals of sample (Procedure D; step D5) and blank (empty buffer elute from the XAD-16 resin column) to the offline mode of ChemStation software. Subtract the blank signal from sample signal and then auto-integrate to automatic identification of peaks. Record the RT (Retention Time) values and peak areas generated in the dialogue box. The pure xanthoferrin signals can be observed while monitoring the missing peak in the HPLC chromatogram of isolated siderophores from ∆xssA mutant of Xanthomonas spp. in comparison to their respective wild-type. From the active fractions of the xanthoferrin, the purest fraction can be isolated, vacuum-dried, weighed and utilized as ‘standard xanthoferrin’ for plotting the standard curve by taking different known amounts for HPLC runs. Draw the standard curve of xanthoferrin by plotting the ‘amounts vs. average peak areas’ obtained after respective HPLC runs (Figure 4). Determine the amount of unknown sample xanthoferrin siderophores using the standard curve. Validate the significance level in the difference, if any exists between the data obtained for two different sets, using a paired Student’s t-test.
Note: For one set of experiments, take at least three independent biological replicates.


Figure 4. Xanthoferrin standard curve; displaying the equation for depicting the amount of siderophores used in the HPLC run for getting respective peak areas

Notes

  1. xssA mutant can be used as negative control. Xanthoferrin peak will be absent or drastically reduced in the ∆xssA mutant strain. Corresponding to the missing peak of HPLC chromatogram for the siderophores isolated from ∆xssA mutant (Pandey et al., 2016a), pure xanthoferrin can be collected from a wild-type strain and used as a standard.
  2. Although the mass of siderophores can be determined directly by weighing dried xanthoferrin, it is preferable to create a standard curve for a large number of samples (> 5).
  3. Since we detected iron (III)-vibrioferrin complexes at 300 nm (retention time ~11 min), the isolated xanthoferrin siderophores are expected to present mostly as iron-siderophore complexes. Therefore it is preferable to prepare the standard curve using the iron-siderophore complex (1:1 ratio).
  4. Bacterial inoculation and CAS-plate assay should be performed in aseptic conditions.
  5. Since long term storage and frequent freeze-thaw cycles may cause degradation of xanthoferrin siderophores, it can be stored at 4 °C for a maximum period of 1 week.
  6. The concentration of 2,2’-dipyridyl can vary slightly (up to ± 25 μM) depending on the availability of iron as impurities in media components.
  7. Column preparation and chromatography should be carried out at room temperature. Chromatography steps (washing, equilibration and elution) should be performed by gravity flow.

Recipes

  1. Peptone-sucrose (PS) medium
    1% peptone (w/v)
    1% sucrose (w/v)
    Dissolve above reagents in MilliQ water
    Adjust pH to 7.4 with 3 N sodium hydroxide and autoclave at 121 °C and 15 psi for 20 min
  2. CAS solution
    1. 0.06 g Chrome Azurol S dye in 50 ml MilliQ water
    2. Fe(III) solution: 10 ml
      1 mM FeCl3
      10 mM HCl
    3. 0.072 g HDTMA in 40 ml MilliQ water
    4. Make all three solutions separately, then mix together and autoclave at 121 °C and 15 psi for 20 min to sterilize
  3. Peptone-sucrose-agar (PSA) medium
    1% BactoTM peptone (w/v)
    1% sucrose (w/v)
    Dissolve above reagents in MilliQ water adjust pH to 7.4 with 3 N sodium hydroxide, add 1.4% BactoTM agar (w/v; final), and autoclave at 121 °C and 15 psi for 20 min
  4. PSA-CAS-DP plate (for 140 mm plate)
    60 ml melted PSA
    2,2’-dipyridyl (75 μM for X. campestris pv. campestris and 50 μM for X. oryzae pv. oryzae and X. oryzae pv. oryzicola)
    7.2 ml CAS solution

Acknowledgments

We acknowledge Dr. Masaki J. Fujita (Hokkaido University, Japan) for providing the pure vibrioferrin for this study. We extend the acknowledgement to a previous study from our lab (Rai, 2015), from where we have modified the siderophore estimation protocol for Xcc. This work was supported by funding to SC from DBT-India, CSIR-HRDG-India, DST-SERB-India and CDFD core funding. SSP was recipient of JRF and SRF from CSIR-India. PS and BS were recipients of JRF and SRF from UGC-India. RKV was recipient of JRF and SRF from DBT-India.

References

  1. Fujita, M. J., Kimura, N., Sakai, A., Ichikawa, Y., Hanyu, T. and Otsuka, M. (2011). Cloning and heterologous expression of the vibrioferrin biosynthetic gene cluster from a marine metagenomic library. Biosci Biotechnol Biochem 75(12): 2283-2287.
  2. Neilands, J. B. (1995). Siderophores: structure and function of microbial iron transport compounds. J Biol Chem 270(45): 26723-26726.
  3. Pandey, A. and Sonti, R. V. (2010). Role of the FeoB protein and siderophore in promoting virulence of Xanthomonas oryzae pv. oryzae on rice. J Bacteriol 192(12): 3187-3203.
  4. Pandey, S. S., Patnana, P. K., Lomada, S. K., Tomar, A. and Chatterjee, S. (2016a). Co-regulation of iron metabolism and virulence associated functions by iron and XibR, a novel iron binding transcription factor, in the plant pathogen Xanthomonas. PLoS Pathog 12(11): e1006019.
  5. Pandey, S. S., Patnana, P. K., Rai, R. and Chatterjee, S. (2016b). Xanthoferrin, the alpha-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage. Mol Plant Pathol.
  6. Rai, R., Javvadi, S. and Chatterjee, S. (2015). Cell-cell signaling promotes ferric iron uptake in Xanthomonas oryzae pv. oryzicola that contribute to its virulence and growth inside rice. Mol Microbiol 96(4): 708-727.
  7. Rey, F., Ferreira, M. A., Facal, P. and Machado, A. A. S. C. (1996). Effect of concentration, pH, and ionic strength on the viscosity of solutions of a soil fulvic acid. Can J Chem 74: 295-299.
  8. Schwyn, B. and Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160(1): 47-56.
  9. Tsuchia, K., Mew, T. W. and Wakimoto, S. (1982). Bacteriological and pathological charecteristics of wild types and induced mutants of Xanthomonas campestris pv. oryzae. Phytopathology 72: 43-46.

简介

黄单胞菌属可以通过分泌铁载体来从细胞外环境中清除铁,这些铁载体由由xss 编码的蛋白质(anthomonas 基因簇。 由于Xanthomonas campestris pv的铁供应有限,铁载体生产因黄托曼德而异。 野蓟菊(Xcc)产生比黄单胞菌(Xanthomonas oryzae)pv更少的铁载体。 米曲霉(Xoo)和黄单胞菌Xanthomonas oryzae pv。 oryzicola(Xoc)。 铁载体生产可以通过HPLC和CAS测量( Chrome azurol S )标准板测定,然而HPLC是在Xanthomonads中用于铁氰菊酯定量的CAS-琼脂平板测定的更准确的方法。 在这里我们描述如何使用高效液相色谱来量化黄单胞菌中的铁载体。
【背景】细菌植物病原体 Xanthomonas 具有(α-羟基羧酸盐型铁载体;类似于弧菌铁蛋白)和编码参与铁载体介导的铁吸收的外膜受体(Pandey和Sonti)所需的操纵子,2010; Pandey等人,2016a和2016b)。铁载体是由细菌分泌以利用不溶性铁的小铁螯合物(Neilands,1995)。在过去三十年中,CAS(( Chrome azurol S ) - 琼脂平板测定法主要用于通过监测细菌菌落周围的晕圈形成来测量细菌铁载体(Schwyn和Neilands,1987; Pandey和Sonti,2010),然而,上述测定不能可靠地用于量化铁载体的铁载体同时也分泌有机酸(例如草酸,柠檬酸和异柠檬酸)的细菌以及铁载体,因为有机酸能够螯合CAS染料中的铁,以损害量铁素体的准确性(Rai等人,2015)由于上述测定法仅定性地提出了关于细菌铁载体生产的想法,因此HPLC介导的铁载体定量是比CAS-琼脂平板测定更好的方法同时产生有机酸的铁载体的细菌,如黄单胞菌素(Xanthomon)作为物种(Rai等人,2015年; Pandey等,,2016a和2016b)。

关键字:黄单胞菌属铁载体, 黄单胞菌铁蛋白, 细菌铁载体估测, HPLC, Amberlite XAD-16树脂柱

材料和试剂

  1. 移液头(Corning,Axygen ®,目录号:TF-300-R-S,TF-200-R-S和TF-1000-R-S)
  2. 文化管(Borosil,目录号:9800U06)
  3. 培养皿(Tarson Products,目录号:460140-140MM)
  4. Stericup ®孔径为0.22μm的过滤单元(EMD Millipore,目录号:SCGPU02RE)
  5. 离心管:500ml,50ml,15ml,2ml和1.5ml(Tarson Products,Kolkata,India)
  6. 软木蛀虫8±0.2毫米(HiMedia实验室,目录号:LA737)
  7. 注射器驱动的具有0.22μm孔隙率的过滤器单元(Millex -GS)(EMD Millipore,目录号:SLGS033SS)
  8. 黄单胞菌(Xanthomonas campestris) pv。 campestris 8004,X。 oryzae pv。 oryzicola BXOR1,X。 oryzae pv。 ae ae ae ynthesis基因A(实验室收集)
  9. 利福平
  10. 2,2'-联吡啶(DP)(Sigma-Aldrich,目录号:14453)
    注意:本产品已停产。
  11. Amberlite XAD16N,20-60目(Sigma-Aldrich,目录号:XAD16-1KG)
  12. 甲醇(Thermo Fisher Scientific,目录号:Q32407)
  13. 标准弧菌素(Fujita等人,2011;见注2和3)
  14. 三氟乙酸(TFA)(Sigma-Aldrich,目录号:T6508-25ML)
  15. 乙腈(CH 3 CN)(Sigma-Aldrich,目录号:34888-1L)
    注意:本产品已停产。
  16. 蛋白胨(HiMedia Laboratories,目录号:CR001)
  17. 蔗糖(HiMedia Laboratories,目录号:GRM601-500G)
  18. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465)
  19. Chrome Azural S(CAS)(默克,目录号:1.02477.0025)
  20. 氯化铁(FeCl 3)(标准试剂,目录号:SRCF005C)
  21. 盐酸(HCl)(Fisher Scientific,目录号:29507)
  22. 十六烷基三甲基溴化铵(HDTMA)(Sigma-Aldrich,目录号:H6269-250G)
  23. Bacto TM琼脂(BD,Bacto TM,目录号:214010)
  24. Bacto TM 蛋白胨(BD,Bacto TM,目录号:211677)
  25. 琼脂(HiMedia Laboratories,目录号:RM026-500G)
  26. 蛋白胨 - 蔗糖(PS)培养基(Tsuchia等人,1982;见食谱)
  27. CAS解决方案(Schwyn and Neilands,1987; see Recipes)
  28. 胨 - 蔗糖 - 琼脂(PSA)培养基(见食谱)
  29. PSA-CAS-DP板(参见食谱)

设备

  1. 锥形瓶:500ml(Vensil Glass Works,目录号:1161)
  2. 锥形瓶:2,000毫升(Borosil,目录号:4980030)
  3. 微量移液器(Eppendorf,目录号:EPPR4396)
  4. 振荡孵化器(Eppendorf,New Brunswick TM ,型号:Innova ® 43)
  5. SORVALL RC-5B PLUS超速离心机(Thermo Fisher Scientific,Waltham,MA,USA)
  6. pH计(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:EC-PH510 / 11S)
  7. Agilent 1100系列HPLC系统(Agilent Technologies,型号:Agilent 1100系列)
  8. Agilent C-18(4.6 mm x 250 mm x 5μm)色谱柱(Agilent Technologies,Santa Clara,CA,USA)
  9. 滴定管(BOROSIL,目录号:2123016),滴定管支架和夹具
  10. 真空浓缩机加(Eppendorf,型号:Concentrator Plus,目录号:5305000304)
  11. Millipore真空,压力泵(EMD Millipore,目录号:XI04 220 50)
  12. 高压灭菌器

软件

  1. 化学工作站软件; Rev. A.10.02 [1757](Agilent,Santa Clara,CA,USA)
  2. Microsoft Office Excel 2013(Microsoft Corporation,Redmond,USA)

程序

  1. 无细胞培养上清液的制备
    1. 在含有利福平(50μg/ ml)的PSA板上,从其甘油储备液中分离细菌菌株,并在28℃的静态培养箱中孵育2-3天。为了开始初级培养,将单个菌落从上述板转移到含有3ml新鲜PS培养基(参见食谱)的高压灭菌培养管中,其中利福平(50μg/ ml)。黄单胞菌属的主要培养物可以在振荡培养箱中在28℃和200rpm下生长至晚指数生长(OD 600〜1.0),持续16-20小时。
    2. 通过使用从上述原代培养物中的0.2%接种物(2ml)开始二次培养,加入补充有适量2,2' - 联吡啶(150μM, pv. campestris str。8004,100μM用于米曲霉米曲霉,50μM用于米曲霉oryzicola)。在28℃的培养箱中以200rpm的振荡孵育第二培养物48小时。
    3. 在4℃下以17,000×g离心细菌培养物1小时以沉淀细胞和外多糖。
    4. 为了从上清液中除去剩余的细胞,用Stericups(孔径0.22μm)和真空压力泵过滤上清液。
    5. 用浓HCl(6N)将pH调节至2.0,以降低铁载体在水中的溶解度(Rey等,1996)。

  2. 使用XAD-16树脂柱的色谱柱
    1. 通过装入220g Amberlite XAD-16聚合物树脂制备2.4×30cm柱。
      注意:在需要色谱柱前一天,将水浸泡过夜,以准备Amberlite树脂。
    2. 用两倍的床体积(440ml)的甲醇洗涤填充柱,并平衡柱体积为三倍体积(660ml)的无菌MilliQ水。
    3. 将无细胞细菌上清液(方法A;步骤A5)通过平衡的XAD-16树脂柱(见注7)。
    4. 用200ml甲醇洗脱,并在2.0ml离心管中收集约100个级分(约2ml)(见附注7;图1)。


      图1. Xad-16树脂柱(在滴定管中制备),带有滴定管支持设备,用于收集黄腐败铁载体的洗脱液的不同部分

  3. CAS平板测定
    1. 准备PSA-CAS-DP板(参见食谱)。
    2. 用无菌软木钻孔器在板上打几个孔,保持孔间距离约2.5厘米。
    3. 将大约250μl的每个级分(根据洗脱顺序,程序B;步骤B4)放入分开的孔中,将盖直立放置在顶部,并在28℃下在静态培养箱中孵育。
    4. 24小时后观察分数的活度。
    5. 特征的黄色晕圈(光环13-24)表示铁载体的存在,而蓝色和红色的晕圈(光环1-12)表示有机酸的存在(图2)。


      图2.具有多个孔的PSA-CAS-DP板,其表现出与洗脱液的不同部分(由XAD-16树脂柱分离)孵育24小时后形成的不同着色晕圈,用于从X离子铁载体分离。 oryzae pv。蓝色和红色晕圈(光环1-12)表示含有机酸的级分,而黄色光环(卤素13-24)表示含有黄铁腐体铁载体的级分。
      注意:
      1. 我们还没有将第二十四分之二的铁载体洗脱液用于HPLC运行。因为我们已经观察到,与在其它杂质的存在相比,存在于这些级分中的铁载体可以忽略不计,在HPLC运行期间产生更多的噪声。添加这些用于HPLC的片段与活性片段一起(第13-20)将使杂质水平增加到更大的程度,而不显着增加样品中铁载体的总量。注意,学生的t检验(配对)用于分析针对HPLC运行的主要活性成分(第13至20号)中这些级分对杂质水平和铁载体量的贡献程度。
      2. 为了最小化由于对应于光晕21-24的部分中存在的其他不需要的成分引起的干扰程度(在加热和试验方法之后),只有对应于光晕13-20的铁载体的活性部分应当从各自剩余的主要库存(每个约1.75毫升)中进行HPLC定量。

  4. HPLC定量 
    1. 从程序B中汇集所有活性级分(对应于光晕13-20);步骤B4进入在PSA-CAS-DP板上显示特征性黄色晕圈的15ml离心管(方法C;步骤C5)。
    2. 为了除去溶剂,使用真空浓缩器(在酒精模式30℃下)将样品完全干燥。
    3. 用1ml甲醇重建,用孔径0.22μm的注射器驱动的过滤器过滤
    4. 注射每个菌株的推荐体积的过滤样品(25μl,用于刺激野生乳杆菌),10μl用于X,oryzae pv。oryzicola和X 。oryzae pv。oryzae)进入Agilent 1100系列HPLC系统(图3A-3B)。
    5. 使用梯度:使用Agilent C-18柱(4.6mm×250mm×5μm)进行分离:A = H 2 O / 0.1%TFA,B = CH 3 CN / 0.1%TFA; 10分钟内为0-30%B,15分钟为30-45%B,20分钟为45-0%B,流速为1 ml / min。
    6. 制作标准曲线,绘制趋势线,并从已知量的弧菌素(Fujita等人,2011)或黄高铁蛋白与通过HPLC色谱图获得的各自的峰面积获得等式(使用Microsoft Excel)(参见注1,2和3)。
    7. 从标准的弧菌铁蛋白或黄高铁蛋白得到的公式计算黄铁烷铁载体(存在于未知样品中)的量。


      图3. HPLC程序和仪器。 A.化学工作站软件中的HPLC程序; Rev. A.10.02 [1757]。 B.一部分HPLC机器,指示样品取向和自动注射针。 

数据分析

将样品(程序D;步骤D5)和空白(XAD-16树脂柱的空缓冲液洗脱液)的HPLC HPLC信号加载到ChemStation软件的离线模式。从样品信号中减去空白信号,然后自动积分,自动识别峰。记录对话框中生成的RT(保留时间)值和峰值区域。可以观察到纯黄素铁蛋白信号,同时监测来自黄单胞菌属(Xenonmonmonasas)的SSX突变体的分离的铁载体的HPLC色谱图中的缺失峰。与其各自的野生型相比。从黄烷铁蛋白的活性级分中,可以分离纯化的级分,真空干燥,称重并用作“标准黄高铁蛋白”,通过采用HPLC运行的不同已知量绘制标准曲线。通过绘制各个HPLC运行后获得的“相对于平均峰面积”绘制黄铁蛋白的标准曲线(图4)。使用标准曲线确定未知样品黄高铁蛋白铁载体的量。验证差异中的显着性水平,如果两个不同组所获得的数据之间存在任何差异,则使用配对的Student's 测试。
注意:对于一组实验,至少需要三次独立的生物重复。


图4. Xanthoferrin标准曲线;显示用于描绘HPLC运行中使用的铁载体量用于获得各个峰值区域的方程

笔记

  1. 可以使用ΔxSA突变体作为阴性对照。在ΔxssA突变株中,黄曲霉素峰将不存在或显着降低。对应于从ΔxssA突变体(Pandey等人,2016a)分离的铁载体的HPLC色谱图的缺失峰,可以从野生型菌株中收集纯黄高铁蛋白并用作标准。
  2. 虽然可以通过称重干黄素铁蛋白直接测定铁载体的质量,但优选为大量样品(> 5)创建标准曲线。
  3. 由于我们在300nm(保留时间〜11分钟)时检测到铁(III) - 亚铁铁氰酸盐络合物,所以预期分离的黄铁锈铁铁载体主要作为铁铁载体配合物存在。因此,优选使用铁铁载体络合物(1:1比例)制备标准曲线。
  4. 细菌接种和CAS平板测定应在无菌条件下进行
  5. 由于长期储存和频繁的冻融循环可能导致黄铁烷铁载体的降解,因此可以在4℃下储存最多1周。
  6. 2,2'-联吡啶的浓度可能稍微变化(高达±25μM),这取决于介质组分中作为杂质的铁的可用性。
  7. 柱制备和色谱法应在室温下进行。色谱步骤(洗涤,平衡和洗脱)应通过重力流程进行。

食谱

  1. 胨 - 蔗糖(PS)培养基 1%蛋白胨(w / v)
    1%蔗糖(w / v)
    在MilliQ水中溶解上述试剂 用3N氢氧化钠将pH调节至7.4,在121℃和15psi下高压灭菌20分钟
  2. CAS解决方案
    1. 0.06克铬Azurol S染料在50ml MilliQ水中
    2. Fe(III)溶液:10 ml
      1mM FeCl 3
      10 mM HCl
    3. 0.072g HDTMA在40ml MilliQ水中
    4. 将所有三种溶液分别混合,然后混合在一起,在121℃和15psi下高压灭菌20分钟以消毒
  3. 胨 - 蔗糖 - 琼脂(PSA)培养基
    1%Bacto TM 蛋白胨(w / v)
    1%蔗糖(w / v)
    在MilliQ水中溶解上述试剂,用3N氢氧化钠将pH调节至7.4,加入1.4%Bacto TM琼脂(w / v;最终),并在121℃和15psi下高压灭菌20分钟< br />
  4. PSA-CAS-DP板(140mm板)
    60 ml熔融PSA
    2,2'-联吡啶基(75μM,野草莓,野菜),50μM用于米曲霉或米曲霉和米曲霉X,oryzae pv。oryzicola)
    7.2 ml CAS溶液

致谢

我们承认Masaki J. Fujita博士(日本北海道大学)为本研究提供纯的弧菌素。我们从实验室(Rai,2015年)向以前的研究提出了确认,从那里我们修改了Xcc的铁载体估计协议。这项工作得到了DBT印度,CSIR-HRDG-印度,DST-SERB-印度和CDFD核心资金的SC的资助。 SSP是来自CSIR印度的JRF和SRF的接收者。 PS和BS是UGC-India的JRF和SRF的接受者。 RKV是DBT-India的JRF和SRF的接收者。

参考

  1. Fujita,MJ,Kimura,N.,Sakai,A.,Ichikawa,Y.,Hanyu,T。和Otsuka,M。(2011)。&lt; a class =“ke-insertfile”href =“http:// www.ncbi.nlm.nih.gov/pubmed/22146715“target =”_ blank“>来自海洋宏基因组文库的弧菌铁蛋白生物合成基因簇的克隆和异源表达 Biosci Biotechnol Biochem 75(12):2283-2287。
  2. Neilands,JB(1995)。&nbsp; 铁载体:结构和功能的微生物铁转运化合物。 J Biol Chem 270(45):26723-26726。
  3. Pandey,A.和Sonti,RV(2010)。&nbsp; FeoB蛋白和铁载体在促进霉菌黄杆菌的毒力中的作用 pv。米饭上的米曲霉。 J Bacteriol 192(12):3187-3203。
  4. Pandey,SS,Patnana,PK,Lomada,SK,Tomar,A.和Chatterjee,S.(2016a)。铁和XibR的铁代谢和毒力相关功能的协调调节,一种新型的铁结合转录因子,在植物病原体黄单胞菌中。 PLoS Pathog 12(11):e1006019。
  5. Pandey,SS,Patnana,PK,Rai,R。和Chatterjee,S.(2016b)。 Mol Plant Pathol 。
  6. Rai,R.,Javvadi,S。和Chatterjee,S。(2015)。细胞信号传导促进黄单胞菌黄单胞菌中的铁铁摄取。有利于其在水稻中的毒力和生长的谷朊病毒。分子微生物 96(4):708-727。
  7. Rey,F.,Ferreira,MA,Facal,P.和Machado,AASC(1996)。&lt; a class =“ke-insertfile”href =“http://www.nrcresearchpress.com/doi/abs/10.1139 /v96-033#.WVNsA2iGNdg“target =”_ blank“>浓度,pH和离子强度对土壤富里酸溶液粘度的影响。 Chem J Chem 74: 295-299。
  8. Schwyn,B.and Neilands,JB(1987)。&nbsp; 用于检测和测定铁载体的通用化学测定。 Anal Biochem 160(1):47-56。
  9. Tsuchia,K.,Mew,TW和Wakimoto,S。(1982)。野毒型和诱导的黄单胞病毒Xanthomonas campestris突变体的细菌学和病理学鉴定。 oryzae。 植物病理学 72:43-46。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pandey, S. S., Singh, P., Samal, B., Verma, R. K. and Chatterjee, S. (2017). Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC. Bio-protocol 7(14): e2410. DOI: 10.21769/BioProtoc.2410.
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