Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

Summary

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

Abstract

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

Introduction

Phycobilisome (PBS) is a huge water-soluble pigment-protein complex that attaches to the cytoplasmic side of the photosystems in the thylakoid membranes of cyanobacteria¹. PBS is primarily composed of colored phycobiliproteins and colorless linker proteins^1,2. The phycobiliproteins can be divided into four major groups: phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin³. The four major groups absorb different wavelengths of light energy in the range of 490-650 nm, which chlorophylls absorbed inefficiently³. The PBS can serve as a light-harvesting antenna for collecting light energy and delivering it to Photosystem II and I⁴.

The structure and composition of PBS vary from species to species. Collectively, three shapes of phycobilisome (hemidiscodial, bundle-shaped, and rod-shaped) have been identified in different cyanobacterial species⁵. Even in the same species, the composition of PBS changes in response to the environment, such as light quality and nutrient depletion^{6,7,8,9,10,11}. Therefore, the experimental procedure to isolate PBS from cyanobacteria has been instrumental in studying PBS¹². Over several decades, many different protocols have isolated PBS and analyzed its structure, composition, and function^{6,7,8,12,13,14,15,16,17}. The wide variety of methods for PBS isolation indeed provides flexibility in isolating the complex in different species with different reagents and instruments. However, it also makes choosing a suitable protocol more difficult for scientists unfamiliar with cyanobacteria and PBS. Therefore, a generalized and straightforward protocol is developed in this work for those interested in starting PBS isolation from cyanobacteria.

The methods for isolating PBS from previous publications are summarized here. Since PBS is a water-soluble protein complex and is readily dissociated, a high ionic strength phosphate buffer is required to stabilize PBS during extraction¹⁸. Several research articles that describe methods for the isolation of PBS from cyanobacterium have been published in the past. Most of the methods require a high concentration of phosphate buffer^{8,14,15,18,19}. However, the procedures for mechanical disrupting of the cells vary, such as glass beads-assisted extraction, sonication²⁰, and French press^6,8,14. Different phycobiliproteins can be obtained by precipitation with ammonium sulfate²⁰ and purified by HPLC²¹ or a chromatographic column²². On the other hand, intact PBS can be easily isolated by sucrose density gradient ultracentrifugation^6,8,15.

In this protocol, one model cyanobacterium and one non-model cyanobacterium were used as the materials for PBS isolation. They are model unicellular glucose-tolerant Synechocystis sp. PCC 6803 (hereafter Syn6803) and non-model filamentous Leptolyngbya sp. JSC-1 (hereafter JSC-1), respectively^7,23,24. The protocol begins by disruption of the unicellular and filamentous cyanobacteria in a high-ionic-strength phosphate buffer. After lysis, the supernatants are collected by centrifugation and then treated with a nonionic detergent (Triton X-100) to solubilize the water-soluble proteins from the thylakoid membranes. The total water-soluble proteins are applied to a discontinuous sucrose density gradient to fractionate the PBS. The discontinuous sucrose gradient in this protocol consists of four sucrose solutions and partitions the intact PBS in the lowest fractions of sucrose layer²⁵. The integrity of PBS can be analyzed by SDS-PAGE, zinc-staining, and 77K fluorescence spectroscopy^6,7,8,26. This method is suitable for scientists who aim to isolate intact PBS from cyanobacteria and study its spectral, structural, and compositional properties.

There are several advantages of this protocol. (1) This method is standardized and can be used for isolating intact PBS from both unicellular and filamentous cyanobacteria. Most of the articles describe the method that applied in either one type of cyanobacteria^{4,7,8,12,13,14,16,18}. (2) This method is performed at room temperature, as PBS dissociates at low temperature^19,27. (3) This method describes using a bead-beater to disrupt the cells; therefore, it is cheaper and safer than high-pressured French press and possible hearing damage from sonicator in other methods^8,13,14,20. (4) This method isolates intact PBS by sucrose gradient ultra-centrifugation. In this way, intact PBS with different sizes and partially dissociated PBS can be separated based on sucrose concentration.

Protocol

The Synechocystis sp. PCC 6803, the model glucose-tolerant strain, was obtained from Dr. Chu, Hsiu-An at Academia Sinica, Taiwan. Leptolyngbya sp. JSC-1, the non-model filamentous,was obtained from Dr. Donald A. Bryant at Pennsylvania State University, USA.

1. Cell culture and harvesting

1. Inoculate Syn6803 or JSC-1 cells using a metallic inoculation loop into a 100 mL conical flask containing 50 mL of B-HEPES medium²⁸. Culture the cells at 30 °C under 50 µmol photons m^-2 s^-1 (white-light LED) in 1% (v/v) CO₂ with constant stirring by a magnetic stirrer (120 rpm) until the culture reaches mid-exponential growth phase (OD₇₅₀ = 0.6-0.8).
   NOTE: Harvest the cell culture when the optical density of the culture at 750 nm (OD₇₅₀) reaches 0.6-0.8 by transferring to a new flask. Optical density was routinely used to estimate microbial cell density in liquid cultures²⁹. The wavelengths from 720-750 nm were used in various studies for measuring cyanobacterial growth^30,31,32. In this protocol, OD₇₅₀ is used because JSC-1 can produce pigments that absorb far-red light⁷. Alternatively, chlorophyll concentration was also used to measure the growth of cyanobacteria³³.
2. Transfer the cell culture (OD₇₅₀ ~0.8) into a 1L conical flask and dilute it to OD₇₅₀ = 0.2 in 500 mL of B-HEPES medium. Grow the culture until the late exponential growth phase (OD₇₅₀ = 0.8-1.0) in the same condition mentioned above (step 1.1) and then harvest the culture by centrifugation.
3. Centrifuge the culture at 10,000 x g for 20 min at room temperature and completely discard the supernatant.
   ​NOTE: The cells pellets can be stored at -80 °C for up to 6 months.

2. Cells lysis

1. Suspend the cell pellets and wash twice with 0.75 M K-phosphate buffer, pH 7.
   NOTE: To make 0.75 M K-phosphate buffer at pH 7, prepare 1.5 M of K₂HPO₄ and 1.5 M of KH₂PO₄ separately. Mix 615 mL of 1.5 M K₂HPO₄ and 385 ml of 1.5M KH₂PO₄ to get 1.5 M K-phosphate buffer at pH 7. Simply dilute it with the same amount of ddH₂O to make 0.75 M K-phosphate buffer at pH 7.
2. Pellet the cells in 50 mL centrifuge tubes by centrifugation at 10,000 x g for 20 min at room temperature. Discard the supernatant and resuspend the cells with 0.75 M K-phosphate buffer. Measure the wet weight of the pellet by an electronic balance and resuspend 1 g wet weight of the pellet in 5 mL of the buffer.
   NOTE: Wet weight of a pellet was measured by subtracting the weight of an empty centrifuge tube from the weight of the centrifuge tube containing the cell pellet.
3. Add 1 mL of cell suspension and 0.4-0.6 g of 0.1 mm glass beads (see Table of Materials) into a 2 mL screw cap vial.
4. Break the cells for 30 s by a bead-beater (see Table of Materials). Allow the tubes to cool in a room temperature water bath for 2 min and repeat the breaking cycle 5 times.
5. After lysis, centrifuge the vials at 500 x g for 10 s at room temperature. Collect the supernatant with a pipette into a 15 mL centrifuge tube. Wash the beads with 0.5 mL of 0.75 M K-phosphate buffer one time, then transfer to the same centrifuge tube.
6. Add Triton X-100 (2% w/v, final concentration) to the lysed cell suspension and incubate on a rocking shaker (40 rpm) at room temperature until the solution becomes homogenous (~20 min).
7. Centrifuge the tubes at 17,210 g for 20 min to remove the intact cells and large cell debris. Store the supernatant without the upper Triton micelle layer at room temperature for up to 1 h.
   ​NOTE: The supernatant contains two separated layers. The upper hydrophobic Triton X layer contains chlorophyll-binding proteins and hydrophobic rod-shaped phycobilisomes⁶. The lower aqueous layer contains water-soluble PBS.

3. Preparation of the sucrose gradient buffers and PBS isolation

1. Make four concentrations of sucrose (2.0 M, 1.0 M, 0.75 M and 0.5 M) in 0.75 M K-phosphate buffer at pH 7.
   NOTE: It is suggested to use 1.5 M K-phosphate buffer at pH 7 to prepare the sucrose gradient because adding sucrose dilutes the concentration of K-phosphate buffer. After sucrose is fully dissolved, add ddH₂O to adjust the concentration to 0.75 M, pH 7.
2. Place 2.8 mL of 2.0 M sucrose buffer at the bottom of the 40 mL centrifuge tube and overlay with three layers of sucrose solutions (8 mL of 1.0 M; 12 mL of 0.75 M and 11 mL of 0.5 M for 40 mL centrifuge tube), and finally PBS-containing the supernatant fraction (3.0 mL) (Figure 1A).
   NOTE: Carefully add the sucrose solution and allow sucrose to drop very slowly inside of the tube. Hold the pipette tip just above the surface of the solution in the tube while loading the solution. Sucrose layers can be observed when they are layered slowly.
3. Centrifuge the resulting gradients at 125,800 x g for ~16h-20 h at 25 °C.NOTE: An ultracentrifuge (see Table of Materials) is required for this step.
4. Upon ultra-centrifugation, condense the fractionated PBS and phycobiliproteins between the layers. Blue bands in Syn6803 (purple bands showed in JSC-1) were observed in the interfaces (Figure 1D).
5. Slowly collect the fractions with a pipette from the top of the sucrose gradients. Remove the sucrose by repeatedly concentrating (3,500 x g for 20 min) and diluting the fractions with 0.75 M K-phosphate buffer in membrane centrifugal filter units (10 K molecular weight cut-off, see Table of Materials).

4. Measurement of PBS fluorescence at 77K

NOTE: A fluorimeter equipped with a liquid nitrogen container (see Table of Materials) is used to measure fluorescence spectra at 77K.

1. Dilute the concentrated PBS sample with 0.75 M K-phosphate buffer to gain at least 500 µL.
   NOTE: The concentrations of phycocyanin of samples were ~4.2 µg mL^-1 based on the formula: (OD₆₁₅ 0.474 x OD₆₅₂)/5.34 [mg/mL]³⁴.
2. Add 500 µL of the PBS sample to a transparent glass tube and freeze the tube in liquid nitrogen until it is completely frozen. Move the frozen tube to a transparent Dewar container (see Table of Materials) pre-filled with liquid nitrogen.
   NOTE: The inner diameter of the glass tube is 3 mm in this protocol. Thin glass tubes were used to minimize re-absorption of short-wavelength emission in the sample.
3. Choose the excitation wavelengths for phycoerythrin and phycocyanin to be 550 nm and 580 nm, respectively.
   ​NOTE: Fluorescence emissions were recorded from 560-800 nm (for 550 nm excitation) or 600-800 nm (for 580 nm excitation).

5. SDS-PAGE analysis of PBS

1. Buffer exchange the concentrated PBS samples in 0.75 M of K-phosphate with 50 mM of Tris buffer (pH 8.0) in membrane centrifugal filter units (3K molecular weight cut-off, see Table of Materials).
2. Mix 50 µL of PBS solution with 10 µL of 6x SDS loading buffer [300 mM Tris pH 6.8, 12% (w/v) Sodium dodecyl sulfate, 0.06% (w/v) Bromphenol blue, 50% (v/v) Glycerol, 6% (v/v) β-Mercaptoethanol] (see Table of Materials) in a microcentrifuge tube and incubate at 95 °C for 10 min in a heat block.
3. Separate the protein samples by SDS-PAGE (8%-20% (w/v) polyacrylamide gel) and continue with zinc and Coomassie staining. The detailed procedure has been described in Reference⁸.
4. For zinc staining, incubate the gel in 50 mM ZnSO₄ solution for 10 min at room temperature, wash the gel with distilled water, and visualize zinc-induced fluorescence under UV irradiation (312 nm).
5. For Coomassie blue staining, incubate the gel in Coomassie Blue staining buffer [0.25% (w/v) of Coomassie R-250, 10% (v/v) of Acetic acid, 50% (v/v) of Methanol, see Table of Materials] for 1 h at room temperature. Wash the gel with distilled water twice to remove the residual staining buffer and incubate the gel with destaining buffer [10% (v/v) Acetic acid, 30% (v/v) Methanol] on a rocking shaker (40 rpm) at room temperature overnight. Visualize the fully-destained gel with a digital camera or a scanner.

Results

The Syn6803 and JSC-1 cells were cultivated in conical flasks with constant stirring in B-HEPES medium at 30 °C, under a LED white light (50 µmol photons m^-2s^-1) in a growth chamber filled with 1% (v/v) CO₂. At the exponential growth phase (OD₇₅₀ = ~0.5), the cells were subcultured into fresh medium with a final optical density OD₇₅₀ = ~0.2. After reaching the late exponential growth phase (OD₇₅₀ = 0.6-0.8), the cultures were collected and centri...

Discussion

This protocol describes a simple and standard method for isolating intact PBS in two types of cyanobacteria, unicellular model Syn6803, and filamentous non-model JSC-1. The critical steps of the protocol are cell homogenization and ultracentrifugation on a discontinuous density gradient of sucrose. Generally, the disruption of filamentous cells is more complicated than unicellular ones. Increasing the amount of the starting material (the wet weight of the cell pellet) and the repetition of bead-beating were helpful ...

Materials

Name	Company	Catalog Number	Comments
0.1 mm glass beads	BioSpec	11079101	for PBS extraction
13 mL centrifugation tube	Hitachi	13PA	ultracentrifugation
40 mL centrifugation tube	Hitachi	40PA	ultracentrifugation
Acetic acid	Merck	8.1875.2500	for Coomassie Blue staining
B-HEPES medium			A modified cyanobacterial medium from BG-11 medium
Brilliant Blue R-250	Sigma	B-0149	for Coomassie Blue staining
Bromophenol blue	Wako pure chemical industries	2-291	protein loading buffer
Electronic balance	Radwag	WLC 2/A2/C/2	for the wet weight measurement of cell pellets
Fluorescence spectrophotometer	Hitachi	F-7000	Spectrophotometer
Glycerol	BioShop	Gly001.500	protein loading buffer
High-Speed refrigerated centrifuge	Hitachi	CR22N	for buffer exchange
Leptolyngbya sp. JSC-1			from Dr. Donald A. Bryant at Pennsylvania State University, USA.
Low temperature measurement accessory	Hitachi	5J0-0112	The accessory includes a transparent Dewar container for 77K fluorescence spectra
Methanol	Merck	1.07018,2511	for Coomassie Blue staining
Microcentrifuge	Thermo Fisher	Pico 21	for PBS extraction
Mini-Beadbeater-16	BioSpec	Model 607	for PBS extraction
Potassium phosphate dibasic	PanReac AppliChem	121512.121	for PBS extraction
Potassium phosphate monobasic	PanReac AppliChem	141509.121	for PBS extraction
Screw cap vial	BioSpec	10832	for PBS extraction
SmartView Pro Imager	Major Science	UVCI-2300	for Znic staining signal detection
Sodium dodecyl sulfate	Zymeset	BSD101	protein loading buffer
Sucrose	Zymeset	BSU101	for PBS isolation
Synechocystis sp. PCC 6803			glucose-tolerant strain from Dr. Chu, Hsiu-An at Academia Sinica, Taiwan
Tris	BioShop	TRS 011.1	protein loading buffer
Triton X-100	BioShop	TRX 506.500	for PBS extraction
Ultra 10 K membrane centrifugal filter	Millipore	UFC901024	for buffer exchange
Ultra 3 K membrane centrifugal filter	Millipore	UFC500324	for buffer exchange
Ultracentrifuge	Hitachi	CP80WX	ultracentrifugation
UV/Vis spectrophotometer	Agilent	Cary 60	Spectrophotometer
Zinc sulfate	PanReac AppliChem	131787.121	for Znic staining
β-Mercaptoethanol	BioBasic	MB0338	protein loading buffer