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 se....

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 consta....

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