A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Summary

Bacterial glycogen structure is greatly impacted by extraction methods which may result in molecular degradation and/or biased sampling. It is essential to develop methods to minimize these problems. Here, four extraction methods have been compared using size distribution and chain length distribution as key criteria for minimizing extraction artifacts.

Abstract

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.

Introduction

Glycogen is a highly branched polysaccharide that consists of glucosyl residues and also a small but significant amount of proteins, in which all glucosyl residues are linked together via α-1,4-glycosidic bonds in linear chains and α-1,6-glycosidic bonds at branching points¹. The structure of glycogen particles is generally divided into three hierarchies: 1) short-chain oligomers, 2) spherical β particles (~20 nm in diameter), and 3) large rosette-shaped α particles aggregated together by β particles, the diameter of which ranges roughly up to 300 nm. Recently, it has been found that glycogen α particles h....

Protocol

1. Bacteria culture and collection

1. Resuscitate E. coli BL21(DE3) from bacterial glycerol stock (-80 °C) by inoculating sterile LB agar plate (10 g/L tryptone,5 g/L yeast extract, 10 g/L NaCl, and 15 g/L agar). Put the plate into a standard incubator and cultivate overnight at 37 °C.
2. Pick up a single colony and inoculate it into a 10 mL sterile LB liquid medium (10 g/L tryptone,5 g/L yeast extract, and 10 g/L NaCl). Mix well via vortexing and culture overn.......

Discussion

Glycogen is an important energy reserve that has been identified in many bacteria¹⁶. To dissect the physiological functions of glycogen particles, it is essential to have a better understanding of the fine structure of glycogen molecules. So far, a variety of methods have been developed to extract glycogen from bacterial culture. However, different size distributions of glycogen particles have been observed from different extraction methods, which suggests damaged glycogen structure. Thus, it is n.......

Materials

Name	Company	Catalog Number	Comments
Equipment
Agilent 1260 infinity SEC system	Agilent	1260 infinity II	Particle size distribution
Analytical column	PSS	10-1000	-
Centrifuge	Eppendorf	5420	-
Filter membrane	Cambio	Km-0220	-
Fluorescence-assisted capillary electrophoresis system	Beckman Coulter	-	Chain length distribution
Freeze dryer	Xinzhi	SCIENTZ-10N	Lyophilization of bacteria and glycogen
Freezer	Thermo Fisher	Forma 900	Sample storage
Guard column	PSS	SUPPERMA	-
Incubator	Thermo Fisher	PR505750R-CN	-
Low-speed large-capacity centrifuge	Hexi	HR/T20MM	Sample centrifugation
Multiskan FC microplate reader	Thermo Fisher	1410101	-
Optima XPN ultracentrifuge	Beckman	XPN-100/90/80	For glycogen
Oscillator	Xinbao	SHZ-82	-
PA-800 Plus System	Beckman Coulter	A66528	-
pH meter	Mettler Toledo	FE28 -TRIS	-
Refractive index detector	Wyatt	Optilab T-rEX	-
Refrigerator	Haier	BCD-406WDPD	-
Thermomixer	Shanghai Jingxin	JXH-100	Sample incubation
Transmission electron microscope	Hitachi Corporation	H-7000	Glycogen particle morphology
Ultracentrifuge tube	Beckman	355651	-
Ultrasonic cell crusher	Ningbo Xinzhi	Scientz-IID	Bacteria disruptor
Ultrasonic oscillating water bath	Jietuo	JT-1027HTD	-
Vortex mixer	Tiangen	OSE-VX-01	-
Water system	Merck Millipore	H2O-MM-UV-T	Deionized water
Material
8-Aminopyrene-1,3,6-Trisulfonic Acid Trisodium Salt	Sigma-Aldrich	196504-57-1	-
Absolute ethanol	Guoyao	10009228	-
Agar powder	Solarbio	A1890	-
Alpha-amylase	Megazyme	E-BLAAM-40ML	-
Amyloglucosidase	Megazyme	E-AMGDF-40ML	-
cOmplete Mini	Roche	4693159001	-
D-(+)Glucose	Sigma-Aldrich	G8270-1kg	-
D-Glucose Assay Kit (GOPOD Format)	Megazyme	K-GLUC	Glycogen quantification
Dimethyl sulfoxide	Vicmed	Vic147	Chaotropic agent
E. coli BL21(DE3)	Tiangen	CB105-02	-
Ethylene diamine tetra-acetic acid	Vicmed	Vic1488	-
Glacial acetic acid	Guoyao	10000218	-
Glycerol	Guoyao	10010618	Bacterial storage
Hydrochloric acid	Guoyao	10011008	-
Hydroxymethyl aminomethane	Sigma-Aldrich	V900483-500g	-
Isoamylase	MegaZyme	9067-73-6	Glycogen debranch
Lithium chloride	Sigma-Aldrich	62476-100g	-
M9, Minimal Salts, 5×	Sigma-Aldrich	M6030-1kg	Bacterial culture
Potassium hydroxide	Guoyao	10017008	-
Pullulan standard	PSS	-	-
Sodium acetate trihydrate	Guoyao	10018718	-
Sodium azide	Sigma-Aldrich	26628-22-8	-
Sodium chloride	Guoyao	10019318	Bacterial culture
Sodium cyanoborohydride	Huaweiruike	hws001297	-
Sodium diphosphate	Sigma-Aldrich	71515-250g	-
Sodium Fluoride	Macklin	S817988-250g	-
Sodium hydroxide	Guoyao	10019762	-
Sodium nitrate	Guoyao	10019928	-
Sodium pyrophosphate	Sigma-Aldrich	V900195-500g	-
Sucrose	Guoyao	10021463	-
Trichloroacetic acid	Guoyao	40091961	-
Tryptone	Oxoid	LP0042	Bacterial culture
Yeast Extract	Oxoid	LP0021	Bacterial culture