Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Summary

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O₂ concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Abstract

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe₃O₄) or greigite (Fe₃S₄) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O₂ concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O₂ concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Introduction

Magnetotactic bacteria (MTB) represent a wide range of Gram-negative prokaryotes ubiquitous in freshwater and marine aquatic habitats¹. These bacteria share the ability to produce magnetic crystals made of either magnetite (Fe₃O₄) or greigite (Fe₃S₄), which are in most cases assembled into chains inside the cells. This particular structural motif is due to the presence of several specific proteins acting both in the cytoplasm of the bacteria and on the lipid membrane that surrounds each crystal². Each individual crystal and its surrounding membrane vesicle is called a magnetoso....

Protocol

1. Installation of the N₂ Station

NOTE: Choose the inner diameter of the tubing so that it can be connected to the gas tank with minimum leakage and so that the cylinder of a 1 mL plastic syringe tightly fits in this tubing. An illustration of the complete N₂ gassing station is provided in Figure 1.

1. Safely install a N₂ gas tank close to a bench on which there is enough space to set up the N₂ station (a length of approximately 50 cm).
2. Connect to the tank a piece of tubing long enough to reach the area where the station will be built. If necessary,....

Results

Successful preparation of the growth media can be assessed as follows. At the end of the process, clear solutions (i.e., free of any precipitate) should be obtained (this is true for both the liquid media and the O₂ gradient semi-solid medium). A picture displaying the expected aspect of MSR-1 liquid medium before inoculation can be seen in Figure 2a. A successful O₂ concentration gradient semi-solid medium is signaled by the fo.......

Discussion

The specific O₂ concentration requirements of MTB make them non-trivial to grow in the laboratory. A key step of the protocol for liquid medium is the initial removal of all O₂ from the medium in order to control the final concentration by adding a definite volume of O₂, just before inoculation. It has been shown that MSR-1 grows under almost fully aerobic conditions, however, the magnetism of the cells is drastically reduced. The results from the same study showed that strains AMB-1 and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AMB-1	American Type Culture Collection (ATCC)	ATCC 700264
MS-1	ATCC	ATCC 31632
MSR-1	Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)	DSM 6361
Ferric citrate	Sigma-Aldrich	F3388-250G
Trace mineral supplement	ATCC	MD-TMS
KH2PO4	EMD	PX1565-1
MgSO4.7 H2O	EMD	MX0070-1
HEPES	BioShop Canada Inc	HEP001.250
NaNO3	Sigma-Aldrich	S5506-250G
Yeast extract	Fischer scientific	DF210929
Peptone	Fischer scientific	DF0436-17-5
Potassium L-lactate solution (60%)	Sigma-Aldrich	60389-250ML-F
D-(-)-Quinic acid	Sigma-Aldrich	138622
FeCl3.6H2O	Fischer scientific	I88-100
Vitamin supplement	ATCC	MD-VS
Sodium succinate hexahydrate	Fischer scientific	S413-500
Sodium L-tartrate dibasic dihydrate	Sigma-Aldrich	228729-100G
Sodium acetate trihydrate	EMD	SX0255-1
Resazurin	Difco	0704-13
Ascorbic acid	Sigma-Aldrich	A4544-25G
K2HPO4	Caledon	6620-1-65
FeCl2 .4H2O	Sigma-Aldrich	44939-250G
Sodium bicarbonate	EMD	SX0320-1
NaCl	Caledon	7560-1
NH4Cl	EMD	1011450500
CaCl2.2 H2O	EMD	1023820500
Agar A	Bio Basic Canada Inc	FB0010
L-cysteine.HCl.H2O	Sigma-Aldrich	C7880-100G
1.0 mL syringes	Fischer scientific	B309659
25G  x 1 needles	BD	305125
125 mL serum bottles	Wheaton	223748
20 mm aluminum seals	Wheaton	224223-01
20mm E-Z Crimper	Wheaton	W225303
Butyl-rubber stoppers	Bellco Glass, Inc.	2048-11800
Hungate tubes	Chemglass (VWR)	CLS-4208-01
Septum stopper, 13mm, Hungate	Bellco Glass, Inc.	2047-11600
Glass culture Tubes	Corning (VWR)	9826-16X
Hydrochloric acid 36.5-38%, BioReagent	Sigma-Aldrich	H1758-100ML	11.6 - 12 N