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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: October 17th, 2018

DOI:

10.3791/58536

1Department of Physics & Astronomy, McMaster University, 2School of Life Sciences, University of Nevada Las Vegas

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.

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.

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....

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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.......

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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.......

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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 .......

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We thank Richard B. Frankel for his help with MTB cultures, Adam P. Hitchcock and Xiaohui Zhu for their support while setting up the MTB cultures at McMaster University, and Marcia Reid for training and access to the electron microscopy facility (McMaster University, Faculty of Health Sciences). This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the US National Science Foundation.

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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

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