Name: growing magnetotactic bacteria of the genus magnetospirillum: strains msr-1, amb-1 and ms-1
Uploaded: 2018-10-17T15:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1 at JoVE.com

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.

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.
<ol>
	<li>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).</li>
	<li>Connect to the tank a piece of tubing long enough to reach the area where the station will be built. If necessary, apply Teflon tape at the output of the tank to avoid any leakage.</li>
	<li>To build a station capable of bubbling five bottles of medium at the same time, cut four pieces of tubing of approximately 5 cm in length.</li>
	<li>Assemble the pieces of tubing in a line with three three-way T-shaped plastic fittings. Connect one end of this line to the piece of tubing at the output of the N2 tank through an extra T-shaped fitting. Add a 90&#176; elbow fitting at the other end.</li>
	<li>Use tape to attach the structure to a horizontal metal rod placed approximately 30 cm above the bench.</li>
	<li>Connect five pieces of tubing (approximately 20 cm in length) to the free outputs of the fittings installed in Step 1.4.</li>
	<li>Remove the pistons from five 1.0 mL plastic syringes and cut the larger end of these syringes (i.e., the opposite side to the needle), keeping only the graduated part. Fill these syringes with cotton, not too tightly.</li>
	<li>Insert the syringes in the 20 cm long vertical pieces of tubing and use soapy water to ensure that there is no leakage when N2 is flowing.</li>
	<li>Remove the caps of five 25 G needles (0.5 mm x 25 mm) and insert these needles into the 10 cm pieces of thin tubing. Ensure that the needles tightly fit in the tubing. 
	CAUTION: There is a risk of stabbing during this step. Do it slowly and carefully.</li>
	<li>Attach the needles prepared in Step 1.9 to the syringes of the N2 station. Ensure that N2 is flowing through all five lines and keep the station on stand-by.</li>
</ol>
2. Growth Medium Preparation
NOTE: It is possible to adjust the amount of medium prepared, as explained in Steps 2.1.2, 2.2.2 and 2.3.8. The amount of water and chemicals used just needs to be proportionally adjusted. The role of all medium components is described in Supplementary Table 1.
<ol>
	<li>Preparation of liquid growth medium for MSR-1
	<ol>
		<li>Prepare a 10 mM ferric citrate solution by adding 0.245 g of ferric citrate to 100 mL of distilled deionized water. Heat and stir to dissolve, until a yellow to orange&#160;clear solution is obtained. Autoclave the solution using a standard cycle (at least 15 min exposure at 121 &#176;C) and then store this stock solution at room temperature in the dark. 
		NOTE: Discard the ferric citrate solution when a precipitate becomes obvious.</li>
		<li>In a beaker containing 1 L of distilled deionized water, add the following in order while stirring: 1.0 mL of the trace mineral supplement solution, 0.1 g of KH2PO4, 0.15 g of MgSO4.7 H2O, 2.38 g of HEPES, 0.34 g of NaNO3, 0.1 g of yeast extract, 3.0 g of soy bean peptone, 4.35 mL of potassium lactate (60% w/w solution) and 5 mL of the 10 mM Fe(III) citrate stock solution. 
		NOTE: Adjust the quantities proportionally if a smaller amount of growth medium is needed. For a N2 station capable of bubbling five bottles at the same time, such as the one built in Step 1 of this protocol, prepare 300 mL of medium. 
		CAUTION: Trace mineral and Fe(III) citrate stock solutions need to be kept sterile. To avoid contamination, use standard sterile technique when using them (flame open tops of bottles using a Bunsen burner) and use sterile pipette tips for dispensing. Store the mineral solution in a refrigerator at 4 &#176;C.</li>
		<li>After the addition of all chemicals, adjust the pH to 7.0 with 1 M NaOH solution. Dispense the freshly prepared medium into 125 mL serum bottles. Pour 60 mL of medium in each bottle.</li>
		<li>Bubble N2 into the medium for 30 min to remove the dissolved O2, using the small tubing connected to the N2 station described in Step 1. Place a butyl-rubber stopper on top of each bottle, leaving a small opening to allow the excess gas to exit the bottle. 
		CAUTION: Foam might form while bubbling with N2. Adjust the gas flow accordingly to avoid foam production.</li>
		<li>Crimp seal each bottle with the prepared stopper and an aluminum seal. The aluminum seal ensures that the bottle remains sealed during the rest of the protocol.</li>
		<li>Disconnect the needles and the thin tubing from the N2 station and replace them with clean needles (1 inch,&#160;&#8804; 23G). Adjust the valves of the N2 tank so that a gentle continuous flow of gas exits the tank (about 50 mL/min). 
		Note: Needles larger than 23G may leave permanent unsealable holes in the stopper.</li>
		<li>Insert one of the needles connected to the N2 tank into a bottle of medium, through the rubber stopper. Immediately insert another clean needle into the same bottle. Repeat this step for the other bottles and let N2 flow for about 30 min to replace the air in the bottles by N2.</li>
		<li>Disconnect one bottle from the N2 station by removing the corresponding needle. Wait for a few seconds until the pressure in the bottle of medium decreases to atmospheric pressure and remove the second needle. Repeat this step for all remaining bottles. 
		CAUTION: To prevent O2 from re-entering the bottles of growth medium after Step 2.1.4, perform Steps 2.1.4 - 2.1.8 in quick succession. If all bottles cannot be connected to the N2 station at the same time, proceed with Steps 2.1.4-2.1.8 for the first set of bottles and then repeat these steps for the remaining bottles.</li>
		<li>Autoclave the bottles. Let them cool down to room temperature overnight and store them at room temperature afterwards.</li>
	</ol>
	</li>
	<li>Preparation of liquid growth medium for AMB-1 and MS-1
	<ol>
		<li>Prepare a 10 mM ferric quinate solution. First dissolve 0.19 g of quinic acid in 100 mL of distilled deionized water, then add 0.27 g of FeCl3.6H2O. Stir to dissolve, until a dark red, clear solution is obtained. Autoclave the solution using a standard cycle (at least 15 min exposure at 121 &#176;C). 
		NOTE: Store the ferric quinate solution at room temperature in the dark as a sterile stock solution. Discard the solution when a precipitate becomes obvious.</li>
		<li>In a beaker containing 1 L of distilled deionized water, add the following in order while stirring: 10.0 mL of the vitamin supplement solution, 5.0 mL of the trace mineral supplement solution, 0.68 g of KH2PO4, 0.848 g of sodium succinate dibasic hexahydrate, 0.575 g of di-Sodium tartrate dihydrate, 0.083g of Sodium acetate trihydrate, 0.45mL of 0.1% aqueous Resazurin, 0.17 g of NaNO3, 0.04 g of ascorbic acid and 3.0 mL of the 10 mM Fe(III) quinate stock solution. 
		NOTE: Adjust the quantities proportionally if a smaller amount of growth medium is needed. For a N2 station capable of bubbling five bottles at the same time, such as the one built in Step 1 of this protocol, prepare 300 mL of medium. 
		CAUTION: Vitamin, trace mineral and Fe(III) quinate stock solutions need to be kept sterile. To avoid contamination, use standard sterile technique and sterile pipette tips when dispensing. Store the mineral and vitamin solutions in a refrigerator at 4 &#176;C.</li>
		<li>After the addition of all chemicals, adjust the pH to 6.75 using 1 M NaOH solution.</li>
		<li>Refer to Steps 2.1.3-2.1.9 for the rest of the protocol.</li>
	</ol>
	</li>
	<li>Preparation of semi-solid growth medium for MSR-1
	<ol>
		<li>Prepare a 0.5 M phosphate buffer solution pH 7.0 by dissolving 3.362 g of K2HPO4 and 4.178 g of KH2PO4 in 100 mL of distilled deionized water. Check if the pH is 7.0 and adjust the pH slightly with KH2PO4 or NaOH if needed. Store the solution in a sealed glass bottle (preferable to plastic in order to avoid oxygen exchange).</li>
		<li>Prepare 100 mL of a 0.02 M hydrochloric solution. Add 0.2 g of FeCl2.4H2O to this solution and stir to dissolve in order to obtain a 10 mM iron chloride solution. Store the solution in the dark in a sealed glass bottle.</li>
		<li>Prepare a 0.8 M sodium bicarbonate solution by dissolving 6.72 g of NaHCO3 in 100 mL of distilled deionized water. Store the solution in a sealed glass bottle.</li>
		<li>Autoclave the solutions prepared in Steps 2.2.1 - 2.2.3 using a standard cycle (at least 15 min exposure at 121 &#176;C). Store them as sterile stock solutions in the dark.</li>
		<li>In a beaker containing 1 L of distilled deionized water, add the following in order while stirring: 5 mL of the trace mineral supplement solution, 0.2 mL of 1% aqueous resazurin solution, 0.4 g of NaCl, 0.3 g of NH4Cl, 0.1 g of MgSO4.7H2O, 0.05 g of CaCl2.2H2O, 1 g of sodium succinate, 0.5 g of sodium acetate, 0.2 g of yeast extract and 1.6 g of agar.</li>
		<li>Cover the beaker with aluminum foil and autoclave the solution prepared in Step 2.3.5.</li>
		<li>Just before the end of the autoclave cycle, prepare a fresh 4% L-cysteine&#183;HCl&#183;H2O solution by dissolving 0.8 g of L-cysteine&#183;HCl&#183;H2O in 20 mL of distilled deionized water. Neutralize the solution to pH 7.0 with 5 M NaOH solution. 
		CAUTION: it is important that the cysteine solution is prepared fresh to avoid oxidation of the cysteine. Store the mineral solution in a refrigerator at 4 &#176;C.</li>
		<li>After autoclaving, let the medium cool down to 50&#8211;60 &#176;C and bring the beaker under the flame of a Bunsen burner. Remove the aluminum foil and quickly add the following in order while gently stirring: 0.5 mL of the vitamin solution, 2.8 mL of the sterile phosphate buffer stock solution, 3 mL of the sterile iron chloride stock solution, 1.8 mL of the sterile sodium bicarbonate stock solution and 10 mL of filter-sterilized cysteine solution. 
		NOTE: Adjust the quantities proportionally if a smaller amount of growth medium is needed. It is usually convenient to prepare a batch of 120 mL of medium. 
		CAUTION: All solutions must remain sterile for future use. Perform Step 2.3.8 using standard sterile technique and sterile pipette tips when dispensing.</li>
		<li>After the addition of all chemicals, transfer the warm medium into 16 mL sterile screw-cap Hungate tubes. Transfer 12 mL of the medium into each tube and seal the tubes. 
		CAUTION: To avoid contamination, perform Step 2.3.9 under the flame of a Bunsen burner. Perform it before the agar solidifies, while the medium is at 40 &#176;C or above.</li>
		<li>Leave the tubes undisturbed for several hours until the agar solidifies and the OAI becomes apparent,&#160;materializing as a pink to colorless interface, approximately 1 to 3 cm below the surface of the medium. 
		NOTE: The medium should slowly turn colorless in the tube. The amount of time needed for this transition is variable and depends on the amount of O2 initially dissolved in the medium.</li>
	</ol>
	</li>
</ol>
3. Inoculation of MTB
NOTE: The cultures of strains AMB-1, MS-1 and MSR-1 can be obtained commercially (Table of Materials).
CAUTION: Perform all the following steps in sterile conditions, under the flame of a Bunsen burner.
<ol>
	<li>Inoculation of strains MSR-1, AMB-1 and MS-1 in liquid medium
	<ol>
		<li>Seal an empty 125 mL serum bottle with a butyl-rubber stopper and an aluminum crimp seal. Insert two needles in the bottle through the stopper, and connect a syringe prepared as in Step 1.7 to one of them. Connect the syringe to a cylinder of O2 through the same type of tubing as the one used for the N2 station.</li>
		<li>Let O2 flow through the bottle for about 30 min, to ensure that all air in the bottle is replaced by O2. Remove both needles, allowing for a slight overpressure in the bottle and then autoclave. Allow the bottle to cool down to room temperature before use. 
		NOTE: To save time, perform Steps 3.1.1 and 3.1.2 during the medium preparation and autoclave the bottle along with the medium or the stock solutions.</li>
		<li>Sterilize the tops of the stoppers of both the fresh medium bottle and the O2 bottle by applying a few droplets of 70% ethanol solution on top of them and passing them through the flame of a Bunsen burner.</li>
		<li>Using a sterile syringe and a needle, extract 1 mL of O2 from the O2 bottle and transfer it into the fresh medium bottle. Make sure that the needle tightly fits on the syringe during this step to avoid any air in the syringe.</li>
		<li>If using the inoculum from another culture grown in a glass bottle, sterilize the stoppers of both the fresh medium bottle and the older culture bottle by applying a few droplets of 70% ethanol solution on top of them and passing them through the flame of a Bunsen burner. If using the inoculum from a tube of frozen culture, just let it warm up to room temperature with the tube sealed under the flame of a Bunsen burner.</li>
		<li>If using the inoculum from another culture grown in a glass bottle, inoculate 1 mL of the older culture into the fresh medium. If using the inoculate from a frozen stock, inoculate only 0.1 mL to dilute the glycerol or dimethyl sulfoxide (DMSO) used in the freezing process. In both cases, use a sterile needle and a sterile syringe.</li>
		<li>Incubate the culture at 32 &#176;C and inoculate it into fresh medium after 4 to 7 days.</li>
	</ol>
	</li>
	<li>Inoculation of MSR-1 in O2 gradient semi-solid medium
	<ol>
		<li>Verify that the tube of fresh medium displays a well-defined OAI, materialized by a pink to colorless interface.</li>
		<li>If the inoculum is coming from another O2 concentration gradient semi-solid culture, harvest the bacteria by pipetting 50 &#181;L of the culture with a sterile pipette tip placed on the band formed by the bacteria. Slowly inoculate these bacteria at the OAI in the fresh medium (pink/colorless interface), avoiding disturbing the interface. If the inoculum is coming from a frozen culture, proceed in the same way with 100 &#181;L of inoculum instead.</li>
		<li>Seal the tube and let the bacteria grow between 25 &#176;C and 30 &#176;C. Transfer into fresh medium using the same procedure before the band of bacteria reaches the surface of the medium.</li>
	</ol>
	</li>
</ol>
4. Observation of the Bacteria
<ol>
	<li>Sterilize the stopper of the culture bottle by applying 70% ethanol solution on top of it and passing it through the flame of a Bunsen burner. For semi-solid medium, open the tube under the flame of a Bunsen burner. Use a sterile needle and a sterile syringe to extract the bacteria.</li>
	<li>Use the hanging drop method8 to ensure that the bacteria are both magnetic and motile. 
	NOTE: Phase contrast microscopy gives great results but is not mandatory. Magnifications ranging from 10X to 60X are suitable.</li>
	<li>Use transmission electron microscopy (TEM) to observe the cell structure and the magnetosomes in detail8.</li>
</ol>

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The authors have nothing to disclose.

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 MS-1 do not grow under fully aerobic conditions6. For the semi-solid culture of MSR-1, all O2 is first reduced by the cysteine solution and then the O2 concentration gradient appears, leading to the formation of the OAI. If a culture does not grow well or is not magnetic, the O2 concentration should be the first thing to check.
Semi-solid growth medium is an interesting choice for isolating unknown MTB or growing the species that are highly sensitive to both O2 and redox gradients, as it ensures the existence of stable gradients and allows the bacteria to position themselves at the location offering the best growth conditions. Liquid medium is more convenient to work with when higher volumes are needed (e.g., for DNA extraction), or when further analysis needs to be conducted on the cells (e.g., magnetosome studies) as it allows for an easier separation of the cells from the growth medium. The agar in semi-solid medium might indeed interfere with the cell harvesting technique.
In some cultures, the cells sometimes become non-motile, likely due to spontaneous mutations. Non-motile cells survive and grow in the growth medium since they do not need to swim to find nutrients necessary for their survival. The standard race-track method10 can then be used to recover a motile culture since only motile cells can swim down the race-track. The loss of the magnetic phenotype can also happen in the culture even if the O2 concentration is optimal, again due to spontaneous mutations11. The best solution in that case is to start a new culture by inoculating frozen stocks of the wild-type bacteria into fresh medium. Alternatively, the race-track technique can also allow the recovery of a magnetic culture.
It is essential to avoid contamination of the culture by other organisms and therefore most of the protocol must be performed using sterile techniques. Manipulating under the flame of a Bunsen burner usually gives satisfactory results in keeping aseptic conditions. However, if the culture is grown in a contamination-prone environment, additional precautions should be taken, such as working in a laminar flow hood when preparing the medium and inoculating the bacteria. It should be noted that the risk of contamination is higher in semi-solid medium as the tubes need to be opened every time the cells are removed.
These protocols enable the growth of enough bacteria to perform many different types of experiments, such as physical studies based on optical microscopy12,13, electron microscopy imaging14,15, X-ray spectromicroscopy analyses16, genomic and protein studies17,18. If necessary, higher concentrations of bacteria in suspension can be achieved by centrifugation. In addition, magnetosomes can be extracted and purified for further applications from cell pellets or dense cell suspensions obtained by centrifugation19.
Growth media formulations are usually based on the same governing principles (see Supplementary Table 1 for a summary of the list of the role of each component in the growth media described here). A carbon source must be available to the bacteria, via organic acids (e.g., succinate, acetate, tartrate, lactate) or inorganic compounds such as bicarbonate. The choice of a suitable carbon source depends on the metabolism of the bacteria. Nitrogen present in DNA and proteins, and phosphorus (DNA, membranes) are also required and provided by NaNO3, NH4Cl and KH2PO4 in the recipes described here. A buffer (HEPES, phosphate buffer) is necessary to resist pH changes. The trace mineral supplement solution contains cofactors for enzymes, and the vitamins can be used by the bacteria as coenzymes or as functional groups for certain enzymes. Yeast extract and soy bean peptone provide a source of amino acids and salts used for protein production. Since MTB are redox sensitive, one or several reducing agents must often be added to the medium (e.g., cysteine, ascorbic acid, lactate). A major source of iron (e.g., ferric citrate, ferric quinate, iron chloride) is needed in the medium for biomineralization. Finally, a small amount of O2 is required for microaerophilic bacteria as terminal electron acceptor. Obligate anaerobic MTB use other compounds such as nitrate or nitrous oxide instead.
The principal limitation of these protocols is that there is no guarantee that they will work for other species of MTB. Strains AMB-1, MS-1 and MSR-1 are all freshwater MTB and therefore the recipes described in this article cannot be used to grow marine magnetospirilla such as&#160;Magnetospira thiophila strain MMS-1, Magnetospira strain QH-2, or other marine MTB, which require higher salt concentrations. However, to grow other strains of MTB, and for the isolation of new strains, the general methods presented in this article for both liquid and semi-solid media can be used to prepare media with different recipes.

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 magnetosome and is ranging in size from about 30 to 50 nm in Magnetospirillum species3. Because of the chain arrangement of magnetosomes, these bacteria possess a permanent magnetic dipole moment that makes them align passively with externally applied magnetic fields. Therefore, these bacteria actively swim along magnetic field lines, acting as self-propelled micro-compasses presumably to more effectively locate the most favorable conditions (e.g., O2 concentration) for growth.
An interesting property of MTB is their ability to regulate both the chemistry and the crystallography of their magnetosome crystals. Most strains produce relatively high purity crystals of either magnetite or greigite, although some biomineralize both minerals4. In all cases, the bacteria are able to precisely control the size and the shape of their single magnetic domain crystals. This explains why a great amount of research is undertaken to develop a better understanding of how MTB perform this biomineralization process. Understanding this process might allow the researchers to tailor-make magnetic nanocrystals for many commercial and medical applications.
A substantial obstacle to extensive research on MTB has been the difficulty of growing them in the laboratory. Most species, including the strains used in this work, are obligately microaerophilic when grown with O2 as a terminal electron acceptor. This explains why these bacteria are most often found at the transition zone between oxic and anoxic conditions (the oxic-anoxic interface, OAI). This clearly shows that MTB have precise O2 concentration requirements which obviously needs to be taken into account when devising growth media for these organisms. Moreover, the great existing diversity of MTB implies that different strains will need different types of chemical gradients and nutrients to achieve optimal growth.
In this work, we describe the methods for growing three of the most widely studied MTB: Magnetospirillum magneticum (strain AMB-1), M. magnetotacticum (MS-1) and M. gryphiswaldense (MSR-1). These species phylogenetically belong to the Alphaproteobacteria class in the Proteobacteria phylum, are helical in morphology and possess a polar flagellum at each end of the cell. We provide the protocols for growing strain MSR-1 in both liquid and O2 concentration gradient semi-solid media, based on previously published medium recipes5,6. We also present a detailed protocol for growing strains AMB-1 and MS-1 in modified Magnetic Spirillum Growth Medium (MGSM)7.

<table><tbody><tr><td>AMB-1</td><td>American Type Culture Collection (ATCC)</td><td>ATCC 700264</td><td></td></tr><tr><td>MS-1</td><td>ATCC</td><td>ATCC 31632&amp;nbsp;</td><td></td></tr><tr><td>MSR-1</td><td>Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)</td><td>DSM 6361</td><td></td></tr><tr><td>Ferric citrate</td><td>Sigma-Aldrich</td><td>F3388-250G</td><td></td></tr><tr><td>Trace mineral supplement</td><td>ATCC</td><td>MD-TMS</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD</td><td>PX1565-1</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>EMD</td><td>MX0070-1</td><td></td></tr><tr><td>HEPES</td><td>BioShop Canada Inc</td><td>HEP001.250</td><td></td></tr><tr><td>NaNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5506-250G</td><td></td></tr><tr><td>Yeast extract</td><td>Fischer scientific</td><td>DF210929</td><td></td></tr><tr><td>Peptone</td><td>Fischer scientific</td><td>DF0436-17-5</td><td></td></tr><tr><td>Potassium L-lactate solution (60%)</td><td>Sigma-Aldrich</td><td>60389-250ML-F</td><td></td></tr><tr><td>D-(-)-Quinic acid</td><td>Sigma-Aldrich</td><td>138622</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fischer scientific</td><td>I88-100</td><td></td></tr><tr><td>Vitamin supplement</td><td>ATCC</td><td>MD-VS</td><td></td></tr><tr><td>Sodium succinate hexahydrate</td><td>Fischer scientific</td><td>S413-500</td><td></td></tr><tr><td>Sodium L-tartrate dibasic dihydrate</td><td>Sigma-Aldrich</td><td>228729-100G</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>EMD</td><td>SX0255-1</td><td></td></tr><tr><td>Resazurin</td><td>Difco</td><td>0704-13</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544-25G</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Caledon</td><td>6620-1-65</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt; .4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>44939-250G</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>EMD</td><td>SX0320-1</td><td></td></tr><tr><td>NaCl</td><td>Caledon</td><td>7560-1</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>EMD</td><td>1011450500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;.2 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>EMD</td><td>1023820500</td><td></td></tr><tr><td>Agar A</td><td>Bio Basic Canada Inc</td><td>FB0010</td><td></td></tr><tr><td>L-cysteine.HCl.H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C7880-100G</td><td></td></tr><tr><td>1.0 mL syringes</td><td>Fischer scientific</td><td>B309659</td><td></td></tr><tr><td>25G&amp;nbsp; x 1 needles</td><td>BD</td><td>305125</td><td></td></tr><tr><td>125 mL serum bottles</td><td>Wheaton</td><td>223748</td><td></td></tr><tr><td>20 mm aluminum seals</td><td>Wheaton</td><td>224223-01</td><td></td></tr><tr><td>20mm E-Z Crimper</td><td>Wheaton</td><td>W225303</td><td></td></tr><tr><td>Butyl-rubber stoppers</td><td>Bellco Glass, Inc.</td><td>2048-11800</td><td></td></tr><tr><td>Hungate tubes</td><td>Chemglass (VWR)</td><td>CLS-4208-01</td><td></td></tr><tr><td>Septum stopper, 13mm, Hungate</td><td>Bellco Glass, Inc.</td><td>2047-11600</td><td></td></tr><tr><td>Glass culture Tubes</td><td>Corning (VWR)</td><td>9826-16X</td><td></td></tr><tr><td>Hydrochloric acid 36.5-38%, BioReagent</td><td>Sigma-Aldrich</td><td>H1758-100ML</td><td>11.6 - 12 N</td></tr></tbody></table>

growing magnetotactic bacteria of the genus magnetospirillum: strains msr-1, amb-1 and ms-1

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.

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 formation of an OAI after a few hours, indicated by the presence of 1&#8211;3 cm of pink medium at the top of the tube (Figure 3, tube A). The rest of the medium should be colorless. An all-pink medium before inoculation is a sign of complete oxidation of the resazurin and therefore of an unsuccessful preparation of the O2 concentration gradient.
Successful growth in liquid medium can be checked about 48 h after inoculation (or after a few days if the inoculum is coming from a frozen culture) simply by examining the cultures for turbidity using a light source or a spectrophotometer. Successful growth is confirmed by turbidity proving that the bacteria actually grew (Figure 2b). If the medium remains clear after 48 h, the bacteria have not grown. For a liquid medium culture growing normally, the presence of bacteria synthesizing magnetosomes can be checked after 48 h by placing the bottle of medium on a magnetic stir plate and by illuminating it with a flashlight. At a low stirring speed, the medium should &#34;flash&#34;, looking alternately darker and brighter depending on the orientation of the stirring magnet with respect to the position of the experimentalist. For a non-magnetic culture, the intensity of light scattered by cells towards the experimentalist will remain constant.
Successful growth in semi-solid medium is indicated by the formation of a microaerophilic band of bacteria at the pink/colorless interface. Due to the consumption of O2 by the bacteria and the changes in the O2 gradient, the band will slowly migrate to follow the OAI (Figure 3, tube B).
The hanging drop method enables the observer to check that the bacteria are both magnetic and motile. After a few minutes, MTB should concentrate at the edge of the hanging drop, proving that they actively swim along the magnetic field lines (Figure 4a). To ensure that the cells are magnetic, flip the orientation of the magnet sitting next to the drop. The bacteria should start swimming towards the opposite edge (Figure 4b). Finally, the shape of the magnetosomes can be checked with TEM. Cuboctahedral crystals of about 30&#8211;45 nm in diameter should be observed9. These crystals are normally arranged in one long chain or multiple shorter chains in the species studied here (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58536/58536fig1.jpg" /> 
Figure 1: Illustration of the N2 gassing station. (a) Schematic representation. (b) Picture of the setup. A successful station will have the appropriate length of large and thin tubing so that the end of the thin tubing described in Step 1.9 of the protocol remains immersed in the medium during the gas bubbling step. The flow of gas should also be adjustable at all times, to ensure that all O2 is removed and that the medium does not spill. <a href="https://www.jove.com/files/ftp_upload/58536/58536fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58536/58536fig2.jpg" /> 
Figure 2: Bottles of liquid growth medium before and after inoculation. (a) Clear MSR-1 medium before inoculation. (b) Turbid MSR-1 medium after 3 days of successful growth. <a href="https://www.jove.com/files/ftp_upload/58536/58536fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58536/58536fig3.jpg" /> 
Figure 3: Tubes of O2 gradient semi-solid medium before (tube A) and 10 days after (tube B) inoculation. The band of bacteria in tube B is indicated by a red arrow.&#160;
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58536/58536fig4.jpg" /> 
Figure 4: Typical results of a hanging drop experiment at 20X magnification using phase contrast microscopy. (a) MTB swim towards the south pole of a magnet and accumulate at the liquid/air interface. (b) 1 min after flipping the magnet, most cells have left the field of view.&#160;
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58536/58536fig5.jpg" /> 
Figure 5: TEM observation of an AMB-1 cell. Magnetosome chains are indicated by red arrows. The two flagella are indicated by black arrows.
Supplementary Table 1.&#160;<a href="https://www.jove.com/files/ftp_upload/58536/SupplementaryTable1-RoleOfIngredients.docx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1 at JoVE.com

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

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.

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

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

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Nanyang Technological University

Research

JoVE Journal

Biology

15.4K Views.  McMaster University. 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. 

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

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Environment

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

Chemistry

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Immunology and Infection

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Bioengineering

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

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

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Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria