Name: identification of fatty acids in bacillus cereus
Uploaded: 2016-12-05T15:00:00
Description: Watch this Scientific Journal Video about Identification of Fatty Acids in Bacillus cereus at JoVE.com

Authors are grateful to Thomas Mison for his technical support, and to Rachel Kopec for revising the manuscript.

1. Bacterial Cultures
<ol>
	<li>Prepare a lawn of the bacteria (Bacillus cereus strain ATCC 14579) by spreading 100 &#181;l of an overnight culture of the strain incubated at 30 &#176;C in LB (Luria-Bertani medium), over the surface of a plate of LB agar medium. Incubate the plate overnight at 30 &#176;C.</li>
</ol>
2. ECL: Equivalent Chain Length
<ol>
	<li>Calculate ECL as follows:&#160; <img alt="Equation" src="/files/ftp_upload/54960/54960eq1.jpg" /> with: 
	i, the solute of i...

<ol>
	<li>Christie, W. W., Han, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lipid Analysis 4th Edition. , (2010).</li><li>H&#220;bschmann, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of GC-MS: fundamental and application. Third edition. , (2015).</li><li>Sasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Bacteria+by+Gas+Chromatography+of+Cellular+Fatty+Acids.">Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids.</a> MIDI Technical note. 101, 1-6 (1990).</li><li>Spitzer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+analysis+of+fatty+acids+by+gas+chromatography+-+Low+resolution+electron+impact+mass+spectrometry+of+their+4,4-dimethyloxazoline+derivatives+-+A+review.">Structure analysis of fatty acids by gas chromatography - Low resolution electron impact mass spectrometry of their 4,4-dimethyloxazoline derivatives - A review.</a> Prog Lipid Res. 35 (4), 387-408 (1996).</li><li>Harvey, D. J., Christie, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in lipid methodology. Volume 1. , 19-80 (1992).</li><li>Diomande, S. E., Nguyen-The, C., Guinebreti&#232;re, M. -. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+CasK/R+two-component+system+in+optimal+unsaturation+of+the+Bacillus+cereus+fatty+acids+during+low-temperature+growth.">Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth.</a> Int J Food Microbiol. 213, 110-117 (2015).</li><li>Berkeley, R. C. W., Heyndrickx, M., Logan, N., De Vos, P., Berkeley, R. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applications and Systematics of Bacillus and Relatives. , 1-7 (2002).</li><li>K&#228;mpfer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limits+and+Possibilities+of+Total+Fatty+Acid+Analysis+for+Classification+and+Identification+of+Bacillus+Species.">Limits and Possibilities of Total Fatty Acid Analysis for Classification and Identification of Bacillus Species.</a> System. Appl. Microbiol. 17 (1), 86-98 (1994).</li><li>Kaneda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty-acids+of+genus+bacillus+-+example+of+branched-chain+preference.">Fatty-acids of genus bacillus - example of branched-chain preference.</a> Bacteriol Rev. 41 (2), 391-418 (1977).</li><li>Chazarreta Cifre, L., Alemany, M., de Mendoza, D., Altabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+Biosynthesis+of+Unsaturated+Fatty+Acids+in+Bacillus+cereus+ATCC+14579+and+Functional+Characterization+of+Novel+Acyl-Lipid+Desaturases.">Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases.</a> Appl Environ Microbiol. 79 (20), 6271-6279 (2013).</li><li>Sasser, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Bacillus+anthracis+from+culture+using+gas+chromatographic+analysis+of+fatty+acid+methyl+esters.">Identification of Bacillus anthracis from culture using gas chromatographic analysis of fatty acid methyl esters.</a> J AOAC Int. 88 (1), 178-181 (2005).</li><li>Schutter, M. E., Dick, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fatty+acid+methyl+ester+(FAME)+methods+for+characterizing+microbial+communities.">Comparison of fatty acid methyl ester (FAME) methods for characterizing microbial communities.</a> Soil Sci Soc Am J. 64 (5), 1659-1668 (2000).</li><li>Destaillats, F., Angers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+methodology+for+the+synthesis+of+FA+picolinyl+esters+from+intact+lipids.">One-step methodology for the synthesis of FA picolinyl esters from intact lipids.</a> J Am Oil Chem Soc. 79 (3), 253-256 (2002).</li><li>Fay, L., Richli, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+double-bonds+in+polyunsaturated+fatty-acids+by+gas-chromatography+mass-spectrometry+after+4,4-dimethyloxazoline+derivatization.">Location of double-bonds in polyunsaturated fatty-acids by gas-chromatography mass-spectrometry after 4,4-dimethyloxazoline derivatization.</a> J Chromatogr. 541 (1-2), 89-98 (1991).</li><li>Zhang, J. Y., Yu, Q. T., Liu, B. N., Huang, Z. 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K., Mikolajczak, K. L., Earle, F. R., Wolff, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatographic+characterization+of+fatty+acids.Identification+constants+for+mono-+and+dicarboxylic+methyl+esters.">Gas chromatographic characterization of fatty acids.Identification constants for mono- and dicarboxylic methyl esters.</a> Anal Chem. 32 (13), 1739-1742 (1960).</li><li>van Den Dool, H., Kratz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generalization+of+the+retention+index+system+including+linear+temperature+programmed+gas-liquid+partition+chromatography.">A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography.</a> J Chromatogr A. 11, 463-471 (1963).</li><li>Stransky, K., Jursik, T., Vitek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+equivalent+chain+length+values+of+monoenic+and+polyenic+(methylene+interrupted)+fatty+acids.">Standard equivalent chain length values of monoenic and polyenic (methylene interrupted) fatty acids.</a> J High Res Chromatogr. 20 (3), 143-158 (1997).</li></ol>

The FAs chromatogram profiles shown in Table 1 correspond to B. cereus ATCC 14579 grown on an agar plate surface. Similar profiles were obtained when the bacterium was grown in aerated liquid media at the same temperature8. In the case of bacteria grown in liquid media, the bacterial biomass is collected by centrifugation of the growth medium and can be washed according to previously described protocols depending on the growth conditions8,19. The identification of the vario...

Fatty acid methyl ester (FAME) gas chromatography (GC) is an essential method for lipid characterization. It rapidly separates and quantifies the various fatty acids (FAs) of a sample after a short extraction step. Derivatives of methyl esters are highly volatile, stable and inert toward the chromatographic column, thereby avoiding tailing peaks. Their identification is rather straightforward when the sample consists of well-known FAs because the chromatographic profiles are either published or compared to standards. In addition, the repeated injection of calibration standards for quantification of various FAs is not required, given their almost constant response to flame ionization detection (FID)1.
In addition to FID, mass spectrometry (MS) detection provides a complementary set of information to confirm FAMEs. However, when FAMEs are charged using electron ionization (EI), the resulting spectra do not always allow for the identification of FA fine structure. For instance, branching position (i.e., a branched methyl group) is difficult to predict because the diagnostic ions are difficult to detect1 and the characteristic change in target ion abundance is machine-dependent, preventing the use of mass spectra libraries2. Another challenge lies in identifying the double bond position because EI causes double bond migration. Thus, FA isomers with varying double bond positions cannot be differentiated by their mass spectra. Fortunately, other tools have been developed for FA identification. For instance, the presence and the position of branching or of double bonds in FAs can be conjectured by calculating the equivalent chain length (ECL)3.
Other derivatization methods result in different mass spectra, dependent on the location of a double bond or a branched methyl group. 4,4-Dimethyl oxazoline derivatives (DMOX)4 allow for easy identification of the position of monounsaturated fatty acid double bonds. 3-pyridylcarbinyl ester (picolinyl ester) derivatives allow for the unambiguous identification of the location of methyl branched FAs5. Combining chromatographic retention (ECL) and mass spectra (DMOX and picolinyl) information allows for the identification of most FAs without the need to use complex methods of purification, as required for nuclear magnetic resonance (NMR) spectrum, the uncontestable method for structural characterization1.
Bacteria of the genus Bacillus, which include some human and animal pathogens, are able to colonize highly diverse niches and are therefore widely distributed in the environment6. Among the Bacillus genus, FA composition is influenced by the ecological niche of the species with modulations in FA patterns to adapt to a wide range of environmental changes (e.g., growth medium, temperature, pH, etc.)7-9. Because of the relative homogeneity of the FA pattern across species of the genus Bacillus during growth in standardized conditions, determination of FA composition is one of the essential criteria used to define the Bacillus species. A unique attribute of the Bacillus genus is the abundance of branched-chain FAs containing 12-17 carbons10-12 with the ratio between iso and anteiso isomers being a key determinant of adaptation to environmental conditions. Bacillus species also adapt to environmental fluctuations by altering the proportion of unsaturated fatty acids. In some species, such as Bacillus cereus, two fatty acid desaturases create double bonds in different positions of the alkyl chain13 with different roles in adaptation9. The example of the Bacillus genus illustrates the importance of precisely identifying the double bond position and FA branching. Collectively, identification of Bacillus FA patterns has several useful applications. Herein, we propose a novel GC-MS approach for Bacillus FA pattern identification that overcomes the inherent limitations of a classical GC-MS analysis.
This innovative approach can be used directly on raw biological material, and consists of a combination of existing techniques: information on retention times (ECL) and mass spectra of different FAs derivatives (FAME, DMOX and picolinyl-ester).
We use the following FA nomenclature. i, a, and n indicate iso, anteiso methyl branched, and straight-chain fatty acid, respectively. Unsaturated FAs were named by C:d where C is the number of carbon atoms in the fatty acid and d is the number of double bonds. &#916;x indicates the position of the double bond, where the double bond is located on the xth carbon-carbon bond, counting from the carboxylic acid end.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GC/MS</td>
			<td>Shimadzu</td>
			<td>QP2010</td>
			<td></td>
		</tr>
		<tr>
			<td>capillary column ZB WAX</td>
			<td>Phenomenex</td>
			<td>7HG-G007-11</td>
			<td>30m x 0.25mm x 0.25&#181;m</td>
		</tr>
		<tr>
			<td>Methanol Lichrosolv</td>
			<td>VWR</td>
			<td>1.06018.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>Aldrich</td>
			<td>P1767</td>
			<td></td>
		</tr>
		<tr>
			<td>THF</td>
			<td>Hipersolv Chromanorm</td>
			<td>28559.320</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Hipersolv Chromanorm</td>
			<td>23373.320</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Hipersolv Chromanorm</td>
			<td>24575.320</td>
			<td></td>
		</tr>
		<tr>
			<td>3-pyridinemethanol</td>
			<td>Aldrich</td>
			<td>P6-680-7</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium tertiobutoxide</td>
			<td>Aldrich</td>
			<td>156671</td>
			<td></td>
		</tr>
		<tr>
			<td>2-amino-2-methyl-1-propanol</td>
			<td></td>
			<td>A-9879</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ Academic</td>
			<td>Millipore</td>
			<td>ZMQS50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Acid Methyl Ester (BAME) Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47080-U Supelco</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of fatty acids in bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

The strategy of FA identification from bacterial cells is presented in Figure 1. Each step provides complementary spectral information or information about chromatographic retention. Step 1 consists of preliminary FA identification using a standard solution. Step 2 allows for the interpretation of FAME EI spectra and their ECL, in order to tentatively identify the products. Step 3 identifies the exact branching location in branched chain-FAs. Finally, step 4 identifies th...

Watch this Scientific Journal Video about Identification of Fatty Acids in Bacillus cereus at JoVE.com

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

The overall goal of this protocol is to identify fatty acids from bacteria and to identify the position of methyl branching and double bonds. This method can help answer key questions in the bacteriology field such as the role of fatty acids in the way bacteria adapt to their environment. The main advantage of this technique is that prolification of fatty acids is not required.The implications of this technique extend toward bacterial identification because of the importance of fatty acid composition in bacterial taxonomy. Though this method was developed on the bacteria Bacillus cereus, it is also very relevant to study other species containing branch chain fatty acids. Generally, in development to this method, we struggled.Because current identification method for fatty acids do not identify methyl branching in double bond position. To begin this procedure, prepare a lawn of the bacteria by spreading 100 microliters of an overnight culture of the strain, incubated at 30 degrees Celsius in LB over the surface of the plate of LB agar medium. Incubate the plate overnight at 30 degrees Celsius.In order to obtain lipid fatty acids, harvest bacterial cells by scraping colonies from the agar plate. And transferring approximately 40 milligrams into a ten milliliter glass tube with a screw cap and PTFE seal. To perform transesterification, add five milliliters of 0.2 molar potassium hydroxide in methanol to the fresh bacterial cells.Vortex, and incubate at 37 degrees Celsius for one hour. Following this, add one milliliter of one molar ascetic acid to lower the pH to seven. Now, add three milliliters of hexane and vortex one minute to extract the fatty acid methyl esters or FAMEs.Transfer the upper phase into clean tubes and concentrate by evaporation at room temperature under a continuous flow of nitrogen to obtain approximately 200 microliters of FAME extract. Then, transfer the extracts into GC vials with inserts. Inject the extracts into a gas chromatography mass spectrometry, or GC-MS system equipped with a capillary column.Set the injection port temperature to 250 degrees Celsius. Hold the oven temperature at 50 degrees Celsius for one minute. Increase to 190 degrees Celsius at a rate of 20 degrees Celsius per minute and increase to a final temperature of 230 degrees Celsius at a rate of two degrees Celsius per minute.For the MS, record the mass spectra by electron ionization, or EI, at 70 electron volts, and set the acquisition of the total ion current between 50 and 400 atomic mass units, or AMU. Dissolve a sample of the dried FAME extract in one milliliter of dry dichloromethane. Then, add the extract solution and 0.2 milliliters of 3-pyridinemethanol to 0.1 milliliters of one molar potassium tert-butoxide in tetrahydrofuran.Heat the solution at 40 degrees Celsius for 30 minutes in a closed vial. After cooling the solution to room temperature, add two milliliters of purified deionized water and four milliliters of hexane. Mix the solution for one minute with a vortex mixer.After allowing the phases to separate, collect the upper organic phase. Next, add anhydrous sodium sulfate until the organic phase is clear. Transfer the organic phase into a clean tube, then evaporate the organic phase under a stream of nitrogen until reaching a volume of approximately 200 microliters.Add 250 milligrams of 2-amino-2-methyl-1-propanol to sample of dried FAME extract. Flush the tube with nitrogen and close it with a stopper. Then, heat the solution at 190 degrees Celsius overnight.After cooling the solution to room temperature, add three milliliters of dichloromethane and five milliliters of purified deionized water. Vortex the mixture and allow the phases to separate. Then, remove the aqueous phase.Following this, wash the organic phase with five milliliters of water. Add anhydrous sodium sulfate until the organic phase is clear. After transferring the organic phase to a new test tube, evaporate it under a stream of nitrogen until reaching a volume of approximately 200 microliters.The I16 picolinal derivative mass spectrum confirms methyl branching. The gap of 28 AMU between ions at 304 and 332 corresponds to fragments created before and after the branched carbon. The diagnostic ions, 113 and 126, characteristic of DMOX derivatives, are shown here.The molecular ion 307 is characteristic of the 16:1 isomer, and a gap of 12 AMU between 196 and 208 indicates a double bond at carbon. The gap of 12 AMU is no longer observed when the double bond is before carbon 7. The intense 153 ion, characteristic of a double bond at carbon 5, and the 307 molecular ion in the DMOX derivative mass spectrum identified this compound as n16 one delta five.The 307 ion of the DMOX derivative and the 345 ion of the picolinal derivative indicate a 16:1 fatty acid isomer. A gap of 28 AMU indicates the branching position, and a gap of 12 AMU for DMOX and 26 AMU for picolinal ester identifies the double bond. Fatty acids and corresponding diagnostic ions identified in Bacillus cereus are listed here.The diagnostic ions identify the methyl branching and double bond positions on the carbon chain for the DMOX and picolinal ester derivatives. Once mastered, this technique can be done in two days, if it is performed properly. Following this procedure, fatty acid can be quantified by GC-MS and the release of the FAME derivatives.After it's development, this technique paved the way for researchers in the field of bacteriology to explore bacterial diversity and mechanisms of bacterial adaptations. After watching this video, you should have a good understanding of how to study fatty acids in bacteria including methyl branched and unsaturated fatty acids. Don't forget that working with reagents can be extremely hazardous, and precautions such as working with a laboratory fume hood and wearing gloves should always be taken while performing this procedure.

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