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>
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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. 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K., Mikolajczak, K. L., Earle, F. R., Wolff, I. 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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 ...

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