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 interest; 
	n, the carbon number of the straight chain saturated fatty acid methyl ester eluting before solute I; 
	n+1, the carbon number of the straight chain saturated fatty acid methyl ester eluting after solute I; 
	Rti, Rtn, Rt(n+1) the retention times of the FAME peaks described above. 
	&#8203;NOTE: Obtain the retention times of straight chain saturated fatty acids methyl esters by injection of a mixture of standards (BAME).</li>
</ol>
3. FAME Preparation and Analysis
<ol>
	<li>In order to obtain lipid fatty acids, harvest bacterial cells by scraping colonies from the agar plate and transfer 40 mg (fresh weight, equivalent to 109 viable cells) of bacteria into a 10 ml glass tube with screw caps and PTFE seals.</li>
	<li>Perform transesterification via the ester link method14,15 detailed below.
	<ol>
		<li>Add 5 ml of 0.2 M KOH in methanol to the fresh bacterial cells and incubate at 37 &#176;C for 1 hr. This reaction consists of alkaline methanolysis, breaking the ester link in the lipid and producing fatty acid methyl esters.</li>
		<li>Add 1 ml of 1 M acetic acid to lower the pH to 7.0. Check the pH with pH test strips.</li>
		<li>Add 3 ml of hexane to extract FAMEs.</li>
		<li>Transfer supernatant (organic phase) into clean tubes and concentrate by evaporation at room temperature under a continuous flow of nitrogen to obtain approximately 200 &#181;l of extract. Transfer the sample into a GC vial with insert.</li>
		<li>Inject extracts into a gas chromatography-mass spectrometry (GC-MS) system.</li>
	</ol>
	</li>
</ol>
4. GC/MS Conditions
<ol>
	<li>Inject FAME samples into a GC-MS instrument equipped with a capillary column ZB-WAX (length, 30 m; diameter, 0.25 mm; film thickness, 0.25 &#181;m).</li>
	<li>Set the injection port (in splitless mode) temperature to 250 &#176;C. Use helium as a carrier gas, with a linear velocity of 37 cm/sec. Hold the oven temperature at 50 &#176;C for 1 min, increase to 190 &#176;C at a rate of 20 &#176;C/min, and increase further to a final temperature of 230 &#176;C at a rate of 2 &#176;C/min.</li>
	<li>For the MS, record the mass spectra by electron ionization (EI) at 70 eV, and set the acquisition of the total ion current between 50 and 400 atomic mass units (amu) (2 scans/sec).</li>
	<li>When required, inject DMOX and picolinyl derivatives under the same condition except the temperature program oven as follows: 
	DMOX: 50 &#176;C (1 min), 20 &#176;C/min until 210 &#176;C and 2 &#176;C/min until 240 &#176;C (5 min); 
	Picolinyl: 6 &#176;C (1 min), 20 &#176;C/min until 220 &#176;C and 2 &#176;C/min until 250 &#176;C (20 min).</li>
</ol>
5. Picolinyl Ester Preparation from FAME16
<ol>
	<li>Evaporate the FAME extract from Section 3 with a flow of nitrogen (at least 10 mg dry material) and dissolve in 1 ml of dry dichloromethane.</li>
	<li>Prepare a 1.0 M solution of potassium tert-butoxide in tetrahydrofuran.</li>
	<li>Add the FAME extract and 0.2 ml 3-pyridinemethanol to 0.1 ml of solution made in step 5.2.</li>
	<li>Heat the solution at 40 &#176;C for 30 min in a closed vial.</li>
	<li>After cooling to room temperature, add purified deionized water (2 ml, see Materials Table) and hexane (4 ml). Mix with a vortex, allow phase to separate, and collect the organic phase.</li>
	<li>Dry it by adding anhydrous sodium sulfate until the organic phase is perfectly clear. Transfer it into a clean tube. Then evaporate to 200 &#181;l. Transfer the sample into a GC vial with insert.</li>
</ol>
6. DMOX Preparation from FAME17
<ol>
	<li>Evaporate the FAME extract from Section 3 with a flow of nitrogen (at least 10 mg dry material).</li>
	<li>To the FAME dry extract, add 250 mg of 2-amino-2-methyl-1-propanol. Flush the vessel with nitrogen, add a stopper, and place it in a heating block overnight at 190 &#176;C.</li>
	<li>After cooling to room temperature, add 3 ml dichloromethane to the tube, and 5 ml purified deionized water (See Materials Table).</li>
	<li>Shake for phase separation and then remove the aqueous phase.</li>
	<li>Wash the organic phase with 5 ml water. Shake for phase separation and then remove the aqueous phase.</li>
	<li>Dry by adding anhydrous sodium sulfate until the organic phase is perfectly clear and transfer it into a clean tube. Evaporate under a stream of nitrogen until reaching a volume of 200 &#181;l. Transfer the sample into a GC vial with insert.</li>
</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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E., Dick, R. 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Y., Yu, Q. T., Liu, B. N., Huang, Z. 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K., Mikolajczak, K. L., Earle, F. R., Wolff, I. 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The authors declare that they have no competing financial interests.

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><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>30 m x 0.25 mm x 0.25&amp;nbsp;&amp;micro;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...

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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 ...

identification-of-fatty-acids-in-bacillus-cereus

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Biochemistry

9.6K Views.  INRA, Université d'Avignon. 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). 

Video: Identification of Fatty Acids in Bacillus cereus

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Environment

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

Chemistry

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Immunology and Infection

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Biology

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and ...

Identification of Fatty Acids in Bacillus cereus

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