<p> This thesis focused on the development of comprehensive, rapid and
simple methodologies for the analysis of fatty acids by gas chromatography mass
spectrometry (GC/MS) and intact lipids by electrospray ionization tandem mass
spectrometry (ESIIMS/MS). The methodologies were applied as a tool for
functional genomics in the soil bacterium Sinorhizobium meliloti. The effects of
inorganic phosphate (Pi)-starvation and acidity on lipid composition were studied. </p> <p> A micro-scale, one-vial method for the analysis of fatty acids as their fatty
acid methyl esters by GC/MS was developed. The method required small sample
sizes, involved minimum handling and avoided tedious extraction steps, which
increased sample throughput. A series of quality controls were included to
measure losses due to handling, derivatization efficiencies and the extent of side
reactions. The method was suitable for the analysis of sensitive bacterial fatty
acids such as cyclopropane fatty acids. </p> <p> A shotgun lipidomics approach was developed for the analysis of intact lipids by ESIIMS/MS. Fatty acid distributions were obtained for eight lipid
classes and up to 58 individual lipids were identified in crude lipid extracts
without sample cleanup or chromatography. For the first time, fatty acid
distributions were provided for non-phosphorus containing lipids using shotgun
lipidomics. Fatty acid distributions within lipid classes suggested that
phospholipids and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine lipids
(TMHSs) were both synthesized from phosphatidic acid while
sulfoquinovosyldiacylglycerol (SLs) had a different biosynthetic origin. </p> <p> The methodologies were applied to study knockout mutants of five genes
thought to participate in lipid metabolism in S. meliloti. It was demonstrated that:
(1) cfa2 gene coded for the main cyclopropane fatty acyl synthase; (2) the plsC
gene coded for a fatty acy 1 transferase specific for C 16 fatty acids in the sn-2
position of phospholipids; (3) a metabolic phenotype was revealed for knockout
mutants of dme and tme genes (DME and TME, malic enzymes) when succinate
was the carbon source. </p> / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/19515 |
| Date | 09 1900 |
| Creators | Saborido Basconcillo, Libia |
| Contributors | McCarry, B.E., Chemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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