Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

December 1993

Abstract

Extraction of the lipids of Legionella pneumophila yields phases unlike those produced from other Gram-negative bacteria. A viscous interface forms between the aqueous (wash) and organic phases. More than half of the hydroxylated fatty acids were found distributed between the aqueous phase and the interfacial material, fractions in which such constituents have not been reported in other Gram-negative species. It was further observed that after the material from the aqueous/interfacial phase was dissolved in methanol or chloroform/methanol (2:1 (V/V)), the addition of acetone would create a white, flocculent precipitate. Analyses showed that the supernatant contained fatty acids that were nonhydroxylated and the precipitate contained both nonhydroxylated and hydroxylated fatty acids. The acetone precipitate could be further purified by column chromatography. Material was eluted from a silicic acid column with sequential additions of chloroform, acetone, and methanol. It was found that the methanol fraction contained the majority of the hydroxylated fatty acid containing material. An improved method for extracting LPS-like material from Legionella pneumophila is presented. This study suggests that LPS-like material can be obtained from L. pneumophila in higher yield (6.4% of total cell weight), of higher purity (as indicated by SDS-PAGE), and by a simpler method than those previously reported. SDS-PAGE profiles of purified (acetone precipitation and column chromatographic separation) LPS-like material extracted with chloroform/methanol (2:1 (V/V)) from L. pneumophila are identical to the previously reported profiles for G. pneumophila LPS. The chemical analyses of the LPS-like material can only account for approximately one-half the isolated material weight. This is suggestive of a moiety that is as of yet undetectable by the means employed to characterize the LPS.

Document Type

Dissertation - Open Access

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