Date of Award:

5-1-1999

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Jon Y. Takemoto

Committee

Jon Y. Takemoto

Committee

Joseph K.-K. Li

Committee

Hiroko Hama

Committee

Ilka Nemere

Committee

Thomas A. Grover

Abstract

The bacterium Pseudomonas syringae pv. syringae produces an antifungal secondary metabolite, syringomycin E. Yeast growth inhibition by this lipodepsipeptide involves sterol and sphingolipids of the plasma membrane. Using a sterol auxotroph of Saccharomyces cerevisiae, strain FY-14, it was shown that the predominant sterol in yeast, ergosterol, is not required for the mechanism of action of this metabolite. With ergosterol, cholesterol, β-sitosterol or stigmasterol as the bulk sterol, strain FY-14 was sensitive to syringomycin E with the last two conferring the highest degree of sensitivity. The type of growth-promoting sterol influenced cellular sensitivity to syringomycin E. Growth-promoting ergosterol afforded the least sensitivity to syringomycin E, whereas cholesterol afforded the highest sensitivity. Studies with planar lipid bilayers showed that addition of ergosterol restores the sensitivity to syringomycin E of cholesterol-containing planar lipid bilayers. The binding of syringomycin E to yeast cells was studied using a [14C]radiolabeled analog of syringomycin E. Syringomycin E-resistant mutants with sterol and sphingolipid biosynthetic defects bound the same amounts of analog as syringomycin E-sensitive wild-type strains. On the other hand, yeast sterol auxotroph strain FY-14 bound different amounts of the analog as the type of bulk sterol was varied. Strain FY-14 cells containing bulk cholesterol or ergosterol bound lower amounts compared to those containing bulk β-sitosterol or stigmasterol. The effects of sterol and sphingolipid biosynthetic defects on syringomycin E-cell interactions were investigated using laser confocal indirect immunofluorescent microscopy. Syringomycin E was localized at the cell periphery of wild-type strains for periods up to 25 min following exposure to the lipodepsipeptide. In contrast, in the resistant mutants with defects in sterol and sphingolipid biosynthesis, syringomycin E was found associated with the plasma membrane and internal structures between 5 and 10 min after exposure. Extended incubation after exposure to syringomycin E results in reduced amounts of the lipodepsipeptide in the lipid mutants. Overall, the research demonstrated that sterols and sphingolipids affect the cellular interaction, mechanism of growth inhibition, and stability of syringomycin E in yeast cells.

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