Isolation, Cloning, And Identification of Syringomycin E Action Genes of Saccharomyces cerevisiae

Author

Stephen D. Stock, Utah State University

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

Gregory J. Podgorski

Committee

Daryll B. DeWald

Committee

Jeff Broadbent

Committee

John M. Stark

Abstract

Members of the syringomycin family of antifungal cyclic lipodepsipeptides are produced by Pseudomonas syringae pv. syringae strains as secondary metabolites. These metabolites are potent antifungal agents effective against a broad range of fungi. The antifungal mechanism of action of these compounds has been investigated using syringomycin E and Saccharomyces cerevisiae as a model fungal system. Mutants resistant to the growth inhibitory effects of syringomycin E were isolated after mutagenesis with nitrosoguanidine. These mutants were screened for the production of ergosterol and by complementation with syr2 mutants to eliminate mutants belonging to known syringomycin E-resistant complementation groups. From a total of 58 syringomycin E-resistant mutants, two strains, SS33 and SS56, were shown to be unrelated to known complementation groups. These two strains were further shown to comprise two new and separate syringomycin E-resistant complementation groups. Genes defective in strains SS33 and SS56 were identified by complementation cloning with a yeast genomic library. Two genes were found to complement the genetic defect of strain SS33. These genes, SYR3/FEN1 and SUR4, are required for the elongation of the very long chain fatty acids of S. cerevisiae sphingolipids. The SYR4/IPT1 gene was identified by complementation of strain SS56. Syr4p was functionally identified as the S. cerevisiae mannosyl-diinositolphosphoryl-ceramide (M(IP)₂C) synthase of sphingolipid biosynthesis. The functional identification of SYR4 allowed tests of the role of M(IP)₂C synthesis in SEC14-dependent protein secretion in yeast. SEC14 is required for vesicular transport from the Golgi to the plasma membrane. Elevated M(IP)₂C synthesis had been proposed to play a role in the sac1 bypass of sec14^ts secretion. This work revealed that M(IP)₂C is not involved in this bypass. Instead another lipid, phosphatidylinositol 4-phosphate, was shown to be elevated in the sec14ts/sac1 bypass strain, and this phosphoinositide may play a significant role in SEC14 dependent protein secretion.

Recommended Citation

Stock, Stephen D., "Isolation, Cloning, And Identification of Syringomycin E Action Genes of Saccharomyces cerevisiae" (1999). Biology. 665.
https://digitalcommons.usu.edu/etd_biology/665