The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygendependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2 gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.Syringomycin E is a member of a family of cyclic lipodepsipeptides produced by strains of the plant bacterium Pseudomonas syringae pv. syringae (1). Traditionally regarded as a virulence factor in a variety of bacterial necrotic diseases of plants (2), syringomycin E and its analogs also possess antifungal properties, and it has been suggested that these metabolites are fungal antagonists that aid survival of the producing bacteria on plants (3, 4).How these compounds produce their toxic effects is unknown, but past physiological studies have shown that syringomycin E targets primarily the plasma membrane (1, 5, 6). To further investigate the molecular mechanisms of action of this bioactive compound, resistant mutants of Saccharomyces cerevisiae were isolated to identify genes that encode proteins necessary for growth inhibition by syringomycin E (7). Several of the mutants were deficient in sterols, and one group was complemented by the gene SYR1 (identical to ERG3), which encodes sterol C-5,6 desaturase of the ergosterol biosynthetic pathway (8). These findings, when combined with results from binding (9) and lipid bilayer (10) studies, indicate that sterols influence the interaction of syringomycin E with the target plasma membrane.Syringomycin E action in yeast was more recently shown to require a second, nonsterol biosynthetic gene, SYR2 (11). SYR2 is identical to SUR2, which was identified in a screen for mutants that suppress the impaired recovery of rvs161 strains from nutritional starvation (12). Syringomycin E-resistant syr2 mutants showed altered glycerophospholipid levels, and the SYR2 gene product was localized to the endoplasmic reticulum (11). Nevertheless, the precise function of Syr2p was ...

Syringomycin E is an antifungal cyclic lipodepsinonapeptide that inhibits the growth of Saccharomyces cerevisiae by interaction with the plasma membrane. A screen conducted to find the yeast genes necessary for its fungicidal action identified two novel syringomycin E response genes, SYR3 and SYR4. A syr3 mutant allele was complemented by ELO2 and ELO3. These genes encode enzymes that catalyze the elongation of sphingolipid very long chain fatty acids. Tetrad analysis showed that SYR3 was ELO2. Strains with deletions of SYR3/ELO2 and ELO3 were resistant to syringomycin E, and lipid analyses of both mutants revealed shortened fatty acid chains and lower levels of sphingolipids. SYR4 was identified by Tn5 inactivation of genomic library plasmids that complemented a syr4 mutant allele. SYR4 was found to be identical to IPT1, which encodes the terminal sphingolipid biosynthetic enzyme, mannosyl-diinositolphosphoryl-ceramide synthase. Deletion ⌬syr4/ ipt1 strains were viable, were resistant to syringomycin E, did not produce mannosyl-diinositolphosphorylceramide, and accumulated mannosyl-inositolphosphoryl-ceramide. Accumulation of mannosyl-inositolphosphoryl-ceramide was not responsible for resistance since a temperature-sensitive secretory pathway mutant (sec14-3 ts ) accumulated this sphingolipid and was sensitive to syringomycin E. Finally, ⌬csg1/sur1 and ⌬csg2 strains defective in the transfer of mannose to inositolphosphoryl-ceramide were resistant to syringomycin E. These findings show that syringomycin E growth inhibition of yeast is promoted by the production of sphingolipids with fully elongated fatty acid chains and the mannosyl and terminal phosphorylinositol moieties of the polar head group.Syringomycin E is a member of a family of small cyclic lipodepsinonapeptides (ca. 1,200 Da) produced by the plant bacterium Pseudomonas syringae pv. syringae (38). Other members include syringomycin A 1 and G, the syringostatins, the syringotoxins, and the pseudomycins (2, 38). All possess a characteristic tetrapeptidyl sequence (dehydroaminobutanoic acid-hydroxyaspartic acid-chlorothreonine-serine) and a ␤-hydroxy fatty acid attached to the N-terminal serine. These metabolites are fungicidal to a broad range of fungi, including yeast and human pathogens (33), and they show relatively low levels of toxicity to plants (21) and cutaneous animal tissues (33). Syringomycin E was recently shown to be partly responsible for the biological control of fungal pathogens on postharvest citrus fruits by certain P. syringae pv. syringae strains (5). Syringomycin E interacts with the fungal plasma membrane, where it causes K ϩ efflux, Ca 2ϩ influx, and changes in membrane potential by processes that are likely related to channel formation (14, 38).Molecular genetic studies with yeast were initiated to more precisely define the antifungal mechanism of action of syringomycin E. Syringomycin E-resistant mutants of Saccharomyces cerevisiae were generated to permit identification of the mutated genes by complementation (39). Two genes, ...

The SEC14 gene in Saccharomyces cerevisiae encodes a phosphatidylinositol transfer protein required for secretory protein movement from the Golgi. Mutation of SAC1, a gene of unknown function, restores secretory flow in sec14-1 ts strains. The existing model for the bypass of the sec14-1 ts defect by sac1-22 involves stimulation of sphingolipid biosynthesis and, in particular, the synthesis of mannosyl-diinositolphosphoryl-ceramide with concomitant increases in Golgi diacylglycerol levels. To test this model, we disrupted IPT1, the mannosyldiinositolphosphoryl-ceramide synthase of S. cerevisiae. Disruption of the IPT1 gene had no effect on the ability of sac1-22 to bypass sec14-1 ts . Furthermore, sphingolipid analysis of sec14-1 ts and sec14-1 ts sac1-22 strains showed that mannosyl-diinositolphosphoryl-ceramide synthesis was not stimulated in the bypass mutant. However, the sec14-1 ts strain had elevated mannosyl-monoinositolphosphoryl-ceramide levels, and the sec14-1 ts sac1-22 strain showed an 8-fold increase in phosphatidylinositol 4-phosphate along with a decrease in phosphatidylinositol 4,5-bisphosphate. Cellular diacylglycerol levels, measured by [ 14 C]acetate incorporation, did not differ between the sec14-1 ts and the sec14-1 sac1-22 bypass strains, although disruption of IPT1 in the bypass strain resulted in reduced levels. These data indicate that phosphatidylinositol 4-phosphate, rather than mannosyl-diinositolphosphoryl-ceramide, accumulates in the sec14-1 ts sac1-22 bypass strain, and that Golgi diacylglycerol accumulation is not required for bypass of the sec14-1 ts growth and secretory phenotypes.

Syringomycin E is an antifungal cyclic lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. To understand the mechanism of action of syringomycin E, a novel resistant Saccharomyces cerevisiae strain, BW7, was isolated and characterized. Lipid analyses revealed that BW7 contained only the hydrophobic subspecies of sphingolipids that are normally minor components in wild type strains. This aberrant sphingolipid composition was the result of lack of K K-hydroxylation of the amide-linked very long chain fatty acids, suggesting a defective sphingolipid K K-hydroxylase encoded by the FAH1 gene. A yeast strain that lacks the FAH1 gene was resistant to syringomycin E, and failed to complement BW7. These results demonstrate that BW7 carries a mutation in the FAH1 gene, and that the lack of K K-hydroxylated very long chain fatty acids in yeast sphingolipids confers resistance to syringomycin E. ß

Saccharomyces cerevisiae genes encoding functions necessary for inhibition by the Pseudomonas syringae pv. syringae cyclic lipodepsipeptide, syringomycin-E, were identified by mutant analyses. Syringomycin-E-resistant mutants were isolated, shown to contain single recessive mutations, and divided into eight gene complementation groups. Representative strains from five groups were resistant to nystatin, and deficient in the plasma membrane lipid, ergosterol. All of the mutant strains were resistant to the related cyclic lipodepsipeptides, syringotoxin and syringostatin. The findings show that: 1) at least eight gene-encoded functions participate in the inhibitory response to syringomycin; 2) ergosterol is important for this response; 3) the three related lipodepsipeptides have similar modes of action.