The effects of fenpropimorph on sterol biosynthesis and growth of Saccharomyces cerevisiae were examined to pinpoint the mode of action of fungicides that inhibit ergosterol biosynthesis. Taking advantage of sterol auxotrophy and sterol permeability in mutant strains, we show that growth inhibition is strongly correlated with inhibition of sterol biosynthesis. We confirm that in vivo and at low concentrations, fenpropimorph inhibits A8-*A7-sterol isomerase, and in addition, when it is used at higher concentrations, it inhibits A14-sterol reductase. We show also that the fungistatic effect of fenpropimorph is not due to the accumulation of abnormal sterols in treated cells but is linked to the specific inhibition of ergosterol biosynthesis, leading to the arrest of cell proliferation in the unbudded G1 phase of the cell cycle.

Cyanate induces expression of the cyn operon in Escherichia coli. The cyn operon includes the gene cynS, encoding cyanase, which catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. A carbonic anhydrase activity was recently found to be encoded by the cynT gene, the first gene of the cyn operon; it was proposed that carbonic anhydrase prevents depletion of bicarbonate during cyanate decomposition due to loss of CO2 by diffusion out of the cell (M. B. Guilloton, J. J. Korte, A. F. Lamblin, J. A. Fuchs, and P. M. Anderson, J. Biol. Chem. 267:3731-3734, 1992). The function of the product of the third gene of this operon, cynX, is unknown. In the study reported here, the physiological roles of cynT and cynX were investigated by construction of chromosomal mutants in which each of the three genes was rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase. The chromosomal mutant carrying cynS::kan expressed induced carbonic anhydrase activity but no active cyanase. The cynS::kan mutant was found to be much less sensitive to cyanate than the delta cynT mutant at a low pCO2, indicating that bicarbonate depletion due to the reaction of bicarbonate with cyanate catalyzed by cyanase is more deleterious to growth than direct inhibition by cyanate. Mutants carrying a nonfunctional cynX gene (cynX::kan and delta cynT cynX::kan) did not differ from the parental strains with respect to cyanate sensitivity, presence of carbonic anhydrase and cyanase, or degradation of cyanate by whole cells; the physiological role of the cynX product remains unknown.

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate to give two CO 2 molecules. The gene for cyanase is part of the cyn operon, which includes cynT and cynS, encoding carbonic anhydrase and cyanase, respectively. Carbonic anhydrase functions to prevent depletion of cellular bicarbonate during cyanate decomposition (the product CO 2 can diffuse out of the cell faster than noncatalyzed hydration back to bicarbonate). Addition of cyanate to the culture medium of a ⌬cynT mutant strain of E. coli (having a nonfunctional carbonic anhydrase) results in depletion of cellular bicarbonate, which leads to inhibition of growth and an inability to catalyze cyanate degradation. These effects can be overcome by aeration with a higher partial CO 2 pressure (M. B. Guilloton, A. F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P. M. Anderson, and J. A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). The question considered here is why depletion of bicarbonate/CO 2 due to the action of cyanase on cyanate in a ⌬cynT strain has such an inhibitory effect. Growth of wild-type E. coli in minimal medium under conditions of limited CO 2 was severely inhibited, and this inhibition could be overcome by adding certain Krebs cycle intermediates, indicating that one consequence of limiting CO 2 is inhibition of carboxylation reactions. However, supplementation of the growth medium with metabolites whose syntheses are known to depend on a carboxylation reaction was not effective in overcoming inhibition related to the bicarbonate deficiency induced in the ⌬cynT strain by addition of cyanate. Similar results were obtained with a ⌬cyn strain (since cyanase is absent, this strain does not develop a bicarbonate deficiency when cyanate is added); however, as with the ⌬cynT strain, a higher partial CO 2 pressure in the aerating gas or expression of carbonic anhydrase activity (which contributes to a higher intracellular concentration of bicarbonate/CO 2 ) significantly reduced inhibition of growth. There appears to be competition between cyanate and bicarbonate/CO 2 at some unknown but very important site such that cyanate binding inhibits growth. These results suggest that bicarbonate/CO 2 plays a significant role in the growth of E. coli other than simply as a substrate for carboxylation reactions and that strains with mutations in the cyn operon provide a unique model system for studying aspects of the metabolism of bicarbonate/CO 2 and its regulation in bacteria.Cyanase (EC 4.3.99.1) catalyzes the reaction of cyanate with bicarbonate to give two molecules CO 2 (15):The synthesis of cyanase in Escherichia coli is induced by addition of cyanate to the growth medium (2). The gene for cyanase is part of the cyn operon, which includes three genes in the order cynT, cynS, and cynX, encoding carbonic anhydrase, cyanase, and a hydrophobic protein of unknown function, respectively (11,(30)(31)(32)(33). The function of carbonic anhydrase appears to be to prevent depletion of cellular bicarb...

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