Specific inhibition of candicidin biosynthesis by the lipogenic inhibitor cerulenin

Cerulenin, an inhibitor of fatty acid synthesis, inhibits specifically the biosynthesis of the polyene macrolide candicidin by resting cells of Streptomyces griseus. 50% inhibition was achieved with a cerulenin concentration of 1.5 μg/ml. Cells in which candicidin synthesis was inhibited for 10 h remained capable of candicidin synthesis after removal of the inhibitor. Cerulenin inhibits specifically the incorporation of [¹⁴C] propionate into candicidin but does not affect total protein or RNA synthesis. The uptake of [¹⁴C] propionate was not inhibited under conditions which totally prevented the incorporation of propionate into candicidin. Incorporation of p-amino[¹⁴C] benzoic acid NH₂[¹⁴C]BzO⁻ into the aromatic moiety of candicidin was also inhibited by cerulenin. The inhibitory action of cerulenin was not reversed by exogenous fatty acids. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, it is concluded that the polyene macrolide candicidin is synthesized via the polyketide pathway by condensation steps similar to those occurring in fatty acid biosynthesis.     

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Phosphate concentration was found to control the biosynthesis of the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [¹⁴C]propionate and [¹⁴C]p-aminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis.     

Effect of exogenous nucleotides on the candicidin fermentation.

Addition of cyclic-AMP (c-AMP) to Streptomyces griseus fermentations inhibited candicidin formation. In a phosphate-free resting cell system, c-AMP inhibited net candicidin formation and incorporation of labeled propionate and p-aminobenzoic acid into the antibiotic but did not inhibit protein synthesis. All nucleotides tested, regardless of the position of the phosphate ester, were effective inhibitors; nucleosides and free bases were not. Inhibition occurred whether the nucleotide was added early or late. The results indicate that inhibition of antibiotic formation by exogenous nucleotides, including cyclic nucleotides, is similar to the effect produced by inorganic phosphate.     

Biosynthesis of candicidin by phosphate-limited resting cells ofStreptomyces griseus

Summary A phosphate-limited resting cell system ofStreptomyces griseus in a synthetic medium has been developed in which biosynthesis of the polyene macrolide, candicidin, is linear for at least 36 h without cell growth. Glucose and to a lesser degree sucrose, but not lactose, support antibiotic synthesis. Glucose is utilized at a constant rate for antibiotic synthesis without affecting mycelial dry weight. Acetate and propionate, the building units of the macrolide aglycone, stimulate candicidin biosynthesis in cultures supplemented with glucose but do not support its synthesis in the absence of glucose. Maximal stimulation of candicidin biosynthesis was produced by 40 mM propionate or 250 mM acetate. The biosynthetic intermediate, methyl malonate, and the analog, 1-propanol, were more stimulatory than propionate at the same concentration.     

Candicidin biosynthesis in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.     

Regulatory control of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase in Streptomyces antibioticus

Streptomyces antibioticus possesses a tryptophan-inhibitable 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthetase whose synthesis is also repressed by L-tryptophan. Studies of the DAHP synthetase obtained by ammonium sulfate fractionation of a crude extract derived from S. Antibioticus revealed that the enzymic activity was only partially inhibited by tryptophan. Inhibition of the DAHP synthetase activity was strongly pH dependent at values below 7.0. A number of tryptophan analogues was noted to inhibit the enzyme; by contrast, other aromatic amino acid end products failed to affect DAHP synthetase activity. Chorismic acid, a key intermediate in aromatic amino acid biosynthesis, was ineffective as an inhibitor when used alone; however, if supplied with L-tryptophan, a further reduction of DAHP synthetase activity (15--25%) was routinely observed.     

Amino acid biosynthesis and sodium-dependent transport in Methanococcus voltae, as revealed by 13C NMR

Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1-4.1 nmol min-1 mg-1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N-ethylmaleimide or 3,3',4',5-tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture. Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I.C.P. & Sprott, G.D. (1983) J. Bacteriol. 156, 316-326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the beta-carbon originating from the carboxyl of acetate and the alpha-carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2-oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2-oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.     

Sequence of candicidin action on yeast célls

The action of the polyene antibiotic candicidin (a heptaene) on yeast cells in growth medium has been studied. Candicidin at the growth-inhibiting concentration produced the following sequence of events. (1) There was an immediate and rapid loss of K⁺. (2) After about 10 min, Mg²⁺ was lost to the medium and protein synthesis and uptake of amino acids decreased rapidly while the intracellular ATP level rose. Candicidin caused a temporary stimulation of amino acid uptake in salt-free buffer. (3) After about 20 min RNA synthesis and glucose consumption declined. Transport of sugars did not appear to be inhibited but energy-dependent accumulation and assimilation were sharply restricted. (4) Candicidin did not cause release of phosphate, amino acids, uracil or uridine from the internal pools, or make the membrane permeable to uridine 5′-phosphate or ATP. Damage to the cell membrane by candicidin appears to be relatively slight and affects primarily membrane components related to ion transport. The various metabolic changes noted probably result from the K⁺ loss and the eventual decline of the energy-generating systems.     

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice.

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Inhibition of Nucleic Acid and Protein Synthesis in Mouse Spleen Cells In Vitro by Azathioprine

The effect of azathioprine on macromolecular biosynthesis was studied in mouse spleen cells cultured in vitro. The rate of incorporation of (3)H-thymidine, (3)H-uridine, and (14)C-leucine into acid-insoluble material was used to measure deoxyribonucleic acid, ribonucleic acid, and protein synthesis. Results indicate that azathioprine inhibited nucleic acid and protein synthesis at levels which did not decrease cell viability.     

Effect of amino acids on thaxtomin A biosynthesis by Streptomyces scabies

The regulatory effect of amino acids on the production of thaxtomin A, a phytotoxin produced by Streptomyces scabies, was investigated. Tryptophan had an important inhibitory effect on the toxin biosynthesis in all five strains of S. scabies tested. Two other aromatic amino acids (tyrosine and phenylalanine) also inhibited thaxtomin A biosynthesis, while aliphatic amino acids did not cause an important decline in thaxtomin A production. Methylation of tryptophan prevented or reduced the inhibitory effect on thaxtomin A biosynthesis. In spite of the inhibitory action of tryptophan and phenylalanine on thaxtomin A production, incorporation of these radiolabeled molecules into thaxtomin A confirmed that they are metabolic precursors for the biosynthesis of the phytotoxin.     

Studies on the inhibition of protein synthesis by selenodiglutathione.

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens.

Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.     

Incorporation of Shikimic Acid into Corynecins and Its Regulation

In the biosynthesis of corynecins by Corynebacterium hydrocarboclastus, it appeared that shikimic acid was one of the efficient precursors, where shikimic acid-U-¹⁴C was incorporated into corynecins in the yield of approximately 15%. Analyses of degradation products of labeled corynecins demonstrated that shikimic acid was incorporated specifically into aromatic ring of corynecins.

The incorporation of shikimic acid was inhibited by several aromatic amines such as p-aminophenylserinol-N-propionamide, although the uptake of shikimic acid was not affected, suggesting that biosynthesis of corynecins might be regulated by p-aminophenyl intermediates. Furthermore, p-ammophenylethylalcohol was found to be a potent inhibitor of biosynthesis of corynecins. In contrast, corynecins and other p-nitro-phenyl derivatives, aromatic amino acids and vitamins related to shikimic acid pathway did not inhibit the biosynthesis of corynecins from shikimic acid.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Continued Expression of the Ribonucleic Acid Control Gene During Inhibition of Escherichia coli Ribonucleic Acid and Protein Synthesis

The effect of the ribonucleic acid (RNA) control (RC) gene on the biosynthesis of viral RNA has been examined in an RC(str) and an RC(rel) host infected with R17 RNA bacteriophage under conditions in which host RNA and protein synthesis were inhibited by the addition of rifampicin. Methionine and isoleucine starvation depressed viral RNA biosynthesis in an RC(str) host but not in an RC(rel) host. However, histidine starvation had little effect on viral RNA and protein synthesis in both RC(str) and RC(rel) cells, although it had a marked effect on host protein and RNA synthesis in an RC(str) host. Chloramphenicol relieved the effect of amino acid starvation on viral RNA synthesis in an RC(str) host. It is concluded that stringent control of viral RNA biosynthesis does not require the continued biosynthesis of the RC gene product (RNA or protein) and that a preformed RC gene product can regulate the biosynthesis of the exogenous RNA. It is suggested that the amino acid dependence of viral RNA biosynthesis is due to its obligatory coupling with the translation of the viral coat protein which lacks histidine. It may be inferred that the amino acid requirement of bacterial RNA is due to its coupling with the translation of a host-specific protein (other than the RC gene product) which requires a full complement of amino acids. Since chloramphenicol is known to permit ribosome movement in the absence of protein synthesis, it is suggested that ribosome movement along the nascent RNA chain is a sufficient condition for the continuation of RNA synthesis.     

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate.