Iron deficiency-induced tetracycline production in submerged cultures by Streptomyces aureofaciens

Streptomyces aureofaciens ATCC 10762 grown in rotary-shaken submerged cultures produced substantial amounts of tetracycline only when the defined medium was deprived of iron. The biosynthesis of tetracycline was inhibited either by free iron at concentrations above 1–2 μmol l⁻¹, or by chelated iron provided by the siderophores of this bacterial strain. Late static iron-containing cultures allowed cell differentiation and sporulation and led to tetracyclines synthesis. A nitrosoguanidine-induced mutant able to synthesize tetracycline in the presence of iron in shaken submerged cultures was isolated and compared to the wild-type strain. However, no constitutive siderophore-mediated iron transport occurred in the mutant. These results suggest the involvement of a putative iron-controlled repressor in the biosynthesis of these secondary metabolites during vegetative growth and primary metabolism of the bacterium.     

The biosynthesis of serine and glycine in Pseudomonas AM1 with special reference to growth on carbon sources other than C1 compounds

1. A mutant, 20S, of Pseudomonas AM1 was obtained that requires a supplement of serine to grow on succinate, lactate or ethanol. This mutant lacks phosphoserine phosphatase and revertants to wild-type phenotype regained this enzymic activity showing that the phosphorylated pathway of serine biosynthesis is necessary for growth on these three substrates. 2. The requirement for supplemental serine by mutant 20S could be met by glycine, suggesting that Pseudomonas AM1 can obtain C(1) units from glycine. 3. Mutant 20S grows on C(1) compounds at a lower rate compared with the wild type. Supplementation with serine stimulated the growth rate of the mutant suggesting that the phosphorylated pathway of serine biosynthesis plays some role, but not an essential role, during growth on C(1) compounds. 4. A mutant, 82G, was obtained that requires a supplement of glycine to grow on succinate, lactate or ethanol. When grown in such supplemented media, the mutant lacks serine hydroxymethyltransferase and revertants to wild-type phenotype regained enzymic activity showing that during growth on succinate, lactate or ethanol, glycine is made from serine via serine hydroxymethyltransferase, and that the organism can obtain C(1) units from glycine. 5. Mutant 82G grew on methanol and then contained serine hydroxymethyltransferase suggesting that this enzyme is necessary for growth on C(1) compounds and that Pseudomonas AM1 may synthesize two such enzymes, one used in growth on C(1) compounds, the other used in growth on other substrates. Mutant 82G might lack the latter enzyme. 6. Phosphoglycerate dehydrogenase is specifically inhibited by l-serine and the regulatory implications of this are discussed.     

Glycine origin of the methyl substituent on C7'-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicating that glycine and/or serine might be involved in the biosynthesis of apramycin. The 13C-NMR analysis of [2-13C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH3 substituent of apramycin on the C7' of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The 15N NMR spectra of [2-13C,15N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the -NH2 substituents of apramycin.     

Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.     

Regulation of serine transhydroxymethylase activity in Salmonella typhimurium

The regulation of serine transhydroxymethylase (EC 2.1.2.1.; l-serine:tetrahydrofolic-5,10-hydroxymethyltransferase) has been investigated in Salmonella typhimurium LT2. Our results indicate that limitation of a methionine auxotroph for methionine does not cause derepression of this enzyme as reported for Escherichia coli. However, a sixfold decrease in specific activity was observed when S. typhimurium cells were grown in glucose minimal medium supplemented with serine, glycine, methionine, adenine, guanine, and thymine. None of these compounds added to the growth medium individually produced more than a 42% reduction of wild-type enzyme activity. This enhanced repression by the combination of compounds suggests a form of cumulative repression of this enzyme. Growth of serine and thymine auxotrophs, with the respective requirement of each limiting, did not result in increased enzyme activity. However, growth of a purine auxotroph with a limiting amount of either guanine or inosine resulted in a five- to sevenfold increase in enzyme activity. A second condition causing significant derepression (fourfold increase) above the levels observed with cells grown in minimal medium was the addition of 0.5 mug of trimethoprim per ml, an inhibitor of the dihydrofolate reductase activity. (A partial report on this work was presented at 1974 meeting of the American Society for Microbiology.)     

Factors affecting the biosynthesis of L-tryptophan by genetically modified strains of Escherichia coli

Derivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (qp) of tryptophan. Supplementation of the growth medium with glycine enhanced qp two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high qp of tryptophan [13.3 g tryptophan (g dry wt cells)-1 h-1], which was correlated to the TS specific activity.     

Serine biosynthesis and its regulation in Bacillus subtilis

Cell-free extracts of Bacillus subtilis strains GSY and 168 convert (14)C-phosphoglycerate to (14)C-serine phosphate and (14)C-serine. These reactions indicate a functional phosphorylated pathway for serine biosynthesis in these cells. The addition of serine to the incubation mixture inhibited the formation of both radioactive products. Extracts of mutant strains that require serine for growth lacked the capacity to synthesize serine phosphate, confirming that the phosphorylated pathway was the only functional pathway available for serine synthesis. Serine phosphate phosphatase and phosphoglycerate dehydrogenase activity were demonstrated in cell extracts, and the phosphoglycerate dehydrogenase was shown to be inhibited specifically by l-serine. The extent of serine inhibition increased when the temperature was raised from 25 to 37 C, and the thermal stability of the enzyme was enhanced by the presence of the inhibitor serine or the coenzyme reduced nicotinamide adenine dinucleotide. At 37 C the curve representing the relationship between phosphoglycerate concentration and enzyme velocity was biphasic, and the serine inhibition which was competitive at low substrate concentrations became noncompetitive at higher concentrations.     

Efficient conversion of glycine to l-serine by a glycine-resistant mutant of a methylotroph using Co2+ as an inhibitor of l-serine degradation

A glycine-resistant mutant, no. 18, which was not lysed by glycine, was obtained from an l-serine-producing mutant, S395 (temperature-sensitive, O-methylserine-resistant), of a facultative methylotroph, Pseudomonas MS31. The mutant stably produced l-serine from glycine. The properties of the enzymes involved in the synthesis and degradation of l-serine were investigated in the wild-type strain MS31 and the l-serine-producing mutants. Mutant derivation had no effect on the activities of methanol dehydrogenase or serine hydroxymethyltransferase, which are involved in l-serine synthesis. On the other hand, the activity of l-serine dehydratase (SDH), which degrades l-serine, was reduced in the mutants. Cobalt (Co²⁺) inhibited SDH activity and its addition (6.5 mM) to the l-serine production culture significantly stimulated l-serine accumulation up to 14.9 mg/ml. The results suggest that blocking of SDH is important for the efficient production of l-serine from glycine by methylotrophs.     

Genetic and physiologic analysis of a formyl-tetrahydrofolate synthetase mutant of Streptococcus mutans.

Previously we reported that transposon Tn917 mutagenesis of Streptococcus mutans JH1005 yielded an isolate detective in its normal ability to produce a mutacin (P. J. Crowley, J. D. Hillman, and A. S. Bleiweis, abstr. D55, p. 258 in Abstracts of the 95th General Meeting of the American Society for Microbiology 1995, 1995). In this report we describe the recovery of the mutated gene by shotgun cloning. Sequence analysis of insert DNA adjacent to Tn917 revealed homology to the gene encoding formyl-tetrahydrofolate synthetase (Fhs) from both prokaryotic and eukaryotic sources. In many bacteria, Fhs catalyzes the formation of 10-formyl-tetrahydrofolate, which is used directly in purine biosynthesis and formylation of Met-tRNA and indirectly in the biosynthesis of methionine, serine, glycine, and thymine. Analysis of the fhs mutant grown anaerobically in a minimal medium demonstrated that the mutant had an absolute dependency only for adenine, although addition of methionine was necessary for normal growth. Coincidently it was discovered that the mutant was sensitive to acidic pH; it grew more slowly than the parent strain on complex medium at pH 5. Complementation of the mutant with an integration vector harboring a copy of fhs restored its ability to grow in minimal medium and at acidic pH as well as to produce mutacin. This represents the first characterization of Fhs in Streptococcus.     

Genetic characterization of a highly efficient alternate pathway of serine biosynthesis in Escherichia coli.

There exists in Escherichia coli a known set of enzymes that were shown to function in an efficient and concerted way to convert threonine to serine. The sequence of reactions catalyzed by these enzymes is designated the Tut cycle (threonine utilization). To demonstrate that the relevant genes and their protein products play essential roles in serine biosynthesis, a number of mutants were analyzed. Strains of E. coli with lesions in serA, serB, serC, or glyA grew readily on minimal medium supplemented with elevated levels of leucine, arginine, lysine, threonine, and methionine. No growth on this medium was observed upon testing double mutants with lesions in one of the known ser genes plus a second lesion in glyA (serine hydroxymethyltransferase), gcv (the glycine cleavage system), or tdh (threonine dehydrogenase). Pseudorevertants of ser mutants capable of growth on either unsupplemented minimal medium or medium supplemented with low levels of leucine, arginine, lysine, threonine, and methionine were isolated. At least two unlinked mutations were associated with such phenotypes.     

Glycine origin of the methyl substituent on C7′-N of octodiose for the biosynthesis of apramycin

Apramycin is unique in the aminoglycoside family due to its octodiose moiety. However, either the biosynthesis process or the precursors involved are largely unknown. Addition of glycine, as well as serine or threonine, to the Streptomyces tenebrabrius UD2 fermentation medium substantially increases the production of apramycin with little effect on the growth of mycelia, indicat-ing that glycine and/or serine might be involved in the biosynthesis of apramycin. The ¹³C-NMR analysis of [2-¹³C] glycine-fed (25% enrichment) apramycin showed that glycine specifically and efficiently incorporated into the only N-CH₃ substituent of apramycin on the C7′ of the octodiose moiety. We noticed that the in vivo concentration of S-adenosyl methionine increased in parallel with the addition of glycine, while the addition of methione in the fermentation medium significantly decreased the productivity of apramycin. Therefore, the methyl donor function of glycine is proposed to be involved in the methionine cycle but methionine itself was proposed to inhibit the methylation and methyl transfer processes as previously reported for the case of rapamycin. The ¹⁵N NMR spectra of [2-¹³C,¹⁵N]serine labeled apramycin indicated that serine may also act as a limiting precursor contributing to the ―NH₂ substituents of apramycin.     

Metabolism of D-serine in Escherichia coli K-12: mechanism of growth inhibition

Without significant killing, d-serine at concentrations greater than 50 mug/ml inhibits growth in minimal media of mutants of Escherichia coli K-12 unable to form d-serine deaminase. The mutants eventually recover at lower concentrations. There is no evidence of d-serine toxicity in rich media. Toxicity is partially reversed by l-serine. d-Serine does not interfere with l-serine activation, one-carbon metabolism, or (Cronan, personal communication) formation of phosphatidylserine. Pizer (personal communication) finds, however, that it is a powerful feedback inhibitor of the first enzyme of l-serine biosynthesis. In the presence of l-serine, the residual toxicity is largely and noncompetitively over come by pantothenate, indicating that d-serine inhibits growth by affecting two targets: pantothenate biosynthesis and l-serine biosynthesis. l-Serine causes transient growth inhibition in E. coli K-12. Contaminating l-serine in d-serine preparations contributes to the d-serine inhibitory response.     

Physiological comparison of L-serine dehydratase and tryptophanase from Bacillus alvei

Tryptophanase from Bacillus alvei also possesses serine dehydratase activity. A comparison of this enzyme with l-serine dehydratase [l-serine hydro-lyase (deaminating), EC 4.2.1.13] in toluene-treated whole cell preparations of the organism was undertaken. Tryptophanase is a constitutive enzyme in B. alvei. The dehydratase undergoes a repression-derepression-repression sequence as the l-serine level in the growth medium is increased from 0 to 0.1 m. Tryptophanase activity is decreased in organisms grown in medium containing glucose. Both enzymes are repressed in organisms grown in glycerol-containing medium. l-Serine dehydratase has a pH optimum of 7.5 in potassium phosphate buffer; tryptophanase functions optimally in this buffer at pH 8.2. Both enzymes lose activity in the presence of tris(hydroxymethyl)aminomethane buffer. Either K(+) or NH(4) (+) is required for full tryptophanase activity, but Na(+) is markedly inhibitory. These three cations are stimulatory to l-serine dehydratase activity. Both enzymes are subject to apparent substrate inhibition at high concentrations of their respective amino acids, but the inhibition of tryptophanase activity can be completely overcome by the removal of indole as it is formed. The dehydratase does not catalyze cleavage of d-serine, l-threonine, or alpha-substituted serine analogues at the concentrations tested. However, activity of the enzyme in cleaving l-serine is competitively inhibited by d-serine, indicating that the d-isomer can occupy an active site on the enzyme. The enzyme catalyzes cleavage of some beta-substituted serine analogues.     

Production of l-serine from glycine by Corynebacterium glycinophilum and properties of serine hydroxymethyltransferase,a key enzyme in l-serine production

Fermentative production of l-serine from glycine by Corynebacterium glycinophilum AJ-3413, an auxotrophic mutant of Leu and Met with increased productivity of l-serine using a one liter jar fermentor was carried out and the properties of serine hydroxymethyltransferase (SHMT), a key enzyme in l-serine synthesis, of the parental strain AJ-3170 were investigated. SHMT was effectively induced by the addition of glycine to the medium at an early stage of cultivation. Under optimal conditions, AJ-3413 produced 16.0 g/l of l-serine from 30 g/l of glycine with a molar yield of 38%. The partially purified SHMT catalyzed the l-allo-threonine degradation in addition to l-serine degradation, but could not catalyze l-threonine degradation. This enzyme showed an absolute tetrahydrofolic acid requirement for l-serine degradation to glycine and formaldehyde, but not for l-allo-threonine degradation. Pyridoxal 5′-phosphate appeared to be required for enzyme activity. The K_m values for glycine and formaldehyde in l-serine synthesis, and for l-serine in l-serine degradation were 1.85, 0.29 and 1.64 mM, respectively.     

Biogenesis of some antibiotics in the presence of 2-chloroethylphosphonic acid.

2-Chloroethylphosphonic acid (CEPA) affected both the growth of and antibiotic production in Streptomyces aureofaciens, S. griseus, S. antibioticus, and Penicillium citrinum. Streptomyces strains seemed to be more sensitive to the presence of CEPA in the medium than did the fungus. A decrease in both growth and antibiotic production was observed with a concomitant increase in the concentration of the ethylene-releasing compound in the medium. Higher concentrations of CEPA completely inhibited the growth of the above microorganisms.     

Biogenesis of some antibiotics in the presence of 2-chloroethylphosphonic acid

2-Chloroethylphosphonic acid (CEPA) affected both the growth of and antibiotic production in Streptomyces aureofaciens, S. griseus, S. antibioticus, and Penicillium citrinum. Streptomyces strains seemed to be more sensitive to the presence of CEPA in the medium than did the fungus. A decrease in both growth and antibiotic production was observed with a concomitant increase in the concentration of the ethylene-releasing compound in the medium. Higher concentrations of CEPA completely inhibited the growth of the above microorganisms.     

The mechanism of action of the herbicide N-(phosphonomethyl)glycine: its effect on the growth and the enzymes of aromatic amino acid biosynthesis in Escherichia coli (author's transl)

N-(Phosphonomethyl) glycine prolongates the lag-phase and inhibits the growth rate of Escherichia coli, Salmonella typhimurium and Pseudomonas aureofaciens. The eucaryotes Saccharomyces cerevisiae and Neurospora crassa are not inhibited. The effect of growth inhibition in an E. coli culture depends on the time of the herbicide addition and no cells showing resistance against it are observed. The inhibitory effect can be overcome completely by a mixture of phenylalanine, tyrosine and tryptophan. N-(Phosphonomethyl)glycine inhibits phospho-2-oxo-3-deoxyheptonate aldolase and 3-dehydroquinate synthase. Both inhibitory effects are removed by addition of CO2. Chorismate mutase, prephenate dehydratase and prephenate dehydrogenase are not influenced by this herbicide. Anthranilate synthase is also inhibited by N-(phosphonomethyl)glycine. This inhibition is removed by addition of Mg2. Phospho-2-oxo-3-deoxyheptonate aldoase is derepressed in E. coli cells grown in minimal medium containing N-(phosphonomethyl)glycine. Under these conditions the tyrosine-sensitive isoenyme is much more strongly derepressed than the phenylalanine-sensitive isoenzyme. 3-Dehydroquinate synthase is not affected. Chorismate mutase, prephenate dehydrogenase, prephenate dehydratase, and anthranilate synthase are derepressed, but to a lesser extent.     

Requirement for Acetate and Glycine (or Serine) for Sporulation Without Growth of Bacillus subtilis

Cells of Bacillus subtilis sporulate when they are transferred, at any time of growth in nutrient sporulation medium, to a potassium-phosphate buffer containing slowly utilizable carbon sources such as l-aspartate, citrate, l-glutamate, or lactate. Transfer to buffer containing more rapidly utilizable carbon sources such as malate or glucose leads to sporulation only when the cells either had reached the end of growth or when the transfer medium also contains glycine. Acetate, which as a sole carbon source does not allow growth, also does not alone permit sporulation; however, the presence of both acetate (0.05 m) and glycine or l-serine (0.01 m) in the buffer medium allows sporulation if the cells are transferred to this medium after they have grown in the nutrient sporulation medium beyond the end of the exponential growth phase (T(0)). The development, required before transfer, does not seem to involve the end of a round of deoxyribonucleic acid duplication, as experiments with tryptophan-starved cells have indicated. Glycine or serine cannot be replaced by any of the known metabolites, which are partially derived from them. Amino acid analysis of nutrient sporulation medium showed that glycine (but not serine) is present at a concentration of 0.3 mm at the beginning of the developmental period, thus allowing, in combination with an acetyl-coenzyme A (CoA) precursor, sporulation but not growth. Acetyl-CoA is required not only for adenosine-triphosphate synthesis but also for some other reactions.     

Effect of serine hydroxamate on the growth of Escherichia coli

The structural analogue of l-serine, l-serine hydroxamate, inhibited the growth of Escherichia coli K-12. Of the other amino acid hydroxamates tested, only l-lysine hydroxamate reduced the rate of growth. Inhibition of growth by l-serine hydroxamate was rapidly reversed by the addition of l-serine to the bacterial culture or by removal of the analogue by filtration. The reversal of inhibition was specific for l-serine. l-Alanine, glycine, or adenine had no effect on an inhibited culture. No evidence for active transport of the analogue was obtained.