Citric acid cycle: gene-enzyme relationships in Bacillus subtilis

The genetic location of mutations affecting the citric acid cycle and the properties of mutants of Bacillus subtilis possessing these mutations have been examined. Genes coding for the component enzymes of the cycle were found to be unlinked to each other and thus do not form an operon. The mutational defect in a mutant lacking fumarase mapped between thr-5 and cysB3. Mutations causing inability to produce isocitrate dehydrogenase and succinate dehydrogenase were found to map between argA11 and leu-1. The alpha-ketoglutarate dehydrogenase mutations were mapped at the terminal end of the B. subtilis chromosome through a weak linkage in phage PBS-1 transduction of one class of these mutations of ilvA2 and metB4. A second class of alpha-ketoglutarate dehydrogenase mutations mapped closer to ilvA2 and metB4 but still terminal with respect to these markers. Aconitaseless mutants possessed mutations that could not be linked to any of the known transducing segments of the chromosome. An effect of mutation conferring loss of one enzyme of the cycle on the specific activity of the other enzymes in the cycle was observed.     

Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis

Mutants have been isolated which correspond to every step concerned with the biosynthesis of the aromatic amino acids in Bacillus subtilis. Each mutant has been characterized, and the lesion it bore was analyzed by deoxyribonucleic acid transformation and PBS-1 mediated transduction. The biochemical analysis revealed that each of the mutations appears to have affected a single enzyme, except for two groups of pleiotropic mutations. All aroF mutants (chorismic acid synthetase) lack dehydroquinic acid synthetase (aroB) activity. The gene that specifies aroB is closely linked to the gene coding for the aroF enzyme. Both genes are a part of the aro cluster. Mutants lacking chorismate mutase activity also lack d-arabino-heptulosonic acid-7-phosphate synthetase and shikimate kinase activity, presumably as a result of these three activities forming a multi-enzyme complex. Another mutant, previously undescribed, had been isolated. The affected gene codes for the tyrosine and phenylalanine aminotransferase activity. All of the mutations have been located on the B. subtilis genome except those in the genes specifying shikimate kinase activity and tyrosine-phenylalanine aminotransferase activity.     

Aromatic amino acid biosynthesis in Trichophyton rubrum

Summary Trichophyton rubrum was assayed for shikimic, quinic, and protocatechuic acids with biological and chemical techniques. Since none of these metabolites were detected, we conclude that the shikimic acid pathway of aromatic biosynthesis is probably not involved in the synthesis of phenylalanine and tyrosine by this organism.     

Aromatic amino acid biosynthesis in Trichophyton rubrum

Summary WhenTrichophyton rubrum is grown in a minimal medium containing glucose, the carbon skeleton of fungal phenylalanine and tyrosine is derived from the glucose carbon. Tracer experiments with variously labeled glucose-C¹⁴ indicate that phenylalanine synthesis is linked to glycolysis, but suggest that the pentose phosphate pathway is not involved. These findings suggest that aromatic amino acid biosynthesis may not be linked to the shikimic acid pathway inT. rubrum.     

An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis.

Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis. One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine. The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids. The other gene has not yet been mapped. Mutants have been isolated that lack one or the other enzyme activity. These mutants are prototrophic for tyrosine and phenylalanine. However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain. The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation. The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways.     

The gene-enzyme relationships of proline biosynthesis in Escherichia coli

A simple chromatographic procedure has been devised to separate gamma-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase, allowing the measurement of the former in crude Escherichia coli extracts. Analysis of a number of strains of E. coli has demonstrated that gene proA codes for gamma-glutamyl phosphate reductase and proB for gamma-glutamyl kinase. Introduction of a ColE1 hybrid plasmid containing the proA,B region into a strain with a chromosomal deletion of proA,B led to 3- and 17-fold increases in the specific activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, respectively.     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16

1. Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the “pin-point” isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type.
2. Mutants accumulating chorismic acid or prephenic acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth.
3. A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A. eutrophus H16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 2 l of medium.

     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H 16

Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated. Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements. During the purification procedure the enzyme was stabilized by 50 mM l-glutamine or during chromatography on DEAE-cellulose and Sephadex G-200 with 30% glycerol, respectively.The glutamine dependent activity of anthranilate synthase was examined; it showed little change between pH 8.4 and pH 9.1. The Arrhenius plot was broken and the activation energy, H, calculated therefrom amounted to 8.9 kcal/mole up to 30°C and 5.5 kcal/mole at higher temperatures. The molecular weight determined by gelfiltration on Sephadex G-200 and by sucrose density gradient centrifugation resulted in 158000 and 126000, respectively. The K _m -values for the two substrates chorismate and glutamine were found to be 5 M and 560 M, respectively.Anthranilate synthase was strongly inhibited by l-tryptophan; the only amino acid that affected enzyme activity. Homotropic interactions for chorismate (Hill coefficient n=1.4) were obtained in the presence of l-tryptophan. 50% inhibition were caused by 10 M l-tryptophan at 100 M chorismate. The inhibition with respect to l-glutamine was noncompetitive.Anthranilate synthase was not associated to phosphoribosyl transferase and easily separable from the latter by different chromatographic methods.Abbreviation TEA triethanolamine     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

Control of aromatic acid biosynthesis in Bacillus subtilis: sequenial feedback inhibition

Nester, E. W. (University of Washington, Seattle), and R. A. Jensen. Control of aromatic acid biosynthesis in Bacillus subtilis: sequential feedback inhibition. J. Bacteriol. 91:1594-1598. 1966.-The three major end products of aromatic acid synthesis, tyrosine, phenylalanine, and tryptophan, were tested for their ability to inhibit the first enzymes of the three terminal branches of the pathway as well as the enzyme common to both tyrosine and phenylalanine synthesis. Tyrosine inhibits the activity of prephenate dehydrogenase and also prephenate dehydratase to a limited extent. Phenylalanine inhibits the activity of prephenate dehydratase and, at much higher concentrations, prephenate dehydrogenase. Tryptophan inhibits the activity of anthranilate synthetase and, to some extent, prephenate dehydrogenase and prephenate dehydratase. Chorismate mutase is not inhibited by either 1 mm tyrosine or 1 mm phenylalanine when these are present singly or together in the reaction mixture. The significance of the feedback control of the terminal branches to the feedback control of that part of the pathway common to the synthesis of all three amino acids is discussed.     

Iron starvation triggers the stringent response and induces amino acid biosynthesis for bacillibactin production in Bacillus subtilis

Iron deprivation in bacteria causes the derepression of genes controlled by the ferric uptake regulator (Fur). The present microarray analysis of iron-starved Bacillus subtilis cells grown in minimal medium unveils additional physiological effects on a large number of genes linked to stringent-response regulation and to genes involved in amino acid biosynthesis associated with pathways essential for bacillibactin production.     

Regulation of leucine biosynthesis in Bacillus subtilis

The biosynthesis of alpha-isopropylmalate (alphaIPM) synthetase, IPM isomerase, and betaIPM dehydrogenase in Bacillus subtilis can be derepressed in leucine auxotrophs by limiting them for leucine. The derepression of the three enzymes is apparently coordinate. A class of mutants resistant to 4-azaleucine excretes leucine and has derepressed levels of all three enzymes. The azaleucine-resistance mutations may lie in a gene (azlA) encoding a repressor. Efforts to find mutations characteristic of a constitutive operator have been unsuccessful. No polar mutations have been found among nine leucine auxotrophs that have characteristics of frameshift mutations. The enzyme catalyzing the first step in leucine biosynthesis, alphaIPM synthetase, is sensitive to feedback inhibition by leucine. We conclude that leucine biosynthesis is controlled by the inhibition of the activity of the first biosynthetic enzyme by leucine, and by the repression of the synthesis of the first three biosynthetic enzymes by leucine. The repression of the three enzymes may be under the control of a single repressor and a single operator, or of a single repressor and a separate operator for each structural gene.     

N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.     

Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.     

Aromatic amino acid biosynthesis in Trichophyton rubrum III. Exogenous studies: Absence of the shikimic acid pathway

Even thoughTrichophyton rubrum is permeable to exogenous shikimic acid, neither shikimic nor quinic acids stimulate the growth of this fungus in a minimal medium deficient in phenylalanine or tyrosine, nor do they serve as substrates for pigmentogenesis in media lacking these amino acids. The respiration of the dermatophyte is unaffected by shikimic or quinic acids and the fungus does not have the capacity to utilize either compound when it is added to the culture medium. Isotope dilution studies with shikimic acid-U-C¹⁴ show that de novo shikimic acid synthesis does not occur. This information supports previous findings that the shikimic acid pathway of aromatic biosynthesis is not involved in the biosynthesis of phenylalanine byTrichophyton rubrum.

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Zusammenfassung ObwohlT. rubrum fur exogene shikimicsäure durchlässig ist, fördern weder Shikimicsäure noch Quinicsäure das Wachstum dieses Pilzes im Falle eines Mangels von Phenykalanine oder Tyrosine, noch dienen sie als Substanzen für Pigmentgenese in Medien ohne diese Aminosäuren. Die Atmung des Pilzes ist durch Shikimicoder Quinicsäure unbeeinflußt und der Pilz ist unfähig, beide Substanzen zu benützen, wenn sie zum Kulturmedium hinzugefügt werden. Isotope Verdünnungen mit Shikimicsäure-U-C¹⁴ zeigten, daß de novo Shikimicsäure-Synthese nicht erfolgt. Diese Erkenntnis unterstüzt vorherige Befunde, daß Shikimicsäure Richtung der aromatischen Biosynthese in der Biosynthese von Phenylalanine durchT. rubrum nicht begangen wird.

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University of Illinois at the Medical Center Department of Microbiology, Chicago, Illinois 60612