Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species

A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.     

Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis.

The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.     

Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis

The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.     

Inactivation of Bacillus subtilis glutamine synthetase by metal-catalyzed oxidation.

Instability of Bacillus subtilis glutamine synthetase in crude extracts was attributed to site-specific oxidation by a mixed-function oxidation, and not to limited proteolysis by intracellular serine proteases (ISP). The crude extract from B. subtilis KN2, which is deficient in three intracellular proteases, inactivated glutamine synthetase similarly to the wild-type strain extract. To understand the structural basis of the functional change, oxidative modification of B. subtilis glutamine synthetase was studied utilizing a model system consisting of ascorbate, oxygen, and iron salts. The inactivation reaction appeared to be first order with respect to the concentration of unmodified enzyme. The loss of catalytic activity was proportional to the weakening of subunit interactions. B. subtilis glutamine synthetase was protected from oxidative modification by either 5 mM Mn2+ or 5 mM Mn2+ plus 5 mM ATP, but not by Mg2+. The CD-spectra and electron microscopic data showed that oxidative modification induced relatively subtle changes in the dodecameric enzyme molecules, but did not denature the protein. These limited changes are consistent with a site-specific free radical mechanism occurring at the metal binding site of the enzyme. Analytical data of the inactivated enzyme showed that loss of catalytic activity occurred faster than the appearance of carbonyl groups in amino acid side chains of the protein. In B. subtilis glutamine synthetase, the catalytic activity was highly sensitive to minute deviations of conformation in the dodecameric molecules and these subtle changes in the molecules could be regarded as markers for susceptibility to proteolysis.     

Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.

The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.     

Effect of Different Nutritional Conditions on the Synthesis of Tricarboxylic Acid Cycle Enzymes

The effect of various nutritional conditions on the levels of Krebs cycle enzymes in Bacillus subtilis, B. licheniformis, and Escherichia coli was determined. The addition of glutamate, alpha-ketoglutarate, or compounds capable of being catabolized to glutamate, to a minimal glucose medium resulted in complete repression of aconitase in B. subtilis and B. licheniformis. The synthesis of fumarase, succinic dehydrogenase, malic dehydrogenase, and isocitric dehydrogenase was not repressed by these compounds. It is postulated that glutamate or alpha-ketoglutarate is the true corepressor for the repression of aconitase. A rapidly catabolizable carbon source and alpha-ketoglutarate or glutamate must be simultaneously present for complete repression of the formation of aconitase. Conditions which repress the synthesis of aconitase in B. subtilis restrict the flow of carbon in the sequence of reactions leading to alpha-ketoglutarate but do not prevent glutamate oxidation in vivo. The data indicate that separate and independent mechanisms regulate the activity of the anabolic and catabolic reactions of the Krebs cycle in B. subtilis and B. licheniformis. The addition of glutamate to the minimal glucose medium results in the repression of aconitase, isocitric dehydrogenase, and fumarase, but not malic dehydrogenase in E. coli K-38.     

Expression in Bacillus subtilis of the 51- and 42-kilodalton mosquitocidal toxin genes of Bacillus sphaericus.

A 3,080-base-pair KpnI-HindIII DNA fragment from Bacillus sphaericus 2362 coding for 51- and 42-kilodalton mosquitocidal proteins was cloned into Bacillus subtilis DB104 by using the vector pUB18. In B. subtilis these proteins were not detected during vegetative growth but were expressed during sporulation at levels comparable to those found in B. sphaericus.     

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.     

Properties of crystalline L-ornithine: alpha-ketoglutarate delta-aminotransferase from Bacillus sphaericus.

The distribution of bacterial L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine:2-oxo-acid aminotransferase [EC 2.6.1.13]) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the enzyme, which was inducibly formed by addition of L-ornithine or L-arginine to the medium. L-Ornithine:alpha-ketoglutarate delta-aminotransferase, purified to homogeneity and crystallized from B. sphaericus, had a molecular weight of about 80,000 and consisted of two subunits identical in molecular weight (41,000) and in amino-terminal residue (threonine). The enzyme exhibited absorption maxima at 278,343, and 425 nm and contained 1 mol of pyridoxal 5'-phosphate per mol of enzyme. The formyl group of pyridoxal 5'-phosphate was bound through an aldimine linkage to the epsilon-amino group of a lysine residue of the protein. The enzyme-bound pyridoxal 5'-phosphate, absorbing at 425 nm, was released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive enzyme, which was reactivated by addition of pyridoxal 5'-phosphate, still had a 343-nm peak and contained 1 mol of a vitamin B6 compound. The holoenzyme showed positive circular dichroic bands at 340 and 425 nm, whereas the inactive form had no band at 425 nm. The enzyme was highly specific for L-ornithine and alpha-ketoglutarate and catalyzed delta-transamination between them to produce L-glutamate and L-glutamate-gamma-semialdehyde, which as spontaneously converted to delta 1-pyrroline-5-carboxylate. The enzyme activity was significantly affected by nonsubstrate amino acids, amines, and carbonyl reagents.     

Inactivation of Aspartic Transcarbamylase in Sporulating Bacillus subtilis: Demonstration of a Requirement for Metabolic Energy

The aspartic transcarbamylase (ATCase) activity of Bacillus subtilis cells disappears rapidly from stationary-phase cells prior to sporulation. ATCase activity does not appear in the culture fluid during the stationary phase; hence the enzyme appears to be inactivated in the cells. The enzyme is inactivated normally in two different mutants lacking proteases; the activity is very stable in crude extracts of cells or in the culture fluid. These results suggest that ATCase is not inactivated by the general proteolysis that occurs in sporulating bacteria. The inactivation of ATCase can be completely inhibited after it has begun by oxygen starvation or addition of fluoroacetate. Inhibitors of oxidative phosphorylation and electron transport also interrupt the inactivation of ATCase. The inactivation of ATCase is very slow in two mutant strains that are deficient in enzymes of tricarboxylic acid cycle. Addition of gluconate to stationary cultures of the mutant strains, which is known to restore depleted adenosine 5'-triphosphate pools in these bacteria, also restores inactivation of ATCase. These experiments support the conclusion that the generation of metabolic energy is necessary for the inactivation of ATCase in stationary cells. ATCase activity is stable in growing cells in which ATCase synthesis is repressed by addition of uracil; the enzyme is inactivated normally, however, when such cells cease growing.     

Expression in Bacillus subtilis of the 51- and 42-kilodalton mosquitocidal toxin genes of Bacillus sphaericus

A 3,080-base-pair KpnI-HindIII DNA fragment from Bacillus sphaericus 2362 coding for 51- and 42-kilodalton mosquitocidal proteins was cloned into Bacillus subtilis DB104 by using the vector pUB18. In B. subtilis these proteins were not detected during vegetative growth but were expressed during sporulation at levels comparable to those found in B. sphaericus.     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ

Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris). Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme. Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases. The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM). The corresponding DD-isomers (3d and 6d) are less effective. The N-modified analogs are the most potent competitive inhibitors. The inhibition constant (Ki) values for B. sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively. These N-modified analogs do not effectively inhibit L-lysine decarboxylase. None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives. Possible mechanisms of inhibition are discussed. In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media. Other analogs (1----3) showed essentially no antibacterial activity.     

Multispecific DNA methyltransferases from Bacillus subtilis phages. Properties of wild-type and various mutant enzymes with altered DNA affinity

Temperate Bacillus subtilis phages SPR, phi 3T, rho 11 and SP beta code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild-type and various mutant methyltransferases were overproduced 1000-fold in Escherichia coli and were purified by three consecutive chromatographic steps. The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (Mr) of about 50,000. The methyl-transfer kinetics of the SPR wild-type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CCATGG). Evaluation of the catalytic properties for DNA and S-adenosylmethionine binding suggested that the NH2-terminal part of the protein is important for both non-sequence-specific DNA binding and S-adenosylmethionine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH-terminal part lead to weaker site-specific interactions of the enzyme. Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the phi 3T, rho 11 and SP beta methyltransferases, bu failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.     

Role of arginine residue in saccharopine dehydrogenase (L-lysine forming) from baker's yeast

The baker's yeast saccharopine dehydrogenase (EC 1.5.1.7) was inactivated by 2,3-butanedione following pseudo-first-order reaction kinetics. The pseudo-first-order rate constant for inactivation was linearly related to the butanedione concentration, and a value of 7.5 M-1 min-1 was obtained for the second-order rate constant at pH 8.0 and 25 degrees C. Amino acid analysis of the inactivated enzyme revealed that arginine was the only amino acid residue affected. Although as many as eight arginine residues were lost on prolonged incubation with butanedione, only one residue appears to be essential for activity. The modification resulted in the change in Vmax, but not in Km, values for substrates. The inactivation by butanedione was substantially protected by L-leucine, a competitive analogue of substrate lysine, in the presence of reduced nicotinamide adenine dinucleotide (NADH) and alpha-ketoglutarate. Since leucine binds only to the enzyme-NADH-alpha-ketoglutarate complex, the result suggests that an arginine residue located near the binding site for the amino acid substrate is modified. Titration with leucine showed that the reaction of butanedione also took place with the enzyme-NADH-alpha-ketoglutarate-leucine complex more slowly than with the free enzyme. The binding study indicated that the inactivated enzyme still retained the capacity to bind leucine, although the affinity appeared to be somewhat decreased. From these results it is concluded that an arginine residue essential for activity is involved in the catalytic reaction rather than in the binding of the coenzyme and substrates.     

Thermostability of Bacillus subtilis neutral protease.

The thermostability of the B. subtilis neutral protease was studied under various conditions. At elevated temperatures the enzyme was inactivated as a result of autolysis. The rate of inactivation did not depend on the enzyme concentration and the enzyme was most stable near its pH optimum. The rate of inactivation was unaffected by the presence of a second protease during the incubation at high temperatures. The results indicate that the rate of thermal inactivation of the neutral protease is determined by the kinetics of local unfolding processes that precede autolysis rather than by the catalytic rate of the autodigestion reaction or an irreversible unfolding step.     

D-amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties.

D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at 280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate, respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate (2.3 muM) and pyridoxamine 5'-phosphate (25 muM).     

Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis

Two modification methylase genes of Bacillus subtilis R were cloned in Escherichia coli by using a selection procedure which is based on the expression of these genes. Both genes code for DNA-methyltransferases which render the DNA of the cloning host E. coli HB101 insensitive to the BspRI (5'-GGCC) endonuclease of Bacillus sphaericus R. One of the cloned genes is part of the restriction-modification (RM) system BsuRI of B. subtilis R with specificity for 5'-GGCC. The other one is associated with the lysogenizing phage SP beta B and produces the methylase M.BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B-derived gene also directs the synthesis in E. coli of a third methylase activity (M.BsuP beta BII), which protects the host DNA against HpaII and MspI cleavage within the sequence 5'-CCGG. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene. No cross-hybridization was detected either between the M.BsuRI and M.BsuP beta B genes or between these and the modification methylase gene of B. sphaericus R, which codes for the enzyme M.BspRI with 5'-GGCC specificity.     

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.     

Induction of citric acid cycle enzymes during initiation of sporulation by guanine nucleotide deprivation

In Bacillus subtilis, conditions causing partial deprivation of guanine nucleotides initiated sporulation and caused the synthesis of citrate synthase, aconitase, and alpha-ketoglutarate dehydrogenase. Alpha-ketoglutarate dehydrogenase could also be induced by acetate, and the specific activity of this enzyme was elevated in mutants that had high intracellular acetyl coenzyme A concentrations because they lacked citrate synthase activity. After deprivation of guanine nucleotides, the intracellular concentration of acetyl coenzyme A also increased, which explained the induction of alpha-ketoglutarate dehydrogenase. Furthermore, the decreases in alpha-ketoglutarate and L-malate concentrations observed during this deprivation accounted for the observed increases in citrate synthase activity (which was repressed by alpha-ketoglutarate and malate) and aconitase activity (which was repressed by alpha-ketoglutarate).     

The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity.

After site-directed mutagenesis, the genes coding for the 42- and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the Bacillus subtilis vector pUE (a derivative of pUB18). The levels of expression of the gene products in B. subtilis DB104 and B. sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots. The results indicated that a higher amount of protein was produced in B. subtilis DB104. Electron microscopic examination of B. subtilis DB104 and B. sphaericus 718 containing the 42- and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B. sphaericus 2362 were produced in the latter. Strains producing only the 42- or the 51-kDa protein were not toxic to larvae of C. pipiens. A mixture of both strains, a single strain producing both proteins, or a fusion of the 51- and the 42-kDa proteins was toxic. The amount of B. subtilis DB104 containing the 42- and the 51-kDa proteins necessary to kill 50% of the larvae of C. pipiens was 5.6 ng (dry weight) of cells per ml. This value was significantly lower than that for B. sphaericus 2362 (14 ng [dry weight] per ml). Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein this protein to primarily 39- and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa. Past studies involving purified proteins from B. sphaericus 2362 indicate an associate of toxicity with the 39-kDa peptide. The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.