Pyridine-2, 6-dicarboxylic acid (dipicolinic acid) formation in Bacillus subtilis. II Non-enzymatic and enzymatic formations of dipicolinic acid from alpha, epsilon-diketopimelic acid and ammonia.

Non-enzymatic formation of dipicolinic acid (DPA) from diketopimelic acid and ammonia was clearly demonstrated using a new method for DPA analysis. The reaction rates of DPA formation were almost the same under aerobic and anaerobic conditions. Nearly equimolecular quantities of DPA and tetrahydrodipicolinic acid were detected in spontaneous reaction mixture. The spontaneous reaction seemed to be due to dismutation of dihydrodipicolinic acid, resulting in DPA and tetrahydrodipicolinic acid. The apparent optimum pH of the spontaneous reaction was 8.2 and the maximal rate of DPA formation was observed with a 1 : 4 molar ratio of diketopimelic acid to ammonia. The rate of the spontaneous reaction was stimulated by ferrous sulfate, FMN, and riboflavin. Dihydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinate, prepared from pyruvate and aspartic beta-semialdehyde, with NADPH as reductant. The reductase was isolated from Bacillus subtilis, and found to stimulate DPA formation from diketopimelic acid and ammonia. The enzymatic DPA formation was absolutely dependent on oxygen, and optimum pH was 6.4. The catalytic action of the enzyme was similar to that of the oxidase. Possible mechanisms of DPA formation from diketopimelic acid and ammonia are proposed.

Acrodermatitis enteropathica and the relation to pellagra.

A patient with a variant form of acrodermatitis enteropathica (AE) without hypozincemia is presented who showed a rise in plasma zinc and partial improvement on a pancreatic enzyme preparation, apparently because of its content of picolinic acid (PA). Complete recovery occurred on 60 mg zinc (1). This patient has now been treated with zinc PA (equal to only 5 mg zinc) and subsequently with PA. Both maintained elevated plasma zinc levels. Because of the similarity of AE with pellagra and the common origin of PA and nicotinic acid from tryptophan, a hypothesis is presented which suggests that skin manifestations in the two disorders are due to PA deficiency since picolinic carboxylase forms NAD preferentially when there is competition for the common precursor.

Relationship between sporulation and synthesis of mycobacillin and dipicolinic acid under condition of catabolite repression in Bacillus subtilis.

Sporulation was repressed in the parent strain by various carbon sources whereas glucose-resistant mutants were resistant to them but not to glycerol 2-phosphate. Both mycobacillin and dipicolinic acid synthesis were repressed in the parent by some of the compounds tested, viz. glucose, pyruvate and glycerol 2-phosphate. However, these syntheses in the glucose-resistant mutants were not repressed by glucose and pyruvate but were repressed by glycerol 2-phosphate. The possible interrelationship between sporulation, dipicolinic acid and mycobacillin synthesis is discussed in light of these findings.

Red pigment in Bacillus megaterium spores.

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation.

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-semialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHDPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of L-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested. In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.

Branched pattern of regulatory interactions between late sporulation genes in Bacillus subtilis.

We measured the synthesis of dipicolinic acid (DPA) during sporulation in spo mutants of Bacillus subtilis by a sensitive biological assay based on cross-feeding of a spoVF mutant strain and also chemically. Many spo mutations, including several that block sporulation at stage III, did not prevent synthesis of DPA but instead prevented its incorporation into the spore. In general, strains with mutations in loci that are expressed in the spore compartment synthesized DPA, whereas strains with mutations in most of the loci that are expressed in the mother-cell compartment did not. Transcription of the gerE gene, as measured by DNA-RNA hybridization, followed a dependence pattern very similar to that of DPA synthesis. However, the dependence patterns of the two operons show that at about stage IV of sporulation there is a branch in the sequence of operon expression in the mother cell. One branch leads through spoVC to synthesis of DPA synthetase, and the other leads through spoVD to expression of gerE.

Biosynthesis and physiological role of homarine in marine shrimp.

Glycine, which contributes 2 carbon atoms and the nitrogen for the biosynthesis of homarine by homogenates of shrimp muscle, reacts metabolically with succinyl coenzyme A to form N-succinylglycine. The latter product is effectively converted by such homogenates to homarine, and it is concluded that N-succinylglycine is on the main pathway of this biosynthetic series of reactions and provides all of the required atoms in homarine, except for the N-methyl carbon. A possible pathway for the complete biosynthesis of homarine is described. Evidence is presented that homarine acts as a transmethylating agent in shrimp muscle homogenates and is capable of transferring its N-methyl group to form mono-, di-, and trimethylamines, trimethylamine oxide, choline, and betaine. In this process, homarine loses its methyl groups to form picolinic acid, and, conversely, picolinic acid can be methylated to yield homarine. It is speculated that homarine is not only a "methyl" donor but may serve as a reservoir of methyl groups in crustacea.

Derepression of sporulation and synthesis of mycobacillin and dipicolinic acid by guanosine 3':5'-cyclic monophosphate under conditions of glucose repression in Bacillus subtilis.

Dibutyryl cyclic GMP, but not dibutyryl cyclic AMP, derepresses sporulation and synthesis of mycobacillin and dipicolinic acid under conditions of glucose repression in Bacillus subtilis strain B34. Neither of these compounds appears to affect sporulation and synthesis of mycobacillin and dipicolinic acid in this strain under normal physiological conditions. Mutants insensitive to glucose repression were indifferent to the addition of either of the nucleotides both in the presence and in the absence of glucose. A role for dibutyryl cyclic GMP in annulling the repressing effect of glucose on sporulation and on synthesis of mycobacillin and dipicolinic acid is thus indicated.

Sporulation in Bacillus subtilis. Morphological changes.

1. When Bacillus subtilis was grown in a medium in which sporulation occurred well-defined morphological changes were seen in thin sections of the cells. 2. Over a period of 7.5hr. beginning 2hr. after the initiation of sporulation the following major stages were observed: axial nuclear-filament formation, spore-septum formation, release of the fore-spore within the cell, development of the cortex around the fore-spore, the laying down of the spore coat and the completion of the corrugated spore coat before release of the spore from the mother cell. 3. The appearance of refractile bodies and 2,6-dipicolinic acid and the development of heat-resistance began between 5 and 6.5hr. after initiation of sporulation. 4. The appearance of 2,6-dipicolinic acid and the onset of refractility appeared to coincide with a diminution of electron density in the spore core and cortex. 5. Heat-resistance was associated with the terminal stage, the completion of the spore coat. 6. The spore coat was composed of an inner and an outer layer, each of which consisted of three or four electron-dense laminae. 7. Serial sections through cells at an early stage of sporulation showed that the membranes of each spore septum were always continuous with the membranes of a mesosome, which was itself in close contact with the bacterial or spore nucleoid. 8. These changes were correlated with biochemical events occurring during sporulation.

Time course of purine nucleoside phosphorylase occurrence in sporulation of Bacillus cereus.

Purine nucleoside phosphorylase (EC 2.4.2.1) from Bacillus cereus T was examined at hourly intervals during growth and sporulation. The enzyme has maximal activity in extracts prepared from cells during stages I and II. The activity during exponential growth is only 6.6% of the maximum and that in free spores is only 3.3%. Conservation of the purine nucleoside phosphorylase during sporulation is apparent as shown by the gradual increase in heat resistance.

Isotopic study of control of the lysine biosynthetic pathway during sporulation of Bacillus cereus.

The extent of incorporation of aspartate into dipicolinic acid and into various amino compounds was determined in Bacillus cereus at various times before, during, and near the end of synthesis of dipicolinic acid. The purpose of this study was to gain further information on the in vivo control of the biosynthesis of amino acids derived from aspartate. Control of the lysine biosynthetic pathway was of particular interest with regard to sporulation, owing to the important role of diaminopimelate and dipicolinate in the structure of the spore. As synthesis of dipicolinate was initiated, incorporation of carbon derived from aspartate was funneled preferentially into this compound as compared with others of the aspartate group. Incorporation into lysine essentially stopped just before the synthesis of dipicolinate began. This is consistent with the previously observed disappearance at this time of diaminopimelic acid decarboxylase in cell-free extracts. Synthesis of diaminopimelate continued during the time of synthesis of dipicolinate. The previous suggestion that diaminopimelate might exert negative control on one of the enzymes between dihydrodipicolinate and diaminopimelate is thus considered unlikely. The possibility is discussed that synthesis of dipicolinate is favored by an increase in the rate of synthesis of dihydrodipicolinate rather than by a block in its rate of utilization.

Ultraviolet sensitivity and photoproducts in spore-like bodies of an excision-repair-deficient and dipicolinic-acid-less strain of Bacillus subtilis.

Bacillus subtilis strain UVS-42DPA is defective in both excision-repair capability and dipicolonic acid(DPA)accumulation. In sporulation medium, it forms spore-like bodies, which are sensitive to ultraviolet light (UV) as the vegetative cells and produce mostly cyclobutane dimers instead of "spore photoproduct" upon UV irradiation. The results suggest that the drastic change in the photochemical reactivity of DNA during sporulation might be induced and(or) maintained by the accumulation of DPA.

Bacterial metabolism of arylsulfonates: role of meta cleavage in benzene sulfonate oxidation by Pseudomonas testosteroni.

Pseudomonas testosteroni H-8 oxidizes certain lower alkylbenzene sulfonates at rates inversely related to the length of the alkyl group. Appreciable Q(O)2 values were observed for benzene sulfonate (BS), toluene sulfonate (TS), and ethylbenzene sulfonate (EBS), but not for propylbenzene sulfonate (PS) and higher homologues. Catechol oxidation was catalyzed by a constitutive catechol-2,3-oxygenase (EC 1.99.2.a). Yellow meta cleavage products accumulated when BS-grown cells were exposed to catechol, 4-methylcatechol, 3-methylcatechol, EBS and PS, but not BS or TS. Traces of a yellow metabolite (probably 2-hydroxymuconic semialdehyde) were detectable during growth on BS. PS completely inhibited growth on BS, but not on L-leucine or nutrient broth. Also, PS antagonized respiration on BS and catechol, but not glutamate, the extent of inhibition being directly related to PS concentration. Formation of a meta cleavage product from PS, and inhibition of catechol oxidation by PS, suggested that the actual inhibitor may not be PS itself, but a metabolite.

Biosynthesis in vitro of homarine and pyridine carboxylic acids in marine shrimp.

Minces and homogenates of muscle obtained from the marine shrimp Penaeus duorarum are capable of synthesizing homarine from [14C]glycine. Glycine carbon atoms are incorporated into homarine but not significantly into picolinate or quinolinate. [2-14C]Acetate is readily incorporated into quinolinate in the in vitro system but only slightly into homarine and not at all into picolinate. Quinolinic acid is rapidly methylated to N-methyl quinolinate which is not decarboxylated to form homarine. Procedures have been developed for the satisfactory separation of N-methyl quinolinate from homarine.

Diaminopimelate decarboxylase of sporulating bacteria.

The meso-diaminopimelate (DAP) decarboxylase of Bacillus licheniformis, a pyridoxal phosphate-requiring enzyme, was stabilized in vitro by 0.15 m sodium phosphate buffer (pH 7.0) containing 1 mm 2,3-dimercaptopropan-1-ol, 100 mug of pyridoxal phosphate per ml, and 3 mm DAP. When the meso-DAP concentration was varied, the enzyme in cell-free extracts of B. licheniformis exhibited Michaelis-Menten kinetics. Pyridoxal phosphate was the only pyridoxine derivative which acted as a cofactor. The enzyme was subject to both inhibition and repression by l-lysine. The inhibitory effect of lysine was on the K(m) (meso-DAP). A maximum repression of about 20% was obtained. No significant inhibition or activation was produced by cadaverine, dipicolinic acid, phenylalanine, pyruvate, ethylenediamine-tetraacetate, adenosine triphosphate, adenosine diphosphate, or adenosine monophosphate. When B. licheniformis was grown in an ammonium lactate-glucose-salts medium, an increase in DAP decarboxylase specific activity occurred during cellular growth with a maximal specific activity at the end of the exponential phase. As soon as growth ceased, the specific activity of the enzyme decreased to approximately one-half of the maximal specific activity and remained at this level thereafter. When B. cereus was grown in complex media, there was an increase in DAP decarboxylase specific activity up to the end of the exponential phase. Thereafter, the specific activity decreased to a nondetectable level in 4 hr. Dipicolinic acid synthesis was first detected 15 min later and was essentially complete after an additional 2.5 hr. The significance of the disappearance of DAP decarboxylase in B. cereus was discussed with regard to control of dipicolinic acid and spore mucopeptide biosynthesis.

Partial purification and characterization of dihydrodipicolinic acid synthetase from sporulating Bacillus megaterium.

Sporulation of Bacillus megaterium Km (ATCC 13632) was synchronized by a technique employing three 10% transfers. The culture was harvested when 60% of the cells contained spore forms. Dihydrodipicolinic acid synthetase was purified 150-fold by ammonium sulfate fractionation at pH 6.5, heating for 15 min at 45 C at pH 6.0, ammonium sulfate fractionation at pH 6.0, and subsequent chromatography on diethylaminoethyl cellulose. During the final stage of the purification procedure, the enzyme exhibited sensitivity to refrigeration temperatures. The enzyme had a pH optimum of 7.65 in imidazole buffer. The apparent K(m) values were 4.6 x 10(-4) and 5.0 x 10(-4)m for beta-aspartyl semialdehyde and pyruvate, respectively. All attempts to demonstrate cofactor requirements were unsuccessful. Sulfhydryl inhibiting reagents and lysine did not inhibit the enzymatic reaction. The enzyme exhibited maximal thermal resistance at pH 10.5. The thermal stability of the enzyme at 75 C was increased more than 1,800-fold by the addition of 0.3 m pyruvate. The E(a) was 67,300 cal/mole for the thermal denaturation of the enzyme. At 60 C, the DeltaF, DeltaH, and DeltaS values for the thermal denaturation of the enzyme were 22,250, 66,700, and 133 cal per mole per degree, respectively.