Dihydrodipicolinic acid synthase of Bacillus licheniformis. Quaternary structure, kinetics, and stability in the presence of sodium chloride and substrates.

Dihydrodipicolinic acid synthase (L-aspartate-beta-semialdehyde hydro-lyase (adding pyruvate and cyclising), EC 4.2.1.52) obtained from Bacillus licheniformis was purified to homogeneity. Its molecular weight was 108 000 to 117 500, depending on the concentration of NaCl and substrates present, and it contained four subunits of identical molecular weight (28000). The Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 and 2.6 mM, respectively. It was previously shown that pyruvate and a high sodium chloride concentration contributed to the stability of the enzyme. The effect of these substances and the other substrate, L-aspartic semialdehyde, on molecular weight was determined. None of these three substances significantly affected the apparent molecular weight. The effect of sodium chloride, pyruvate, and L-aspartic semialdehyde on enzyme structure was studied by determining the effect of their presence on inactivation of the enzyme by several chemical denaturants and heat. Pyruvate dramatically protected against inactivation by all of the denaturants. Sodium chloride protected against inactivation by sodium dodecyl sulfate, guanidine-HCl, urea, and heat, but somewhat facilitated inactivation by ethanol. L-Aspartic semialdehyde had no significant effect on inactivation by sodium dodecyl sulfate and ethanol; it rendered the enzyme slightly more sensitive to inactivation by guanidine-HCl and urea. The thermal melting curve obtained for the enzyme in the presence of L-aspartic semialdehyde was biphasic. The activity was reduced approximately 50% by heating for 30 min at temperatures between 50 and 80 degrees C. Only by heating at temperatures above 80 degrees C did the inactivation become complete. The partially inactivated enzyme could be reactivated by heating after removal of the L-aspartic semialdehyde. Pyruvate prevented the partial inactivation and facilitated reactivation. The only difference detected between the native enzyme and the partially inactivated form of the enzyme was that the latter had a reduced V. It is known that in other spore-formers, dihydrodipicolinate synthase increases in activity late in sporulation. This increase may be important for normal sporulation to occur. The possibility is discussed that the intracellular pool sizes of pyruvate and L-aspartic semialdehyde might have an influence on the level of dihydrodipicolinate synthase activity, by controlling the amount of partial inactivation of the enzyme that occurs in vivo.

Medium Promoting Sporulation of Bacillus larvae and Metabolism of Medium Components.

A new medium, designated TMYGP broth, was developed that allowed the honeybee pathogen Bacillus larvae NRRL B-3650 to produce up to 5 x 10 spores per ml of culture (microscopic count). This species normally sporulates poorly, if at all, in artificial broth media. An aeration rate lower than that normally used to cultivate other Bacillus species was required for sporulation. During the exponential growth phase, acids were produced by catabolism of yeast extract components, causing a decrease in pH of the medium. Thereafter, the pH began to increase, probably because of derepression of the citric acid cycle and consumption of the acids. Only after this time did usage of glucose from the medium occur. Thus, glucose usage seems to be regulated by catabolite repression. The presence of glucose was needed for one or more of the later events of sporulation. Of many substances tested, only gluconic acid and glucosamine partially substituted for glucose as a requirement for sporulation. Pyruvate was also required for good sporulation. It was metabolized during the late-exponential phase of growth.

Protection of Bacillus larvae from Oxygen Toxicity with Emphasis on the Role of Catalase.

Sporulation of Bacillus larvae NRRL B-3650 occurred only at aeration rates lower than those used for cultivation of most Bacillus species. One possible explanation for the requirement for a low level of aeration in B. larvae is that toxic forms of oxygen such as H(2)O(2) and superoxide are involved. The superoxide dismutase levels of strain B-3650 were similar to those of Bacillus subtilis 168 during sporulation, and no NADH peroxidase was present. Catalase activity was absent during exponential growth and first appeared near the start of the stationary phase. The catalase activity was 2,700 times less than that in B. subtilis 168 at the same stage of development. Therefore, the relative deficiency of catalase (and NADH peroxidase) might be the cause of the apparent O(2) toxicity. It was postulated that B. larvae might accumulate H(2)O(2) in the medium and exhibit more than normal sensitivity to H(2)O(2). Experimental results did not verify either postulate, but the possibilities of intracellular accumulation of H(2)O(2) and unusual sensitivity to endogenous H(2)O(2) were not excluded. The catalase present in early-stationary-phase cells was soluble, heat labile, and inhibited by cyanide, azide, and hydroxylamine. An increase in catalase activity also occurred at the time of appearance of refractile spores in both B. larvae NRRL B-3650 and B. subtilis 168. The level of catalase activity in strain B-3650 was 5,400 times less than that in B. subtilis 168 at this stage. In B. larvae, this second increase occurred primarily within the developing endospore. The activity in spore extracts was particulate, heat stable, and inhibited by hydroxylamine but not by azide or cyanide. Synthesis of catalase in B. larvae was unaffected by H(2)O(2), O(2), or glucose.

Studies on transfection and transformation of protoplasts of Bacillus larvae, Bacillus subtilis, and Bacillus popilliae.

Protoplasts of Bacillus larvae NRRL b-3555 and Bacillus subtilis RM125 (restrictionless, modificationless mutant) were transfected with DNA from the B. larvae bacteriophage PBL1c in the presence of polyethylene glycol. B. subtilis 168 and Bacillus popilliae NRRL B-2309M protoplasts could not be transfected with PBL1c DNA. Protoplasts of B larvae NRRL B-3555 were transformed with plasmids pC194 and pHV33 in the presence of polyethylene glycol. The frequency of transformation was much higher when the plasmids were isolated from B. larvae NRRL B-3555 transformants than when they were isolated from B. subtilis 168. These results indicate that the restriction-modification systems found in B. larvae NRRL B-3555 and B. subtilis 168 may be different. Conditions for protoplast formation and cell wall regeneration were developed for B. popilliae NRRL B-2309S. However, no transformation occurred with plasmids pC194 and pHV33 (isolated from B. subtilis 168).

Ultrastructure of Sporulating Bacillus larvae in a Broth Medium.

An electron microscopic study of sporulation of Bacillus larvae, a honeybee pathogen, in TMYGP broth (D. W. Dingman and D. P. Stahly, Appl. Environ. Microbiol. 46:860-869, 1983) was conducted. No parasporal structures were evident in the sporangial cytoplasm. The stages of sporulation were similar to those observed in other sporeformers. A rather unusual inner coat layer consisting of seven lamellae was apparent.

Prodiginine (prodigiosin-like) pigments from Streptoverticillium rubrireticuli, an organism that causes pink staining of polyvinyl chloride.

Red pigments were extracted from Streptoverticillium rubrireticuli strain 100-19, an organism frequently incriminated in pink staining of polyvinyl chloride. These pigments were identified as undecylprodiginine and butylcycloheptylprodiginine.

Polysaccharide that may serve as a carbon and energy storage compound for sporulation in Bacillus cereus.

An intracellular, glucose-containing polysaccharide accumulates in Bacillus cereus early in sporulation and is degraded at the time of spore maturation. This pattern of accumulation and degradation occurred when growth was limited by glucose or a component of yeast extract. These data suggest that the polysaccharide may be serving as a carbon and energy storage compound for sporulation. A somewhat similar pattern of accumulation and degradation of poly-beta-hydroxybutyric acid (PHB) was shown earlier by Kominek and Halvorson (1965) to occur in Bacillus cereus. When cells were grown in a medium buffered strongly at pH 7.4, however, very little accumulation of PHB occurred. We have found that polysaccharide accumulates in cells grown in both the strong and weakly buffered media. Perhaps polysaccharide is the major carbon and energy storage compound when cells are grown under conditions preventing significant accumulation of PHB. The lack of polysaccharide accumulation during the exponential phase of growth may be an indication that the needed biosynthetic enzymes are controlled by catabolite repression during growth. The polysaccharide was purified and found to consist of glucose. The iodine absorption spectrum suggests a degree of branching between that of glycogen and amylopectin.

Repression of diaminopimelate decarboxylase by L-lysine in different Bacillus species.

Synthesis of diaminopimelate decarboxylase in 7 of 11 different Bacillus species tested was subject to l-lysine repression.

Control of diaminopimelate decarboxylase by L-lysine during growth and sporulation of Bacilluscereus.

l-Lysine caused repression of diaminopimelate decarboxylase synthesis in Bacillus cereus when grown in either a minimal defined medium (CDGS medium) or a complex defined medium (a modified lysine assay medium). When cells were grown in either of the two media, variations in the specific activity of the enzyme as a function of time were found to be correlated with the intracellular lysine pool size during growth. From all of the data presented, it seems reasonable to conclude that during growth the synthesis of diaminopimelate decarboxylase is probably regulated by the intracellular lysine pool size. The relationship between lysine pool concentration and the specific activity of the enzyme did not occur in sporulating cells. The specific activity of diaminopimelate decarboxylase started to decrease at the end of exponential growth and continued to decline until it became nondetectable at the time of dipicolinic acid synthesis and development of spore refractility. Throughout this time, the intracellular lysine pool size remained below that which allowed derepression of enzyme synthesis during exponential growth. The mechanism(s) responsible for the observed decrease in the specific activity of the enzyme at the end of exponential growth is unknown. A threefold rise in the intracellular diaminopimelic acid concentration occurred when there was little or no detectable enzyme activity at the time of dipicolinic acid synthesis. This accumulation of diaminopimelic acid may exert positive control on the synthesis of spore peptidoglycan, the major component of the spore cortex.

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.

Regulation of dihydrodipicolinate synthase during growth and sporulation of Bacillus cereus.

A four- to sixfold increase in specific activity of dihydrodipicolinic acid synthase was observed during sporulation of Bacillus cereus. The enzyme from cells harvested before and after the increase in specific activity appeared to be very similar as judged by pH optima, heat denaturation kinetics, apparent Michaelis constants, chromatography on diethylaminoethyl-cellulose and Sephadex G-200, and polyacrylamide gel electrophoresis. Studies with various combinations of amino acids and one of the enzyme substrates, pyruvate, failed to give evidence for control of the enzyme by activation, inhibition, repression, induction, or stabilization. Omission of calcium from the sporulation medium had no significant effect on the specific activity pattern of the enzyme as a function of age of culture.

Sporulation and regulation of homoserine dehydrogenase in Bacillus subtilis.

Homoserine dehydrogenase in dialyzed cell extracts of Bacillus subtilis 168 was studied, particularly with regard to inhibition, repression, and level of activity as a function of stage of development (growth and sporulation). It was assayed in the "forward direction" using L-aspartic semialdehyde and NADPH as substrates. Of the potentials inhibitors tested, only cysteine and NADP were found to be effective. Both L- and D-cysteine were equally effective. Therefore, the physiological significance of cysteine as an inhibitor is somewhat questionable. Amino acids involved in repression of homoserine dehydrogenase included methionine, isoleucine, possibly threonine, and one or more unidentified components of Casamino acids. The specific activity of homoserine dehydrogenase was highest during the exponential phase of growth and declined steadily during the stationary phase of growth. The low specific activity during late sporulation may favor preferential funnelling of L-aspartic semialdehyde into the lysine pathway, where it is needed for synthesis of large amounts of dipicolinic acid and diaminopimelic acid.

Effect of 8-azaguanine on the transition from vegetative growth to presporulation in Bacillus cereus.

Stahly, D. P. (University of Illinois, Urbana), V. R. Srinivasan, and H. Orin Halvorson. Effect of 8-azaguanine on the transition from vegetative growth to presporulation in Bacillus cereus. J. Bacteriol. 91:1875-1882. 1966.-The guanine analogue, 8-azaguanine (azaG), was found to inhibit sporulation of Bacillus cereus strain T when added to proliferating cells, but not to inhibit when added after the transition to presporulation. When azaG was added to vegetative cells, the growth rate was reduced, but no immediate bactericidal effect was demonstrated. Azaguanine was shown to be incorporated solely into ribonucleic acid (RNA). All of the natural purine bases and nucleosides were found to prevent azaG inhibition by blocking incorporation of the analogue into the RNA. Addition of a subinhibitory level of C(14)-azaG to proliferating cells resulted in an increase in incorporation paralleling the increase in number of cells. At the time of transition from growth to presporulation, a rapid removal of the azaG label from the cells occurred in the absence of net RNA breakdown. If differentiation was inhibited by increasing the concentration of azaG, then no expulsion took place. Instead, at the end of growth, net incorporation ceased, and a steady-state condition was established in which incorporation equaled breakdown. No azaG degradative enzymes are present in presporulating cells. The possibility is discussed that an increase in the ratio of natural purines to azaG occurred at the time of transition, and that the natural purine derivatives then were reincorporated into RNA preferentially to azaG. The data are consistent with the hypothesis than an increased rate of RNA turnover occurs at the time of transition from vegetative growth to presporulation. Addition of phosphate buffer (pH 7.0, 0.1 m) to azaG-inhibited vegetative cells caused reversal of inhibition, the reversal being accompanied by expulsion of the azaG. At least a partial explanation of this effect is that phosphate causes a decrease in the azaG intracellular pool size.

Isolation of two bacteriophages from Bacillus larvae, PBL1 and PBL0.5, and partial characterization of PBL1.

Two temperate bacteriophages have been isolated from Bacillus larvae: PBL1 and PBL0 .5. Strains lysogenic for either of these phages are immune to lysis by the same phage but are sensitive to the other phage. PBL1 has an oval head, a non-contractile tail, and a base plate with a pin structure but no apparent tail fibres. The genome of PBL1 consists of double-stranded DNA with a molecular weight of 24.1 (+/-0.6) X 10(6), a G + C content (derived from melting temperature) of 41.5%, and cohesive ends. Restriction enzyme analysis permitted construction of a physical map of the genome.

Selective Medium for Quantitation of Bacillus popilliae in Soil and in Commercial Spore Powders.

A medium consisting of MYPGP agar supplemented with vancomycin was found to be highly selective for Bacillus popilliae, especially for strains originally isolated from Japanese beetle larvae. The medium has proven to be useful for the quantitation of B. popilliae spores in commercial spore powder and in soil.

Bacterial sporulation and regulation of dihydrodipicolinate synthase in ribonucleic acid polymerase mutants of Bacillus subtilis.

The activity of dihydrodipicolinate synthase increased late in sporulation in Bacillus subtilis. Mutants blocked at several stages of sporulation due to having an altered ribonucleic acid polymerase failed to exhibit this increase.

An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene.

McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.

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.