Sporulation-associated activation of Bacillus sphaericus larvicide

Preparations of the larvicidal crystal from 46-h cultures of Bacillus sphaericus 2362 contain 125-, 110-, 63-, and 43-kilodalton (kDa) proteins (P. Baumann, B. M. Unterman, L. Baumann, A.H. Broadwell, S.J. Abbene, and R.D. Bowditch, J. Bacteriol. 163:738-747, 1985). The 63- and 43-kDa proteins, which have been purified, are not immunologically cross-reactive, and only the 43-kDa protein is toxic to mosquito larvae. Since antigenic determinants of the two smaller proteins have been detected in the higher-molecular-weight proteins (125 and 110 kDa), it has been suggested that the latter are precursors of the 43- and 63-kDa peptides. In the present study, purified 110-kDa protein was found to be toxic to the larvae of Culex pipiens (50% lethal concentration = 115 ng/ml). A luciferase-luciferin assay for intracellular ATP as well as an assay based on the exclusion of Trypan Blue by live cells indicated that the 110-kDa protein had no effect on tissue-culture-grown cells of C. quinquefasciatus, while cells exposed to the 43-kDa protein rapidly lost viability (50% lethal concentration = 54 microgram(s)/ml by the intracellular ATP assay). These findings suggested that the 110-kDa protein and, by extension, the 125-kDa protein are protoxins which are activated during sporulation by cleavage to a 43-kDa toxin. To further investigate the origins and relationships of the crystal proteins of B. sphaericus, we analyzed samples during the growth and sporulation of the culture. Synthesis of crystal proteins was initiated at the end of exponential growth and was completed after about 7 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sporulation-associated activation of Bacillus sphaericus larvicide.

Preparations of the larvicidal crystal from 46-h cultures of Bacillus sphaericus 2362 contain 125-, 110-, 63-, and 43-kilodalton (kDa) proteins (P. Baumann, B. M. Unterman, L. Baumann, A.H. Broadwell, S.J. Abbene, and R.D. Bowditch, J. Bacteriol. 163:738-747, 1985). The 63- and 43-kDa proteins, which have been purified, are not immunologically cross-reactive, and only the 43-kDa protein is toxic to mosquito larvae. Since antigenic determinants of the two smaller proteins have been detected in the higher-molecular-weight proteins (125 and 110 kDa), it has been suggested that the latter are precursors of the 43- and 63-kDa peptides. In the present study, purified 110-kDa protein was found to be toxic to the larvae of Culex pipiens (50% lethal concentration = 115 ng/ml). A luciferase-luciferin assay for intracellular ATP as well as an assay based on the exclusion of Trypan Blue by live cells indicated that the 110-kDa protein had no effect on tissue-culture-grown cells of C. quinquefasciatus, while cells exposed to the 43-kDa protein rapidly lost viability (50% lethal concentration = 54 microgram(s)/ml by the intracellular ATP assay). These findings suggested that the 110-kDa protein and, by extension, the 125-kDa protein are protoxins which are activated during sporulation by cleavage to a 43-kDa toxin. To further investigate the origins and relationships of the crystal proteins of B. sphaericus, we analyzed samples during the growth and sporulation of the culture. Synthesis of crystal proteins was initiated at the end of exponential growth and was completed after about 7 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone side-chain cleavage by Bacillus sphaericus

The side-chain of progesterone was cleaved by Bacillus sphaericus to produce two C-19 keto androstene steroids. The structures of these metabolites were androstenedione and 1-dehydroandrostenedione. High concentrations of glucose in the culture medium inhibited conversion of progesterone to these two metabolites.     

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.     

Type II restriction endonucleases from Bacillus sphaericus

Abstract The galactophilic lectin of the bacterium Pseudomonas aeruginosa (PA-I) was used for mitogenic stimulation of peripheral bloodlymphocytes from cancer-bearing patients and healthy subjects. This lectin, which preferentially stimulates sialidase-treated lymphocytes, was shown to be useful in the detection of an impairment in the mitogenic response of the patients' lymphocytes. Its efficiency was at least as that of the Phaseolus vulgaris lectin (PHA), which is widely used for the diagnosis and prognosis of deficient immunocompetence states.     

Purification of larvicidal protein from Bacillus sphaericus 1593

Coat proteins from the spores of Bacillus sphaericus 1593 were separated by preparative polyacrylamide gel electrophoresis. Neutralising antibodies were raised against a single protein band exhibiting toxicity to mosquito larvae. IgG was purified and coupled to CNBr-activated Sepharose 4B to be used as an immunoaffinity matrix. The larvicidal protein was purified to electrophoretic homogeneity using this immunoaffinity column. The single protein species resolved into four peptides of molecular weights 42.6, 44.1, 50.7 and 51.3 KDa on polyacrylamide gel electrophoresis under denaturing conditions. This protein contained 12% carbohydrates. The purified protein exhibited an LC50 value of 8.3 +/- 1.6 ng/ml when tested against early third instar larvae of the mosquito Culex pipiens var quinquefasciatus.     

Structure of the mosquitocidal toxin from Bacillus sphaericus

The catalytic domain of a mosquitocidal toxin prolonged by a C-terminal 44 residue linker connecting to four ricin B-like domains was crystallized. Three crystal structures were established at resolutions between 2.5A and 3.0A using multi-wavelength and single-wavelength anomalous X-ray diffraction as well as molecular replacement phasing techniques. The chainfold of the toxin fragment corresponds to those of ADP-ribosylating enzymes. At pH 4.3 the fragment is associated in a C(7)-symmetric heptamer in agreement with an aggregate of similar size observed by size-exclusion chromatography. In two distinct crystal forms, the heptamers formed nearly spherical, D(7)-symmetric tetradecamers. Another crystal form obtained at pH 6.3 contained a recurring C(2)-symmetric tetramer, which, however, was not stable in solution. On the basis of the common chainfold and NAD(+)-binding site of all ADP-ribosyl transferases, the NAD(+)-binding site of the toxin was assigned at a high confidence level. In all three crystal forms the NAD(+) site was occupied by part of the 44 residue linker, explaining the known inhibitory effect of this polypeptide region. The structure showed that the cleavage site for toxin activation is in a highly mobile loop that is exposed in the monomer. Since it contains the inhibitory linker as a crucial part of the association contact, the observed heptamer is inactive. Moreover, the heptamer cannot be activated by proteolysis because the activation loop is at the ring center and not accessible for proteases. Therefore the heptamer, or possibly the tetradecamer, seems to represent an inactive storage form of the toxin.     

The ADP-ribosylating mosquitocidal toxin from Bacillus sphaericus: proteolytic activation, enzyme activity, and cytotoxic effects.

The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is a approximately 97-kDa protein sharing sequence homology within the N terminus with the catalytic domains of various bacterial ADP-ribosyltransferases. Here we studied the proteolytic activation of the ADP-ribosyltransferase activity of MTX. Chymotrypsin treatment of the 97-kDa MTX holotoxin (MTX(30-870)) results in a 70-kDa putative binding component (MTX(265-870)) and a 27-kDa enzyme component (MTX(30-264)), possessing ADP-ribosyltransferase activity. Chymotryptic cleavage of an N-terminal 32-kDa fragment of MTX (MTX(30-308)) also yields MTX(30-264), but the resulting ADP-ribosyltransferase activity is much greater than that of the processed MTX(30-870). Kinetic studies revealed a K(m) NAD value of 45 microm for the processed 32-kDa MTX fragment, and a K(m) NAD value of 1300 microm for the processed holotoxin. Moreover, the k(cat) value for the activated MTX(30-308) fragment was about 10-fold higher than that for the activated holotoxin (MTX(30-870)). Precipitation analysis showed that the 70-kDa proteolytic fragment of MTX remains noncovalently bound to the N-terminal 27-kDa fragment, thereby inhibiting ADP-ribosyltransferase and NAD glycohydrolase activities. Glu(197) of MTX(30-264) was identified as the "catalytic" glutamate that is conserved in all ADP-ribosyltransferases. Whereas mutated MTX(30-264)E197Q has neither ADP-ribosyltransferase nor NAD glycohydrolase activity, mutated MTX(30-264)E195Q possesses glycohydrolase activity but not transferase activity. Transfection of HeLa cells with a vector encoding a fusion protein of MTX(30-264) with a green fluorescent protein led to cytotoxic effects characterized by cell rounding and formation of filopodia-like protrusions. These cytotoxic effects were not observed with the catalytically inactive MTX(30-264)E197Q mutant, indicating that the MTX enzyme activity is essential for the cytotoxicity in mammalian cells.     

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.     

Larvicidal toxin from Bacillus sphaericus spores: Isolation of toxic components

Meiotic maturation of amphibian oocytes induced by progesterone is known to be regulated by protein phosphorylation. To investigate a possible role for protein phosphatase-1 in this process, the effect of phosphatase inhibitor-2 was determined on the in vivo rate of dephosphorylation of phosphorylase a and on the rate of oocyte maturation. Dephosphorylation of microinjected phosphorylase a was inhibited up to 40% in the presence of inhibitor-2, with half-maximal inhibition at an intracellular concentration of 0.6 μM. Inhibitor-2 also caused over a 3-fold increase in the half-time for maturation, suggesting a possible role for protein phosphatase-1 in the regulation of meiosis.     

Clearance of Bacillus sphaericus and Bacillus thuringiensis ssp. israelensis from mammals

The maximum recovery period following topical ocular instillation and intraperitoneal injection of two preparations of Bacillus thuringiensis ssp. israelensis de Barjac and two preparations of Bacillus sphaericus 2362 was evaluated in rabbits and mice. B. sphaericus 2362 persisted for 8 wk after administration to the conjunctival cul-de-sac of rabbits; B. thuringiensis ssp. israelensis persisted for 1 wk. Infection was not evident, but both entomopathogens were recovered from flushed and unflushed eyes. High doses of B. sphaericus 2362 (greater than or equal to 10(8) colony-forming units) were toxic to CD-1 mice, and the toxic factor was heat stable. Injection of 10(7) colony-forming units of B. sphaericus 2362 resulted in clearance from the spleens of euthymic and athymic mice. Recovery occurred up to 67 d after injection. Mice failed to remove one preparation of B. thuringiensis ssp. israelensis from their spleen, and a constant number of colony-forming units were recovered for 80 d. B. sphaericus 2362 and B. thuringiensis ssp. israelensis were recovered from heart blood; their disappearance from heart blood coincided with their clearance from the spleen. There was no evidence that either organism was infectious. We conclude that these organisms can be used safely in environments where human exposure might occur.     

Effect of potassium phosphate on Bacillus subtilis competence mutants

Abstract We have previously described the isolation and characterization of four Bacillus subtilis competence-deficient mutants (J. Bacteriol. (1984) 157, 152–157). Further experiments, reported here, have shown that the transformation frequency of two of the mutants (FB92 and FB94) can be increased by the addition of high concentrations of potassium phosphate buffer present in the concentrated supernatant. This buffer stimulates up to 40–50 times the transformation frequency of FB92 and FB94 strains, while it has an inhibitory effect on the other two mutants and on the wild-type strain. Potassium phosphate inhibits DNA binding to competent cells but, at the same time, activates a second much less efficient binding system which partly restores the capacity of FB92 and FB94 mutants to take up DNA.     

Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus.

Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.     

Recovery of Bacillus sphaericus 2362 spores from growth medium by flocculation/sedimentation

Bacillus sphaericus 2362 spore sedimentation was evaluated through batch settling experiments, using tannin as the flocculating agent. The flocculation was demonstrated to be efficient up to the tannin concentration of 150 ppm. The floc diameter averaged 208 micro m, and its density was found to be 1.09 g/mL. The sedimentation process using tannin as a flocculating agent is promising, and a spore concentrate was obtained without loss of biological activity, presenting a LC(50) equal to 0.22 ppm. The result indicates that the larvicide activity is appropriate for controlling populations of Culex quinquefasciatus.