Immunological Homology Between Crystal and Spore Protein of Bacillus thuringiensis

Spore suspensions containing about 0.3% crystals and crystal suspensions containing about 0.1% spores were obtained from cultures of Bacillus thuringiensis by extraction with a two-phase system. Both preparations were tested for the presence of contaminating material from vegetative cells and were judged to be clean. Solutions of spore protein were obtained by extracting broken spores with phosphate buffer followed by extraction with either alkali- or urea-mercaptoethanol. The alkali spore or urea spore extracts had the same isoelectric point as crystal protein solubilized with these reagents. An antiserum prepared against alkali crystal solution precipitated alkali or urea spore extracts and crystal solutions but not phosphate spore extracts or extracts of whole cells. Lines of identity between spore and crystal precipitates were observed by using the Ouchterlony double-diffusion technique. Absorption of the antiserum with an excess of urea spore extract caused a disappearance of the precipitin bands originating from the spore protein and the homologous bands from the crystal protein. The results suggest that the crystal and endospore contain one or more common proteins.     

Chemical and Morphological Studies of Bacterial Spore Formation : II. Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The development of both the spore and parasporal protein crystal of Bacillus cereus var. alesti was followed using chemical and cytological techniques. The changes which led to the formation of the fore-spore were similar to those already described for Bacillus cereus. However, adjacent to the developing fore-spore a small inclusion became discernible in phase contrast. This protein inclusion during its growth was differentiated from the chromatin and lipid-containing inclusions by sequential staining techniques. During spore and crystal formation no net synthesis of either nucleic acid was detected. Tracer studies with radioactive phosphorus confirmed that the spore chromatin was derived from that in the vegetative cell. These same studies also indicated that a turnover of ribonucleic acid occurred during the sporulation process. During their formation both the spore and crystal incorporated methionine-³⁵S from the medium and from cellular material into a bound form. Sequential extractions with alkali and with alkaline-thioglycollate reagent revealed that the solubility characteristics of the mature crystal were possibly related to the presence of intermolecular disulphide bonds which developed after the major synthesis of the crystal was complete. The synthetic nature of sporogenesis and crystal formation is discussed with reference to the concept of "endotrophic" sporulation.     

Protein crystals in Adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors.     

Unique regulation of crystal protein production in Bacillus thuringiensis subsp. yunnanensis is mediated by the cry protein-encoding 103-megadalton plasmid.

In sporulating cultures of Bacillus thuringiensis subsp. yunnanensis HD977, two cell types are observed: cells forming only spores and cells forming only crystals. Curing analysis suggested that the crystal proteins are plasmid encoded. Through plasmid transfer experiments, it was established that a 103-MDa plasmid is involved in the crystal production. Conjugal transfer of this plasmid to Cry- recipient cells of Bacillus thuringiensis subsp. kurstaki HD73-26 conferred the ability to produce crystals exclusively on asporogenous cells of the recipient, indicating that the 103-MDa plasmid mediates the unique regulation of Cry protein production. When the dipteran-specific cryIVB gene was introduced into wild-type (Cry+) and Cry- backgrounds of B. thuringiensis subsp. yunnanensis by phage CP51ts45-mediated transduction, similar to all other B. thuringiensis strains, irregular crystals of CryIVB protein were produced by spore-forming cells in both backgrounds. However, the synthesis of the bipyramidal inclusions of B. thuringiensis subsp. yunnanensis was still limited only to asporogenous cells of the transductant. Thus, it appears that the unique property of exclusive crystal formation in asporogenous cells of B. thuringiensis subsp. yunnanensis is associated with the crystal protein gene(s) per se or its cis acting elements. As the crystals in B. thuringiensis subsp. yunnanensis were formed only in asporogenous cells, attempts were made to find out whether crystal formation had any inhibitory effect on sporulation. It was observed that both Cry+ and Cry- strains of B. thuringiensis subsp. yunnanensis (HD977 and HD977-1, respectively) exhibited comparable sporulation efficiencies. In addition, the Cry- B. thuringiensis subsp. kurstaki host (HD73-26) and its Cry+ transconjugant (HD73-26-16), expressing the B. thuringiensis subsp. yunnanensis crystal protein, were also comparable in their sporulation efficiencies, indicating that production of the crystal proteins of B. thuringiensis subsp. yunnanensis does not affect the process of sporulation.     

Protein synthesis and breakdown in the mother-cell and forespore compartments during spore morphogenesis in Bacillus megaterium

Recently developed techniques for isolating forespores from bacilli at all stages of spore morphogenesis have been exploited to investigate the contribution of each of the two compartments of the sporulating cell to the overall pattern of protein synthesis and degradation during sporulation in Bacillus megaterium. These studies have shown: (1) that protein synthesis continues in both compartments throughout spore morphogenesis; (2) that the degradation of proteins made at all times during vegetative growth and sporulation is confined to the mother-cell compartment; (3) that proteins synthesized in the mother-cell compartment during sporulation are subsequently degraded more rapidly than proteins synthesized during vegetative growth. This rate of degradation increases the later the proteins are synthesized in the sporulation sequence. Mature spores were disrupted, and the percentage of the total protein in soluble and particulate fractions was determined. Pulse-labelling experiments were performed to investigate the extent to which the proteins of these two fractions are newly synthesized during sporulation. These data were used to calculate the extent of capture of vegetative cell protein at the time of formation of the forespore septum. The value obtained is consistent with evidence from electron micrographs and supports a model for the origin of spore protein in which there is no protein turnover in the developing forespore.     

Functional significance of the crystal cells in the larva of Drosophila melanogaster

Cytoplasmic crystalline inclusions are found in some larval haemocytes of Drosophila melanogaster. Blackening can be experimentally induced in these cells, and previously it was suggested that either the substrate or enzyme for the tyrosine-tyrosinase system leading to melanin production in Drosophila larvae is found in these inclusions in the crystal cells. The present report is an attempt to further localize the enzyme and substrate. Larvae have been fed on food containing alpha-C(14)-tyrosine and autoradiographs of the blood cells taken from these larvae subsequently prepared. The C(14) activity in the crystal cells is restricted to the crystal inclusions in the cells and is significantly higher than that found in the other type of haemocytes, the plasmatocytes. When samples of the blood cells are incubated in DOPA solution, the extra-crystalline cytoplasm becomes blackened while the crystals themselves remain colorless. These observations are consistent with the hypothesis that the substrate is localized in the crystal inclusions whereas enzyme is found in the surrounding cytoplasm. An in vivo structural isolation would serve to separate enzyme and substrate rather than an inhibition by dehydrogenases as postulated by previous authors. In vitro examination with the phase microscope has shown that the crystal cells rupture easily and the crystals dissolve in the haemolymph. Therefore any treatment which tends to disrupt the structural integrity of the cell will allow the enzyme and substrate to come together. Humoral factors preceding metamorphosis might account for the in vivo release of the enzymatic reaction by initially altering the permeability of the cell.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.     

A surface net on parasporal inclusions of Bacillus thuringiensis

Protease digestion of parasporal inclusions from several subspecies of Bacillus thuringiensis revealed by electron microscopy a delicate protein net residue on the crystal surfaces. Pretreatment of inclusions with a catechol-ascorbic acid reagent potentiated the subsequent digestion of the crystals and the sharpness of the nets. The net structure maintained the overall shape of the digested crystal as a hexagonally assembled sheet but expanded somewhat from the original crystal size. Each hexagon of the expanded net was some 20 nm in diameter. A mesh size of some 10 nm was occasionally seen in the small amounts of net residue remaining after dissolution of crystals at elevated pH in the presence of thiol reducing agents. The net could also be seen in thin sections and on freeze-etched crystals. Net formation appeared to be associated with the sporulation (stage VI) uptake of cystine. They were rich in hexose (11%) and absent on developing crystals as well as on those in a Sp^? Cry⁺ mutant blocked at stage II of sporulation. Toxicity analyses, with silkworm (Bombyx mori) larvae, indicated that the net residue was more toxic than the protease-digested contents but less toxic than the original crystal. Crystal and net protein showed complete identity against crystal antiserum.     

Functional Significance of the Crystal Cells in the Larva of Drosophila melanogaster

Cytoplasmic crystalline inclusions are found in some larval haemocytes of Drosophila melanogaster. Blackening can be experimentally induced in these cells, and previously it was suggested that either the substrate or enzyme for the tyrosine-tyrosinase system leading to melanin production in Drosophila larvae is found in these inclusions in the crystal cells. The present report is an attempt to further localize the enzyme and substrate. Larvae have been fed on food containing α-C¹⁴-tyrosine and autoradiographs of the blood cells taken from these larvae subsequently prepared. The C¹⁴ activity in the crystal cells is restricted to the crystal inclusions in the cells and is significantly higher than that found in the other type of haemocytes, the plasmatocytes. When samples of the blood cells are incubated in DOPA solution, the extra-crystalline cytoplasm becomes blackened while the crystals themselves remain colorless. These observations are consistent with the hypothesis that the substrate is localized in the crystal inclusions whereas enzyme is found in the surrounding cytoplasm. An in vivo structural isolation would serve to separate enzyme and substrate rather than an inhibition by dehydrogenases as postulated by previous authors. In vitro examination with the phase microscope has shown that the crystal cells rupture easily and the crystals dissolve in the haemolymph. Therefore any treatment which tends to disrupt the structural integrity of the cell will allow the enzyme and substrate to come together. Humoral factors preceding metamorphosis might account for the in vivo release of the enzymatic reaction by initially altering the permeability of the cell.     

Chemical and Morphological Studies of Bacterial Spore Formation : III. The Effect of 8-Azaguanine on Spore and Parasporal Protein Formation in Bacillus cereus var. Alesti

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.     

Detection of membrane protein two-dimensional crystals in living cells

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation.     

A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis.

The effect of a 20-kDa protein on cell viability and CytA crystal production in its natural host, Bacillus thuringiensis, was studied by expressing the cytA gene in the absence or presence of this protein. In the absence of the 20-kDa protein, B. thuringiensis cells either were killed during sporulation (strain cryB) or produced very small CytA crystals (strain 4Q7). Expression of cytA in the presence of the 20-kDa protein, however, preserved cell viability, especially in strain cryB, and in both strains yielded bipyramidal crystals of the CytA protein that were larger than those of wild-type B. thuringiensis. These results suggest that the 20-kDa protein promotes crystal formation, perhaps by chaperoning CytA molecules during synthesis and crystallization, concomitantly preventing the CytA protein from interacting lethally with the bacterial host cell.     

Sporulation in Bacillus subtilis. The role of exoprotease

1. Intracellular turnover of protein was measured in wild-type Bacillus subtilis, which produces exoprotease at stage I in the sporulation process. Protein is degraded at a rate of 8–10%/hr. 2. As a result of this turnover, the proteins of the mother cell are extensively degraded and resynthesized by about 6hr., so that the later stages of spore formation occur in a cytoplasm containing mainly `new' protein. 3. The same protease appears to be responsible both for the intracellular turnover of protein and for extracellular proteolytic activity. In mutants that have lost the exoenzyme the intracellular protein is stable for many hours. In addition, these mutants fail to produce antibiotic and are asporogenous. When the exoprotease is regained as a result of back-mutation all the lost capacities of the cell are restored together. 4. Protease activity also accounts for the change in antigenic pattern of extracts of cells sampled during sporulation. Immunoelectrophoresis shows that, in the wild-type, the antigens characteristic of the vegetative cell have largely disappeared after a few hours; in the proteaseless mutants the vegetative-cell pattern is conserved. Apart from changing the protein pattern of the cell the protease could also have the function of removing protein inhibitors of sporulation. Other possible interpretations of the results are discussed.     

Biochemical Studies of Bacterial Sporulation and Germination XIII. Adenylate Kinase of Vegetative Cells and Spores of Bacillus subtilis

The spore and vegetative cell adenylate kinases of Bacillus subtilis, purified about 1,000-fold, proved indistinguishable by several physical and functional tests, including polyacrylamide gel electrophoresis, DEAE cellulose chromatography, and specificity toward substrates. Adenylate kinase activity in cell extracts, followed throughout growth and sporulation, was found to reach a maximum near the end of exponential growth, remain at that level during sporulation, until shortly before the appearance of refractile forms, and then decline, along with total protein, during the subsequent maturation of the spores. The enzyme, stable in extracts of exponential growing cells, was unstable in extracts of sporulating cells, presumably as a result of degradation by protease(s) appearing after the end of exponential growth.     

Discovery of crystalline inclusions in Bacillus licheniformis that resemble parasporal crystals of Bacillus thuringiensis

Crystalline inclusions were discovered in stationary and sporulating cells of the spore-forming bacterium Bacillus licheniformis ATCC 9945a. As detected by electron microscopy, dying or sporulating bacterial cells contain a single crystal of strikingly large size. The crystals in sporulating cells are located next to nascent spores and can be several times larger than the spores. Morphologically, most crystals are rhomboid with uniformly spaced grids. These newly discovered crystalline inclusions of B. licheniformis closely resemble parasporal crystals of Bacillus thuringiensis that are formed by insecticidal toxin proteins and used widely as biopesticides. The taxonomic identity of this strain was verified by its 16S rRNA gene sequence and its fatty acid profile. The finding of crystal proteins in B. licheniformis may lead to the discovery of new protein toxins and may expand our pool of biopesticides.     

The crystallization of beef heart mitochondrial adenosine triphosphatase.

Conditions are described under which crystals are formed with the ATPase enzyme from beef heart mitochondria. Enzyme activity is retained during the crystallization process. Some unit cell parameters have been determined by electron microscopy of negatively stained crystals; comparison with the unit cell crystalline matris inclusions indicates that such inclusions could be ATPase crystals.