RESUMEN

The Bacillus subtilis spore coat is composed of at least 15 polypeptides plus an insoluble protein fraction arranged in three morphological layers. The insoluble fraction accounts for about 30% of the coat protein and is resistant to solubilization by a variety of reagents, implying extensive cross-linking. A dodecapeptide was purified from this fraction by formic acid hydrolysis and reverse-phase high-performance liquid chromatography. This peptide was sequenced, and a gene designated cotX was cloned by reverse genetics. The cotX gene encoding the dodecapeptide at its amino end was clustered with four other genes designated cotV, cotW, cotY, and cotZ. These genes were mapped to 107 degrees between thiB and metA on the B. subtilis chromosome. The deduced amino acid sequences of the cotY and cotZ genes are very similar. Both proteins are cysteine rich, and CotY antigen was present in spore coat extracts as disulfide cross-linked multimers. There was little CotX antigen in the spore coat soluble fraction, and deletion of this gene resulted in a 30% reduction in the spore coat insoluble fraction. Spores produced by strains with deletions of the cotX, cotYZ, or cotXYZ genes were heat and lysozyme resistant but readily clumped and responded more rapidly to germinants than did spores from the wild type. In electron micrographs, there was a less densely staining outer coat in spores produced by the cotX null mutant, and those produced by a strain with a deletion of the cotXYZ genes had an incomplete outer coat. These proteins, as part of the coat insoluble fraction, appear to be localized to the outer coat and influence spore hydrophobicity as well as the accessibility of germinants.

Asunto(s)

Bacillus subtilis/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Familia de Multigenes , Factor sigma , Esporas Bacterianas/química , Factores de Transcripción , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Secuencia de Bases , Western Blotting , Mapeo Cromosómico , Clonación Molecular , Microscopía Electrónica , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional , Oligodesoxirribonucleótidos/química , Mapeo Restrictivo

RESUMEN

The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Esporas Bacterianas/fisiología , Secuencia de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , División Celular , Eliminación de Gen , Immunoblotting , Datos de Secuencia Molecular , Muramidasa/farmacología , Homología de Secuencia de Aminoácido , Esporas Bacterianas/química , Supresión Genética

RESUMEN

Endospores of Bacillus subtilis are encased in a two-layer protein shell known as the coat, which consists of a lammellar-like inner layer and an electron-dense outer layer. We report the cloning of the structural gene (designated cotE) for an alkali-soluble coat protein of 24 kD and show that the cotE gene product is a morphogenic protein required in the assembly of the outer coat. The nucleotide sequence of cotE reveals an open reading frame capable of encoding a 181-residue-long polypeptide of 21 kD. A cotE mutant was created by replacing the chromosomal gene, which was located at 145 degrees on the chromosome, with an in vitro constructed, deletion-mutated gene. The resulting cotE mutant formed normal-looking (optically refractile) spores that were heat resistant but were sensitive to lysozyme and somewhat impaired in germination. Ultrastructural analysis indicated that the mutant spores lacked the electron-dense outer layer of the coat but retained a normal-looking inner coat. The mutant spores were pleiotropically deficient in several coat proteins, including the product of cotE and the products of previously cloned cot genes A-C. Based on experiments in which expression of the cotA and cotC genes was found to be unimpaired in cotE mutant cells, we infer that the cotE gene product is involved in the assembly of the products of cotA-cotC, and certain other proteins into the electron-dense outer layer of the coat.

Asunto(s)

Bacillus subtilis/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Bacteriano/genética , Genes , Datos de Secuencia Molecular , Esporas Bacterianas/genética

RESUMEN

Bacillus thuringiensis subsp. finitimus produced at least two parasporal inclusions. One inclusion was formed within the exosporium and remained with the spore after mother cell lysis. A second inclusion formed somewhat later exterior to the exosporium. Each inclusion contained a major polypeptide of about 135,000 daltons with unique antigenic determinants. This subspecies contained only two plasmids, of 98 and 77 megadaltons (MDa). Strains cured of these plasmids produced only the free inclusion. Since the plasmid-cured strains did not contain DNA sequences homologous to plasmid DNA, the gene for the free-inclusion protein must be encoded in the chromosome. In contrast, the enclosed parasporal inclusion was produced only when the plasmid of 98 MDa was present. In addition, transfer of the 98-MDa plasmid to Bacillus cereus resulted in transcipients that produced small inclusions enclosed within the exosporium, and the protein extracted from these inclusions reacted with antibody specific for enclosed inclusion protein of B. thuringiensis subsp. finitimus. Genes in both the chromosome and a plasmid function in the synthesis of distinct parasporal proteins in this subspecies.

Asunto(s)

Bacillus thuringiensis/ultraestructura , Cuerpos de Inclusión/análisis , Bacillus thuringiensis/análisis , Bacillus thuringiensis/genética , Proteínas Bacterianas/análisis , Secuencia de Bases , ADN Bacteriano/análisis , Plásmidos , Esporas Bacterianas/ultraestructura

RESUMEN

The multisegmented ovoidal inclusion of Bacillus thuringiensis subsp. israelensis was found to be composed of two structurally and biochemically distinct components. Electron microscopy of the inclusion revealed it to be composed mainly of osmiophobic or lightly stained segments crystallized in a lattice showing a repeat of approximately 4.3 nm. These light segments of the inclusions were shared by osmiophylic darkly stained segments with a crystal lattice repeat of approximately 7.8 nm. The lightly stained segments were soluble at pH 9.2 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The extracts of lightly stained segments were lytic to mammalian erythrocytes, and the precipitate obtained by lowering the pH to 5.2 was toxic to the larvae of Aedes egypti. The dark inclusion segments remaining, besides being much less toxic to larvae, were nonlytic to erythrocytes and were soluble at pH 10.5 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The light segment was composed of two major polypeptide doublets with molecular weights of 145,000 and 135,000, and 27,000 and 26,500, and the dark segments were composed of a single major polypeptide with a molecular-weight of 70,000. Hence, the inclusion of B. thuringiensis subsp. israelensis is more complex than previously reported, and we conclude that the toxin may be the polypeptide with a molecular weight of 27,000 and 26,500.

RESUMEN

Two major classes of polypeptides were extracted from the spore surface of Bacillus thuringiensis subsp. kurstaki: the 134,000-dalton protoxin that is the major component of the crystalline inclusion and spore coat polypeptides very similar to those found on Bacillus cereus spores. The quantity of spore coat polypeptides produced was reduced when compared with that produced by certain acrystalliferous mutants or by B. thuringiensis subsp. israelensis. The latter organism produced an inclusion toxic to mosquito larvae, but deposited very little of the inclusion protein on the spore surface. The reduction in spore coat protein in B. thuringiensis subsp. kurstaki was also seen in freeze-etched electron micrographs of spores. B. thuringiensis subsp. kurstaki spores germinated rather slowly when compared with related species, a property previously correlated with a deficiency or defect of the spore coat. Many mutants of B. thuringiensis subsp. kurstaki unable to form a crystalline inclusion were nontoxic and lacked a well-defined spore coat. Other mutants isolated either directly from the wild type or from coat-deficient mutants produced spores that were identical to those produced by the closely related species. Bacillus cereus, on the basis of morphology, germination rate, and the size and antigenicity of the spore coat polypeptides. Most of the protein extractable from the inclusion produced by B. thuringiensis subsp. israelensis was about 26,000 daltons, considerably smaller than the major polypeptide extractable from other inclusions. Some of the B. thuringiensis subsp. israelensis inclusion protein was found on the spore surface, but the majority of the extractable spore coat protein was the same size and antigenicity as that found on B. cereus spores. The B. thuringiensis subsp. israelensis spores germinated at a rate close to that of B. cereus, especially when the spores were formed at 37 degrees C, and the morphology of the spore surface was very similar to that of B. cereus.

Asunto(s)

Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/biosíntesis , Bacillus thuringiensis/ultraestructura , Proteínas Bacterianas/aislamiento & purificación , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Electroforesis en Gel de Poliacrilamida , Grabado por Congelación , Cinética , Microscopía Electrónica , Esporas Bacterianas/metabolismo , Esporas Bacterianas/ultraestructura

RESUMEN

We removed by recombinant deoxyribonucleic acid (DNA) techniques a small DNA segment from within a cloned gene (the 0.4 kb gene) in which transcription in under sporulation control in Bacillus subtilis. These deletion mutation was introduced into the B. subtilis chromosome by transformation with cloned DNA. Competent cells bearing a mutation (tms-26) that is closely linked to the 0.4 kb gene were transformed with linearized plasmid DNA containing the truncated 0.4 kb gene and the wild-type allele of the tms locus. Selection for Tms+ transformants yielded oligosporogenous mutants of unusually dark-brown colony pigmentation. This phenotype was caused by a mutation which mapped at or very near the site of the 0.4 kg gene deletion, whose presence and position in chromosomal DNA was confirmed by Southern hybridization analysis. Phase-contrast microscopy and electron microscopy showed that the mutation, which we designated as spoVG, impaired sporulation at about the fifth stage; bacteria harboring the spoVG mutation proceeded normally through stage IV of development but frequently lysed thereafter, apparently as a result of disintegration of an immature spore cortex. This identifies the 0.4 kb gene (or DNA in its immediate vicinity) as a new sporulation locus and shows that its product functions at a late stage in development.

Asunto(s)

Bacillus subtilis/genética , Genes Bacterianos , Esporas Bacterianas/genética , Bacillus subtilis/fisiología , Mapeo Cromosómico , Cromosomas Bacterianos , Clonación Molecular , Mutación , Esporas Bacterianas/fisiología , Transformación Bacteriana

RESUMEN

Heat activation (70 degrees C for 20 min) resulted in alteration in structural proteins and enzymes found in Bacillus cereus spore coats. The three notable changes were increased glycosylation of coat proteins, alteration in polypeptide pattern on sodium dodecyl sulfate - polyacrylamide gels, and an increase in free SH groups of proteins. About three polypeptides leaked out in small quantities from the spore coats during heat activation. The extraction of five spore coat associated enzyme activities was followed during the coat stripping procedures, which left the cortex and core intact. Two of these activities, L-alanine dehydrogenase and purine nucleoside hydrolase, were solubilized when the undercoat was extracted by 1,4-dithioerythritol (DTE) at pH 9.8. Three other activities, a protease, a corticolytic enzyme, and purine nucleoside phosphorylase, were solubilized by both DTE alone and DTE plus urea at pH 9.8. The DTE plus urea extraction removed the two more insoluble coat layers, the outer cross-patch, and the inner pitted layers. Mutants deficient in the cross-patch layer contained normal amounts of the protease, corticolytic, and purine nucleoside phosphorylase activities suggesting their association with the pitted layer. In intact spores all five enzymes were found to be stable to the heat activation treatment. However, extracted and partially purified preparations of protease, purine nucleoside phosphorylase, and L-alanine dehydrogenase were heat sensitive. Similar preparations of corticolytic enzyme and purine nucleoside hydrolase were stable to the heat activation conditions.

Asunto(s)

Bacillus cereus/enzimología , Alanina-Deshidrogenasa , Aminoácido Oxidorreductasas/metabolismo , Bacillus cereus/fisiología , Activación Enzimática , Calor , N-Glicosil Hidrolasas/metabolismo , Péptido Hidrolasas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Esporas Bacterianas/enzimología

RESUMEN

Spores of Bacillus cereus mutants selected for slow response to germinants and sensitivity to lysozyme were found to be deficient in coat, but were heat-resistant and contained the same quantity of dipicolinic acid as the wild-type. While the average coat protein content of the spores was 25% of the wild-type, many spores were coatless with large whorls of coat deposited in the cytoplasm. These coat deposits were isolated in Renografin gradients and found to cross-react immunologically with wild-type coat. The proteins extractable from these deposits were virtually identical to those extracted from wild-type spores. The morphology of the coat deposits was very similar to coats of wild-type spores, but with a deficiency of the outermost cross-patched layer. The sites of formation and deposition were altered. Since the mutant reverted to a phenotype identical to the parental strain with a frequency consistent with an initial point mutation, apparently a single defect resulted in alteration of the deposition of the spore coats on to the outer forespore membrane. Despite this defect, mutant cells were able to synthesize and process spore coat precursors into an array of morphological layers very similar to the wild-type. There are apparently distinct morphogenetic pathways for the formation of the spore body and coat layers.

Asunto(s)

Bacillus cereus/genética , Bacillus cereus/fisiología , Bacillus cereus/ultraestructura , Proteínas Bacterianas/análisis , Electroforesis , Calor , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Microscopía de Contraste de Fase , Muramidasa/farmacología , Mutación , Esporas Bacterianas/análisis , Esporas Bacterianas/genética , Esporas Bacterianas/ultraestructura

Asunto(s)

Bacillus thuringiensis/fisiología , Bacillus thuringiensis/clasificación , Bacillus thuringiensis/ultraestructura , Proteínas Bacterianas/análisis , Proteínas Bacterianas/fisiología , Toxinas Bacterianas/análisis , Fenómenos Químicos , Química , Cristalización , Glicoproteínas/análisis , Glicoproteínas/fisiología , Esporas Bacterianas/fisiología , Esporas Bacterianas/ultraestructura

RESUMEN

Bacillus medusa was found to carry three phages or phagelike structures named phi med-1, phi med-2, and phi med-3. phi med-1 is a minute, 25-nm-diameter particle without a tail. It was extracted from the sporulation lysate of a phi med-2-minus strain of B. medusa and purified by differential centrifugation. The nucleic acid from this structure was shown to be orcinol positive, alkali sensitive, RNase sensitive, and DNase resistant. An RNase-resistant core of nucleic acid was not found, indicating that it was single-stranded RNA. A host strain has not yet been found for phi med-1. Phage phi med-3 was induced with mitomycin C or UV light and consisted of empty heads of 57 nm in diameter, whereas phi med-2 induced with mitomycin was a phage of 60-nm head diameter and 220-nm tail length. The sporulation sequence proceeded faster in those mutants lacking phi med-2, and when the phage was reintroduced to B. medusa the extended wild-type sporulation sequence was observed. B. thuringiensis var. schwetzova was sensitive to phi med-2 and yielded small turbid plaques. B. medusa produced small numbers of phi med-2 during growth. The other phage may be produced at the same time but were not detected. Phi med-1 was found in sporulating cells by electron microscopy techniques. Its release from these was demonstrated by both electron microscopy techniques and a radioactive assay. It appears to participate in the formation of a surface layer on the parasporal inclusion of B. medusa.

Asunto(s)

Bacillus , Bacteriófagos , Bacillus/crecimiento & desarrollo , Bacteriófagos/análisis , Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/ultraestructura , Cuerpos de Inclusión Viral , Lisogenia , Mitomicinas/farmacología , ARN Viral/análisis , Efectos de la Radiación , Esporas Bacterianas/crecimiento & desarrollo , Rayos Ultravioleta , Replicación Viral

RESUMEN

Two classes of spore mutants have been selected in Bacillus cereus T, those producing lysozyme-sensitive spores, and those producing spores dependent upon lysozyme for germination. One mutant from each class was studied in detail and found to have defective packing of the spore coat layers. The major spore coat poplypeptide appeared to be altered on the basis of gel electrophoretic profiles and/or peptide maps of half-syctine-containing peptides. The spores of the mutants of both classes were sensitive to lysozyme and failed to respond to the germinants L-alanine plus adenosine. The spores were also more sensitive to octanol than the parental strain, but contained the same amount of dipicolinic acid and were equally heat resistant. The reversion frequencies in both cases were consistent with an initial point mutation, suggesting that an alteration in the major coat polypeptide accounted for the phenotypic properties studied.

Asunto(s)

Bacillus cereus/ultraestructura , Mutación , Adenosina/farmacología , Alanina/farmacología , Bacillus cereus/análisis , Bacillus cereus/efectos de los fármacos , Proteínas Bacterianas/análisis , Grabado por Congelación , Muramidasa/farmacología , Péptidos/análisis , Fenotipo , Esporas Bacterianas/análisis , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/ultraestructura

RESUMEN

Spontaneous release of the temperate bacteriophage T (phiT), carried by Bacillus megaterium 899a, occurred during early growth of the host cells. Rejuvenated cells (accomplished by a 5x dilution in fresh medium) and unrejuvenated cells were induced by mitomycin C during the course of sporulation and subsequent phage phiT production measured by burst size. Induction of sequential samples of unrejuvenated cells resulted in burst sizes that fell to zero as T(0) sporulation time in the main culture was approached. This drop in burst size was not considered a sporulation event, as it also occurred during analogous stages of growth in an asporogenous mutant. Rejuvenated, induced portions of the culture of sporulating cells of B. megaterium 899a gave large burst sizes until T(+2), when the burst sizes fell to zero. The stage I asporogenous mutant, treated in a similar manner, gave lower, but still substantial, burst sizes; thus, the sharp decline in burst size of induced rejuvenated sporulating cells appeared to be a sporulation event. Sporulating cells induced at times shortly after T(1.5) formed spores in which the induced phage were trapped until germination of the spores, which formed infectious centers. This induced phage-trapping was maximal when the sporulating cultures were induced at T(2.25). Commitment to sporulation could be defined by our system as that point beyond which rejuvenated sporulating cells were unable to support the replication of the phage. This point also correlated with the increase in induced phage-trapping by spores. Two other methods gave a similar commitment time. Commitment to sporulation, in spite of added glucose or fresh complex medium, occurred at the same time. Electron micrography showed that the committed cell was still undergoing engulfment. The fate of induced phage phiT was determined at different points during growth and sporulation. Induction at times prior to T(0), which were longer than the eclipse period of the phage, resulted in a burst size of approximately 50. At times prior to T(0), shorter than the phage eclipse period, induction led to lysis with low burst sizes, approaching zero. The pattern of spontaneous phage release during growth was similar. From T(0) up to the point of commitment to sporulation, induction resulted in the blocking of spore formation without lysis. At the commitment point, induced phage were trapped and carried into spores which germinated to give infectious centers. The spontaneous derepression of phage at a time which blocked spore formation led to 7 x 10(4) infectious centers per ml and would not normally be noticed. Derepression at the time of phage entrapment was not observed to occur without induction with mitomycin C.

Asunto(s)

Bacillus megaterium/crecimiento & desarrollo , Bacteriófagos/aislamiento & purificación , Esporas Bacterianas/crecimiento & desarrollo , Bacillus megaterium/citología , Bacteriófagos/crecimiento & desarrollo , Membrana Celular , Medios de Cultivo , Glucosa , Lisogenia , Microscopía Electrónica , Microscopía de Contraste de Fase , Mitomicinas/farmacología , Mutación , Espectrofotometría , Factores de Tiempo , Replicación Viral

RESUMEN

Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.

Asunto(s)

Bacillus megaterium/crecimiento & desarrollo , Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/análisis , Bacteriófagos/efectos de los fármacos , Bacteriófagos/aislamiento & purificación , Cloruro de Calcio/farmacología , Centrifugación por Gradiente de Densidad , Cloroformo/farmacología , ADN Viral/análisis , Farmacorresistencia Microbiana , Calor , Lisogenia , Microscopía Electrónica , Mitomicinas/farmacología , Mutación , Ácido Fosfotúngstico , Esporas Bacterianas/crecimiento & desarrollo , Coloración y Etiquetado , Ensayo de Placa Viral , Proteínas Virales , Replicación Viral

RESUMEN

Although untreated phiT may be dried on grids without damage, they were disrupted when negatively stained with potassium phosphotungstate. The potassium phosphotungstate reacted with phage suspended in phosphate buffer, and the altered but still-viable phage was disrupted only on drying. Stability of altered phage was not recovered by dialysis against 10(-3) M Mg(2+).

Asunto(s)

Bacillus megaterium , Bacteriófagos/efectos de los fármacos , Ácido Fosfotúngstico/farmacología , Tampones (Química) , Centrifugación por Gradiente de Densidad , Diálisis , Magnesio , Microscopía Electrónica , Fosfatos , Potasio , Coloración y Etiquetado , Proteínas Virales

RESUMEN

At 47 C, a temperature-sensitive mutant of Bacillus subtilis 168 accumulates membrane-associated protein inclusions and exhibits a pleiotropic phenotype indicative of a defect in lipid synthesis. The mutant bacteria cease growing at 47 C, and the turbidity of the culture gradually declines. The lack of growth is not due to the death or lysis of the cells, since viability does not decrease for about 1 hr and the "lysis" can be delayed for several hours by increasing the osmotic pressure of the medium. Synthesis of deoxyribonucleic acid and ribonucleic acid stops at 47 C although a residual synthesis of protein occurs. When the temperature is raised, the mutant fails to increase the proportion of 17:0 branched-chain fatty acids and to decrease the proportion of 18:0 and 18:1 fatty acids. The membrane-associated inclusions can be seen by phase-contrast or electron microscopy and remain attached to protoplast membranes during isolation. The inclusions are mostly protein and are digested with Pronase.

Asunto(s)

Bacillus subtilis/citología , Proteínas Bacterianas/análisis , Cuerpos de Inclusión/análisis , Mutación , Temperatura , Bacillus subtilis/análisis , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Bacteriólisis , Isótopos de Carbono , Membrana Celular , Centrifugación por Gradiente de Densidad , Cromatografía de Gases , Medios de Cultivo , Ácidos Grasos/análisis , Genética Microbiana , Histocitoquímica , Cuerpos de Inclusión/efectos de los fármacos , Lípidos/biosíntesis , Microscopía Electrónica , Microscopía de Contraste de Fase , Presión Osmótica , Péptido Hidrolasas/farmacología , Fenotipo , Coloración y Etiquetado , Tritio

RESUMEN

The spore coat layers of Bacillus cereus T can be at least partially restored by mixing spores stripped of their coat with solubilized coat protein.

Asunto(s)

Bacillus cereus/citología , Pared Celular , Esporas/citología , Proteínas Bacterianas , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Cistina , Diálisis , Diatrizoato , Ditiotreitol , Concentración de Iones de Hidrógeno , Isoleucina , Microscopía Electrónica , Microscopía de Contraste de Fase , Muramidasa , Esporas Bacterianas/citología , Tritio , Urea

RESUMEN

Refractility as indicated by light microscopy, electron microscopy of thin sections, and freeze fracture etching was increased and maintained in a cortexless mutant, A(-)1, of Bacillus cereus var. alesti by the addition during sporulation stage 4 of actinomycin D, which prevents the terminal lysis of spore core associated with sporulation in this organism. (45)Calcium uptake levels and dipicolinic acid (DPA) content were similarly maintained. The location of these components appears to be in the spore protoplast. In the parent A(-), treated with actinomycin D during stage 4, spore particles with similar morphology to the mutant, that is without a cortex and with the characteristics of refractility, were obtained. A major difference in sensitivity to actinomycin D between the processes of (45)Ca uptake and DPA synthesis was observed. Some heat resistance in A(-) made cortexless by actinomycin D could be observed. These studies indicate that the role of the cortex is not to produce the dehydrated refractile spore state but to maintain it.

Asunto(s)

Bacillus cereus/citología , Dactinomicina/farmacología , Esporas/citología , Bacillus cereus/efectos de los fármacos , Bacillus cereus/crecimiento & desarrollo , Bacillus cereus/metabolismo , Técnicas Bacteriológicas , Isótopos de Calcio/metabolismo , Membrana Celular , Medios de Cultivo , Citoplasma , Grabado por Congelación , Genética Microbiana , Calor , Microscopía Electrónica , Microscopía de Contraste de Fase , Mutación , Ácidos Picolínicos/biosíntesis , Esporas Bacterianas/citología , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/metabolismo

RESUMEN

Coat-stripped spores suspended in hypertonic solutions and supplied with two essential cations can be converted into viable protoplasts by lysozyme digestion of both cortex and germ cell wall. Calcium ions are necessary to prevent membrane rupture, and magnesium ions are necessary for changes indicative of hydration of the core, particularily the nuclear mass. Since remnant spore coat covered such protoplasts of Bacillus subtilis and the germ cell wall of B. cereus spores is not lysozyme digestible, coatless spores of B. megaterium KM were more useful for these studies. Lysozyme digestion in cation-free environment produced a peculiar semi-refractile spore core free of a cortex but prone to rapid hydration and lytic changes on the addition of cations. Strontium could replace Ca(2+) but Mn(2+) could not replace Mg(2+) in these digestions. When added to the spores, dipicolinic acid and other chelates appeared to compete with the membrane for the calcium needed for stabilization during lysozyme conversion to protoplasts. It is argued that calcium could function to stabilize the inner membrane anionic groups over the anhydrous dipicolinic acid-containing core of resting spores.

Asunto(s)

Bacillus megaterium , Protoplastos , Esporas , Bacillus megaterium/citología , Bacillus megaterium/efectos de los fármacos , Técnicas Bacteriológicas , Tampones (Química) , Calcio/farmacología , Membrana Celular , Pared Celular , Centrifugación , Quelantes/farmacología , Medios de Cultivo , Detergentes , Soluciones Hipertónicas , Manganeso/farmacología , Microscopía Electrónica , Microscopía de Contraste de Fase , Muramidasa/metabolismo , Ácidos Picolínicos/farmacología , Protoplastos/efectos de los fármacos , Protoplastos/crecimiento & desarrollo , Sodio , Espectrofotometría , Esporas/citología , Esporas/efectos de los fármacos , Estroncio/farmacología , Sacarosa , Sulfatos

RESUMEN

A stage 4 sporulation mutant of a strain of Bacillus cereus var. alesti fails to synthesize a cortex although all other structural components appear normal. With terminal lysis the spore core as well as the sporangium is lysed. Both the uptake of (45)Ca and the synthesis of dipicolinic acid (DPA) are similar to these activities in the parent strain, but these components (DPA and Ca) are lost to the medium with the drastic lysis. The first stage of diaminopimelic acid incorporation, that into germ cell wall mucopeptide, is intact in the mutant; the second stage, that into cortical mucopeptide, is absent. These biochemical studies as well as phospholipid metabolism and freeze-etch analysis suggest the lesion lies in the outer forespore membrane.

Asunto(s)

Bacillus cereus/crecimiento & desarrollo , Acetatos/metabolismo , Bacillus cereus/citología , Bacillus cereus/metabolismo , Isótopos de Calcio/metabolismo , Isótopos de Carbono , Membrana Celular , Pared Celular , Cromatografía DEAE-Celulosa , Gránulos Citoplasmáticos