Immunocytochemical Localization of Spore Specific Antigens in Ultrathin Sections

Short, J. A.; Walker, P. D.; Hine, Paul M.; Thomson, Robert

doi:10.1111/j.1365-2672.1977.tb00724.x

Cited by 6 publications

(8 citation statements)

References 32 publications

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“…The distribution of radioactivity in the forespore (Table 2) and the analysis of the membranous-soluble protein on the SDS-polyacrylamide gel (Fig. 5) confirm the results of Short et al (21). These results also imply that coat components (mainly the low-molecular-weight polypeptides) synthesized in the forespore compartment are probably transported across the cortex and two unit membranes of the forespore during assembly of the spore coat layer.…”

Section: Discussionsupporting

confidence: 86%

“…A recent immunocytochemical study indicates that the outer layer of the spore coat in B. cereus and B. subilis is synthesized in the mother-cell cytoplasm, and that, conversely, the inner layer of the spore coat is synthesized in the forespore compartment (21). Electron microscopic observation indicated that the t5 forespore of B. subtilis 60015 is at an early stage V and has a normal morphology of the cortex layer and immature spore coat layers.…”

Section: Discussionmentioning

confidence: 99%

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Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Watabe

Iida

Nakamura

et al. 1981

Microbiology and Immunology

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Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse-labeling experiments indicated that [14C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t5 (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [14C]phenylalanine was observed when the isolated forespores at ts were incubated with the corresponding mother-cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t5 was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the 14C-labeled cytoplasmic protein had a-molecular weight of about 20,000, and that a protein having the same molecular weight was present in the ts forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the 14C-labeled membranous-soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.After a final round of chromosomal replication, bacterial sporulation proceeds through a sequence of biochemical and morphological changes in the cell (20). Stationary phase cells are asymmetrically partitioned into two compartments by the rapid growth of a folded mother-cell double-membrane septum. The smaller of these compartments (forespore) is then engulfed as a discrete cell within the larger compartment (mother cell). Many important questions about the biochemical events and gene expression taking place in both compartments throughout the development of sporulation have remained unanswered because of the absence of reliable methods for isolating the forespore from the mother cell. Extensive protein turnover has been shown to occur during sporulation (4,12,13,22); at least 70% of the spore protein in Bacillus thuringiensis and B. subtilis has been shown to be newly synthesized during sporulation. These results were, however, obtained from the whole cells of an earlier stage of sporulation, which did not contain a discrete cell (forespore) within the mother-cell cytoplasm.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Watabe

Iida

Nakamura

et al. 1981

Microbiology and Immunology

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“…protein, the precursor may be very easily extracted from the forespores during isolation procedures. The only previously published evidence for the site of spore-coat synthesis reported immunocytochemical localization of purified coatspecific antigen within the forespore compartment (Short et al, 1977). The spore-coat material used for antibody preparation in this latter study still contained the spore outer membrane, which forms part of the integument fraction (Crafts-Lighty & Ellar, 1980), and the possibility exists therefore that the antiserum was preferentially directed towards forespore-specific membrane proteins rather than the structural components of the spore coat that are under examination in the present paper.…”

Section: Pulse For 5min With L-[35slmethionine At Stage V and Subsequmentioning

confidence: 99%

Precursor processing during the maturation of a spore-coat protein in Bacillus megaterium KM.

Stewart¹

1983

Biochemical Journal

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A protein of apparent mol.wt. 35000 that is extractable from the purified coat fraction of Bacillus megaterium KM spores is synthesized during sporulation as a precursor protein from which a 12-13 amino acid peptide is removed. Cleavage of this small peptide is delayed until 60-90 min after precursor synthesis and is concomitant with the morphological appearance of stage VI. The addition of chloramphenicol, subsequent to precursor synthesis, prevents the appearance of this late processing event. Two-dimensional non-equilibrium pH-gradient gel electrophoresis of the integument extract of forespores isolated at stage V from sporangia pulse-labelled with L-[35S]methionine 1 h before isolation, revealed both unprocessed and processed components. Similar analysis of total protein from the corresponding mother cells revealed only the unprocessed component in relatively small amounts, suggesting that, although the protein may be synthesized in the mother-cell compartment, processing may be restricted to the forespore. Peptide analysis by limited proteolysis was used to examine the relationship between the 35000- and a 17500-mol.wt. coat protein. The possible implications of limited proteolytic processing to maturation of the spore coat are discussed.

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“…A little more DPA found in the coat region, which cannot be overlooked, may correspond to a completely dissociated form of DPA which is released in small amounts during heat activation (5,18). Short et al (20) also observed small deposits of DPA in the coat region of B. cereus dormant spores stained with DPA-conjugated antiserum and peroxidase-antiperoxidase, but they regarded them as negligible. More deposits of DPA in the coat region of autoclaved spores seem to be caused by a flux of DPA located in the core, owing to the unusual heat treatment.…”

Section: Resultsmentioning

confidence: 99%

“…24showed DPA to be located in the cortical region, particularly just beneath the spore coat. Subsequently, however, they reported, by the use of the soluble peroxidase-antiperoxidase complex and unlabeled antibody to DPA, that DPA is located predominantly in the core of mature spores (20). Beta-attenuation analysis (11) and UV irradiation (2) have suggested that DPA is located primarily in the spore core.…”

mentioning

confidence: 99%

Ultrastructural localization of dipicolinic acid in dormant spores of Bacillus subtilis by immunoelectron microscopy with colloidal gold particles

Kozuka¹,

Yasuda²,

Tochikubo³

1985

J Bacteriol

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The localization of dipicolinic acid in dormant spores of Bacillus subtilis was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were distributed mainly in the core regions of dormant spores and were not observed in those of germinated or autoclaved spores. This result clearly demonstrates that dipicolinic acid is localized in the cores of dormant spores.

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Immunocytochemical Localization of Spore Specific Antigens in Ultrathin Sections

Cited by 6 publications

References 32 publications

Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Precursor processing during the maturation of a spore-coat protein in Bacillus megaterium KM.

Ultrastructural localization of dipicolinic acid in dormant spores of Bacillus subtilis by immunoelectron microscopy with colloidal gold particles

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