Fractionation and Biochemical Properties of the Mother Cell and Forespore Fractions from Sporulating Cells of Bacillus subtilis

Nakayama, Tatsuo; Kurogi, Yumiko; Irikura, Maki; Matsuo, Hisayuki; Sakai-Wada, Aiko

doi:10.1093/oxfordjournals.jbchem.a132977

Cited by 7 publications

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“…Glucose dehydrogenase is a forespore-specific enzyme (5), while alkaline phosphatase, a membrane-bound and secreted enzyme (7), is associated with the mother cell compartment (14). The forespore portion of our extract contained 90% of the total glucose dehydrogenase activity that was present in the preparation but only 25% of the alkaline phosphatase activity and 32% of the total protein (Table 1).…”

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confidence: 98%

The sigma E subunit of Bacillus subtilis RNA polymerase is present in both forespore and mother cell compartments

Carlson

Haldenwang

1989

J Bacteriol

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Bacillus subtilis cells harvested 3.5 h after the onset of sporulation (t3.5) were fractionated into extracts enriched in either mother cell or forespore components and were analyzed immunologically for ffE and its precursor protein, P3. We determined by Western blot (immunoblot) analysis that equivalent amounts of p3l and CJE were present in both mother cell and forespore extracts. This result implies that, although C.E is not synthesized until a stage in development when the cell is partitioned into progenitor forespore and mother cell compartments, it probably directs the transcription of genes that are expressed in both of these structures.Endospore formation by Bacillus subtilis is a simple developmental process that requires the activation of sporulation-specific genes in a precise temporal sequence. Although the mechanism by which sporulation genes are properly expressed is still unclear, novel RNA polymerase sigma factors are believed to be important elements of spore gene transcription (12). At least four sporulation-essential genes (sigH, spoIIAC, sigE, and spoIIIG) encode sigma-like proteins (2, 4,6,10,17,19 Conf., abstr. 28). UE, the sigE gene product, is the best characterized of these proteins. One of the more intriguing aspects of 'E iS its complex and highly regulated mode of synthesis.Synthesis of CuE iS controlled both at the level of transcription and by a posttranslational modification of the sigE gene product (9, 11). The primary translation product of sigE is not the sigma factor itself but rather an inactive precursor protein (P") (20). P31 is processed into SE by the removal of 29 amino acids from the P" amino terminus (11). This reaction is catalyzed by a developmentally regulated proteolytic activity which is first detected in B. subtilis between 1 h (t1) and 2 h (t2) into sporulation (11,21). The stage of development at which the processing of P31 occurs corresponds to that period during which the forespore septum is synthesized (1). This structure partitions the developing cell into progenitor mother cell and forespore compartments. It had previously been reported that a sporulation-specific RNA polymerase subunit (81), which is probably identical to UE, is selectively associated with the RNA polymerase fraction that can be extracted from forespores but not from mother cells (13). If this is true, then it is possible that the coincidence of P3" processing with the formation of the spore septum is a consequence of a forespore-specific activity that only appears once compartmentalization occurs. Assuming that this notion is true, we would anticipate that P31, whose synthesis begins before septation occurs, would be present in both mother cell and forespore compartments, while a.E would be present only in the forespore fraction. To test this possibility, we separated sporulating B. subtilis into mother * Corresponding author. cell and forespore fractions and then assayed the resulting extracts for the presence of p31 and UE in a Western blot (immunoblot) assay.B. subtilis SMY was grown i...

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confidence: 98%

The sigma E subunit of Bacillus subtilis RNA polymerase is present in both forespore and mother cell compartments

Carlson

Haldenwang

1989

J Bacteriol

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“…Recently, procedures for isolating the forespores of B. cereus (1,2), B. megaterium KM (6), and B. subtilis (8,15) have been developed, and we also succeeded in isolating the forespore of B. subtilis 60015 and described some biochemical and functional properties of both compartments (23). Ellar and Posgate studied the protein turnover in the sporulating cells of B. megaterium KM and indicated that protein synthesis continued in both forespore and mother-cell compartments, while protein turnover was not detectable in the forespore compartment (6).…”

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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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“…Procedures for isolation of intact forespores have been developed in many species of Bacillus (Andreoli et al, 1973;Ellar & Posgate, 1974;Fujita et al, 1977;Watabe et al, 1980;Nakayama et al, 1980) and some of the biochemical properties closely related to the developing forespores have been investigated (Eaton & Ellar, 1974;Ellar & Posgate, 1974;Andreoli et af., 1975Andreoli et af., , 1978Ellar et al, 1975;Fujita et al, 1977;Nakayama et al, 1980;Watabe et al, 1980Watabe et al, , 1981Setlow, 198 1). We have developed a [ Clphenylalanine-incorporation system using isolated forespores of Bacillus subtilis 60015, and found continuous protein synthesis in the young forespore throughout sporulation.…”

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confidence: 99%

Purification and Characterization of Cytoplasmic Proteins Synthesized in the Developing Forespores of Bacillus subtilis during Sporulation

et al. 1983

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Forespores of Bacillus subtilis 60015 were isolated from sporulating cells at t5 and were incubated with an amino acid mixture containing [14C]phenylalanine. Three species of 14C-labelled cytoplasmic proteins synthesized in the forespores were purified to homogeneity by gel filtration in the presence of detergents and by ion-exchange chromatography. The molecular weights of the purified proteins, called A, B and C , were estimated by SDS-PAGE as 24200, 1 1 500 and 12 700, respectively. Protein B contained remarkably high amounts of tyrosine (2343%) and alanine (22.8%), and proteins A and C contained relatively high amounts of glutamate + glutamine, glycine, and alanine (10.4 to 13.3%). The synthesis of these proteins can provide markers for the control events in the forespore compartment during sporulation.

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Fractionation and Biochemical Properties of the Mother Cell and Forespore Fractions from Sporulating Cells of Bacillus subtilis

Cited by 7 publications

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The sigma E subunit of Bacillus subtilis RNA polymerase is present in both forespore and mother cell compartments

The sigma E subunit of Bacillus subtilis RNA polymerase is present in both forespore and mother cell compartments

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

Purification and Characterization of Cytoplasmic Proteins Synthesized in the Developing Forespores of Bacillus subtilis during Sporulation

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