Synthesis and Deposition of Spore Coat Proteins during Sporulation of Bacillus megaterium

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It was proved that three spore coat proteins of 48, 36, and 22 kDa (P48, P36, and P22) were the components of the outermost layer (OL) of Bacillus megaterium ATCC 12872 spore by analysis of the isolated OL. And it was indicated that these proteins were deposited not by disulfide bond, but by ionic and/or hydrophobic bonds on the spore. Among them, P36 and P22 were expected to be located on the very surface of the spore by immunological analysis. In the OL deficient mutant of B. megaterium ATCC 12872, MAE05, whose spore was lacking in these OL proteins and galactosamine-6-phosphate polymer, both P36 and P22 were present in the mother cell cytoplasm and deposited on the forespores, but they disappeared with the lysis of mother cells. An OL protein-releasing factor having proteolytic activity was detected in the culture supernatant at the late sporulating stage of both the wild-type and the mutant strains. But the factor could not act on the proteins of the mature spores and the forespores at tin (tn indicates n hr after the end of exponential growth) of the wild-type strain. Moreover, P36 and P22 were found in the spores of a revertant of MAE05 which could form galactosamine-6-phosphate polymer, suggesting that this sugar polymer played the role in protecting the OL proteins against the protease-like substance after the deposition.

“…For visualization, gels were stained with Coomassie brilliant blue R-250 or immunologically. Immunoblot analysis was carried out by the method of Imagawa et al (9). IN …”

Section: Methodsmentioning

confidence: 99%

Characterization and Deposition of the Proteins in the Outermost Layer of Bacillus megaterium Spore

Takubo

Okuda

Takemura

et al. 1989

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“…1(B)), In our previous paper (5) , using the antibody to whole proteins extracted from B. megaterium ATCC 12872 spore coat preparation, we did immunoblot analysis and the enzyme immunoassay. The coat proteins were detected from 3 hr after the end of exponential growth (t3) in the mother cell cytoplasmic fraction, while in the forespore coat protein fraction, they increased from t3 and reached a plateau at tio (5).…”

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confidence: 99%

“…Twenty-two kilodalton protein was purified by cutting and extracting the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (5). SDS-PAGE revealed the single band of 22K protein in Fig.…”

mentioning

confidence: 99%

Immunological Detection of 22K Protein in Sporulating Cells of Bacillus megaterium ATCC 12872

Nishihara

Sakagami

Tani

et al. 1988

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The synthesis and deposition of 22,000-dalton (22K) spore coat protein were examined immunochemically on the sporulating cells of Bacillus megaterium ATCC 12872 using the antibody to purified 22K spore coat protein. This antibody cross-reacted with 44K and 25K proteins in immunoblot analysis of dormant spore coat proteins. Immunoblot analysis on the sporulating cells showed that 22K protein was detected from t8 in forespore coat protein fractions. Sandwich enzyme immunoassay revealed that 22K protein in the spore coat protein fraction appeared at t6 and reached a plateau at t9, and 22K protein in the mother cell cytoplasmic fraction was detected at only t7 and t8 at a very low level.

“…The procedure for ELISA was as described previously (15,16). A sample of 0.25 ml was mixed with 0.25 ml of Buffer A in a small test tube and then a polystyrene bead coated with anti-toxin IgG was put into the mixture.…”

mentioning

confidence: 99%

“…The concentration of F(ab')2 was determined assuming 1.48 A280=1 mg/ml (18 Coating of polystyrene beads with anti-toxin IgG. Polystyrene beads were coated with anti-toxin IgG as described previously (16). Polystyrene beads (6.5 mm in diameter) were immersed in a solution of 100 eug of anti-toxin IgG per ml of 0.1 M sodium phosphate buffer (pH 7.5) containing 0.1 % NaN3 at 4 C overnight.…”

mentioning

confidence: 99%

Development of a Highly Sensitive Bead‐ELISA to Detect Bacterial Protein Toxins

Oku

Uesaka

Hirayama

et al. 1988

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A highly sensitive sandwich enzyme-linked immunosorbent assay to detect bacterial toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The entire assay could be completed within 3.5 hr. The sensitivity of this bead-ELISA was found to be quite high with various bacterial toxins : less than 20 pg/ml for thermostable direct hemolysin of Vibrio parahaemolyticus, less than 60 pg/ml for Shiga toxin, less than 20 pg/ml for VT2 (Shiga-like toxin II) of Escherichia coli, less than 200 pg/ml for heat-labile enterotoxin of E. coli, and less than 6 pg/ml for cholera enterotoxin.