Vol. 126, No. 3
JOURNAL OF BACTERIOLOGY, June 1976, p. 1342-1343 Copyright © 1976 American Society for Microbiology
Printed in U-SA.
Appearance of Spore Coat Protein in the Cell Extracts of Bacillus subtilis Asporogenic Mutants ARITSUNE UCHIDA,* HAJIME KADOTA, AND PIERRE SCHAEFFER
Laboratory of Microbiology, Department ofFisheries, Faculty of Agriculture, Kyoto University, Kyoto, Japan,* and Institute of Microbiology, Faculty of Science, University of Paris-Sud, 91-Orsay, France Received for publication 22 March 1976
By use of the antigen-antibody techniques we have studied whether asporogenic mutants of Bacillus subtilis can synthesize the spore coat protein. Antibody specific to spore coat protein was prepared and used to demonstrate that the spore coat protein was synthesized at the early stage of sporulation. We report here that asporogenic mutants synthesize the spore coat protein.
Bacillus subtilis 3610 (ATCC 6051) and 168
trp- were used as the wild-type strains. Asporo-
genic mutant strains used were classified according to the cytological stage at which the sporulation process was blocked by mutation (3). Cells were grown in the Schaeffer liquid nutrient broth sporulation medium (3) and harvested at the intervals of every 2 h after the end of exponential growth. Portions of the 500-ml culture at various stages were centrifuged, washed twice with TMS buffer containing 0.01 M Tris-hydrochloride (pH 7.5), 0.06 M NH4Cl, 0.01 M magnesium acetate, and 0.007 M (8-mercaptoethanol, and suspended in 4 ml of TMS buffer, and the cells were disintegrated by sonication. The extracts were centrifuged in a refrigerated centrifuge at 10,000 x g for 10 min. The supernatant fluids were again centrifuged and frozen at -20 C. Antiserum was prepared in rabbits immunized with purified spore coat protein which was emulsified in Freund complete adjuvant. The antiserum obtained was stored at 0 C. The detection of the spore coat protein antigen was carried out by use of the Ouchterlony technique (2). After a 48-h incubation in a moist chamber, appearance of the precipitin band was determined. The results are shown in Table 1. All of the spoO and spoIl mutants do not form the spore coat protein antigen. On the other hand,. the 94U (spoIII) and liT (spoIV) mutants synthesize spore coat protein as early as do wild-type strains. Strains liT has been characterized morphologically; it forms a complete cortex but does not produce a spore coat (4). Strain 94U is a spolII mutant blocked even earlier at the stage before cortex development is seen in the electron microscope. Yet, the results in Table 1 show that both strains produce soluble spore coat protein. Quantitative analyses with these
mutants are now in progress. It is thought that these two mutants produce the spore coat protein in a soluble form in the mother cells, but that the coat protein cannot be deposited on the surface of spores. Based on morphological observations, Balassa and Yamamoto (1) and Waites et al. (5) also reported that late stage mutants of B. subtilis, in which the cortex was normally formed, sometimes showed incomplete formation of the spore coat. Wood (6) reported the ability of asporogenic mutants to produce the alkali-extractable spore coat protein as early as stage II of sporulation. These results suggest that the biosynthesis of spore coat protein is an earlier event in the sporulation of B. subtilis than is formation of the coat. TABLE 1. Appearance of soluble spore coat protein antigens in the extracts prepared from B. subtilis cells at different stages of sporulation Presence of spore coat protein in cell extracts
Strains Vegetative
cells and
t,
t2 through truhtt8
+ subtilis 3610 (ATCC 6051) + B. subtilis 168 trp11ONA(spoOA)a 3U(spoOB) 9V(spoOC) 4Z(spoII) + 94U(spoIII) + 11T(spoIV) a Additional spoOA mutants (3NA, 5NA, and 14UL), spoOB mutants (6Z, 9NA, and 17NA), unclassified mutants(sp-98-8 and sp-C14-1), and spoll mutant (26V) all failed to produce soluble spore coat antigens at any stage of post-logarithmic phase growth. 1342
B.
VOL. 126, 1976 LITERATURE CITED 1. Balassa, G., and T. Yamamoto. 1970. Biochemical genetics of bacterial sporulation. III. Correlation between morphological properties of sporulation mutants. Mol. Gen. Genet. 108:1-22. 2. Crowle, A. J. 1960. Immunodiffusion. Academic Press Inc., New York. 3. Ionesco, H., J. Michel, B. Cami, and P. Schaeffer. 1970. Genetics of sporulation in Bacillus subtilis Marburg. J. Appl. Bacteriol. 33:13-24.
NOTES
1343
4. Millet, J., and A. Ryter. 1972. Mutants de Bacillus subtilis. Marburg bloques tardivement dans leur sporulation. Ann. Inst. Pasteur Paris 122:395-406. 5. Waites, W. M., D. Kay, I. W. Dawes, D. A. Wood, S. C. Warren, and J. Mandelstam. 1970. Sporulation in Bacillus subtilis. Correlation of biochemical events with morphological changes in asporogenous mutants. Biochem. J. 118:667-676. 6. Wood, D. A. 1972. Sporulation in Bacillus subtilis. Properties and time of synthesis of alkali-soluble protein of the spore coat. Biochem. J. 130:505-514.
JOURNAL OF BACTERIOLOGY, June 1976, p. 1342-1343 Copyright © 1976 American Society for Microbiology
Printed in U-SA.
Appearance of Spore Coat Protein in the Cell Extracts of Bacillus subtilis Asporogenic Mutants ARITSUNE UCHIDA,* HAJIME KADOTA, AND PIERRE SCHAEFFER
Laboratory of Microbiology, Department ofFisheries, Faculty of Agriculture, Kyoto University, Kyoto, Japan,* and Institute of Microbiology, Faculty of Science, University of Paris-Sud, 91-Orsay, France Received for publication 22 March 1976
By use of the antigen-antibody techniques we have studied whether asporogenic mutants of Bacillus subtilis can synthesize the spore coat protein. Antibody specific to spore coat protein was prepared and used to demonstrate that the spore coat protein was synthesized at the early stage of sporulation. We report here that asporogenic mutants synthesize the spore coat protein.
Bacillus subtilis 3610 (ATCC 6051) and 168
trp- were used as the wild-type strains. Asporo-
genic mutant strains used were classified according to the cytological stage at which the sporulation process was blocked by mutation (3). Cells were grown in the Schaeffer liquid nutrient broth sporulation medium (3) and harvested at the intervals of every 2 h after the end of exponential growth. Portions of the 500-ml culture at various stages were centrifuged, washed twice with TMS buffer containing 0.01 M Tris-hydrochloride (pH 7.5), 0.06 M NH4Cl, 0.01 M magnesium acetate, and 0.007 M (8-mercaptoethanol, and suspended in 4 ml of TMS buffer, and the cells were disintegrated by sonication. The extracts were centrifuged in a refrigerated centrifuge at 10,000 x g for 10 min. The supernatant fluids were again centrifuged and frozen at -20 C. Antiserum was prepared in rabbits immunized with purified spore coat protein which was emulsified in Freund complete adjuvant. The antiserum obtained was stored at 0 C. The detection of the spore coat protein antigen was carried out by use of the Ouchterlony technique (2). After a 48-h incubation in a moist chamber, appearance of the precipitin band was determined. The results are shown in Table 1. All of the spoO and spoIl mutants do not form the spore coat protein antigen. On the other hand,. the 94U (spoIII) and liT (spoIV) mutants synthesize spore coat protein as early as do wild-type strains. Strains liT has been characterized morphologically; it forms a complete cortex but does not produce a spore coat (4). Strain 94U is a spolII mutant blocked even earlier at the stage before cortex development is seen in the electron microscope. Yet, the results in Table 1 show that both strains produce soluble spore coat protein. Quantitative analyses with these
mutants are now in progress. It is thought that these two mutants produce the spore coat protein in a soluble form in the mother cells, but that the coat protein cannot be deposited on the surface of spores. Based on morphological observations, Balassa and Yamamoto (1) and Waites et al. (5) also reported that late stage mutants of B. subtilis, in which the cortex was normally formed, sometimes showed incomplete formation of the spore coat. Wood (6) reported the ability of asporogenic mutants to produce the alkali-extractable spore coat protein as early as stage II of sporulation. These results suggest that the biosynthesis of spore coat protein is an earlier event in the sporulation of B. subtilis than is formation of the coat. TABLE 1. Appearance of soluble spore coat protein antigens in the extracts prepared from B. subtilis cells at different stages of sporulation Presence of spore coat protein in cell extracts
Strains Vegetative
cells and
t,
t2 through truhtt8
+ subtilis 3610 (ATCC 6051) + B. subtilis 168 trp11ONA(spoOA)a 3U(spoOB) 9V(spoOC) 4Z(spoII) + 94U(spoIII) + 11T(spoIV) a Additional spoOA mutants (3NA, 5NA, and 14UL), spoOB mutants (6Z, 9NA, and 17NA), unclassified mutants(sp-98-8 and sp-C14-1), and spoll mutant (26V) all failed to produce soluble spore coat antigens at any stage of post-logarithmic phase growth. 1342
B.
VOL. 126, 1976 LITERATURE CITED 1. Balassa, G., and T. Yamamoto. 1970. Biochemical genetics of bacterial sporulation. III. Correlation between morphological properties of sporulation mutants. Mol. Gen. Genet. 108:1-22. 2. Crowle, A. J. 1960. Immunodiffusion. Academic Press Inc., New York. 3. Ionesco, H., J. Michel, B. Cami, and P. Schaeffer. 1970. Genetics of sporulation in Bacillus subtilis Marburg. J. Appl. Bacteriol. 33:13-24.
NOTES
1343
4. Millet, J., and A. Ryter. 1972. Mutants de Bacillus subtilis. Marburg bloques tardivement dans leur sporulation. Ann. Inst. Pasteur Paris 122:395-406. 5. Waites, W. M., D. Kay, I. W. Dawes, D. A. Wood, S. C. Warren, and J. Mandelstam. 1970. Sporulation in Bacillus subtilis. Correlation of biochemical events with morphological changes in asporogenous mutants. Biochem. J. 118:667-676. 6. Wood, D. A. 1972. Sporulation in Bacillus subtilis. Properties and time of synthesis of alkali-soluble protein of the spore coat. Biochem. J. 130:505-514.
