Changes in membrane-associated proteins during sporulation in Bacillus subtilis.

Membrane proteins from vegetative and sporulating cells of Bacillus subtilis were separated by the two-dimensional gel electrophoresis system using isoelectric focusing and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (O'Farrell technique). Membrane proteins were isolated according to published procedures. The gels were stained with Coomassie blue. Three different concentrations of proteins were analyzed to detect even minor constituents. Over two hundred different membrane proteins were identified in vegetative cells by their isoelectric point (pI) and molecular weight (Mr). Analysis of membrane proteins from cells harvested during and at the end of logarithmic growth (A600 approximately equal to 0.8; T0) and every hour thereafter until T4 showed that in the wild-type strain 55 proteins are degraded mostly at the beginning or sporulation. Many others (76 proteins) are newly synthesized during sporulation. About 16 proteins are synthesized at times during sporulation but again degraded within 1 h or less. Others (uncertain proteins, 65) are degraded and resynthesized again. This observation is in agreement with experiments previously published by Andreoli et al. [Andreoli, A. J., Kao, M., Chui, R., Cabrera, J., and Wong, S. K. S (1981) in Sporulation and Germination (Levinson, H. S., Sonenshein, A. L., and Tipper, D. J., eds) pp. 168-173, American Society for Microbiology, Washington] using Bacillus cereus. Experiments with the early blocked asporogenous mutant JH 649 (spoOF) showed that few proteins (40%) are degraded and even fewer (30%) are newly synthesized between A600 approximately equal to 0.8 and T4. Protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline) have no effect on the protein patterns. The experiments presented here show that proteins involved in differentiation in B. subtilis can be identified by the two-dimensional gel electrophoresis system and with the aid of asporogenous mutants. In order to assure that no cytoplasmic proteins are contaminating the membrane preparations, several cytoplasmic enzyme activities have been measured. Their concentration was found to be always below 0.005% of total protein, which is below the level of detection by Coomassie blue staining.