Flow cytometric study of differentiating cultures of Bacillus subtilis

Abstract

We report on (1) the development of aflow cytometry-based technique for detecting β-galactosidase in differentiating cultures of Bacillus subtilis and (2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-β-D-galactopyranoside (C⁸-FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C⁸-FDG staning mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C⁸-fluorescein and PKH26 allow nonspecific C⁸-FDG staining in this control population to be monitored using two-color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulationg genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.