Regulation of expression of genes coding for small, acid-soluble proteins of Bacillus subtilis spores: studies using lacZ gene fusions

Abstract

We constructed in-frame translational fusions of the Escherichia coli lacZ gene with four genes (sspA, sspB, sspD, and sspE) which code for small, acid-soluble spore proteins of Bacillus subtilis, and integrated these fusions into the chromosomes of various B. subtilis strains. With single copies of the fusions in wild-type B. subtilis, beta-galactosidase was synthesized only during sporulation, with the amounts accumulated being sspB much greater than sspE greater than or equal to sspA greater than or equal to sspD. Greater than 97% of the beta-galactosidase was found in the developing forespore, and the great majority was incorporated into mature spores. Less than 2% of the maximum amount of beta-galactosidase was made when these fusions were introduced into B. subtilis strains blocked in stages 0 and II of sporulation, as well as in some stage III mutants. Other stage III mutants, as well as stage IV and V mutants, had no effect on beta-galactosidase synthesis. Increasing the copy number of the sspA-, sspD-, or sspE-lacZ fusions (up to 17-fold for sspE-lacZ) in wild-type B. subtilis resulted in a parallel increase in the amount of beta-galactosidase accumulated (again only in sporulation and with greater than 95% in the developing forespore), with no significant effect on wild-type small, acid-soluble spore protein production. Similarly, the absence of one or more wild-type ssp genes or the presence of multiple copies of wild-type ssp genes had no effect on the expression of the lacZ fusions tested. These data indicate that these ssp-lacZ fusions escape the autoregulation seen for the intact sspA and sspB genes. Strikingly, the kinetics of beta-galactosidase synthesis were identical for all four ssp-lacZ fusions and paralleled those of glucose dehydrogenase synthesis. Similarly, all asporogenous mutants tested had identical effects on both glucose dehydrogenase and ssp-lacZ fusion expression.