No abstract

The spoOA gene of Bacilus subtilis is critical for the initial stages in the developmental cycle leading to the formation of an endospore. We show that one function of the SpoOA protein is to negatively regulate another regulatory locus, abrB, which controls the expression of many genes associated with the onset of sporulation. Purified SpoOA protein binds to a specific region of the abrB promoter and functions as a repressor of transcription in an in vitro assay. The binding of the SpoOA protein is independent of the binding of the AbrB protein, which is known to autoregulate its expression. This independence mirrors the temporal sequence of events in abrB control.The products of the Bacillus subtilis spoO genes are required for the initiation of sporulation in this organism (1, 2). Mutations of spoO in B. subtilis block sporulation at its earliest stage and exhibit pleiotropic effects on the expression of a wide variety of gene products associated with the end of exponential growth and the onset of sporulation: the transition stage (1, 3). The most pleiotropic of the spoO mutations occur at the spoOA locus.It is believed that SpoOA is the main component through which the environmental signals for sporulation are relayed to the transcriptional machinery of the cell (4, 5). The spoOA gene codes for a protein of 29,700 Da (6), which exhibits amino acid sequence homology with the regulator class of molecule (e.g., OmpR, NtrC, CheB, CheY, etc.) in prokaryotic two-component regulatory systems (7-10). The SpoOA protein regulates the onset of stationary phase and sporulation, in part, by controlling the expression of the abrB gene. The product of the abrB gene is a repressor that functions to prevent the expression of transition stage-specific genes during vegetative growth (11,12). Among the genes regulated by abrB are some that are necessary for normal sporulation: spoOE (5), spoOH (13), and spoVG (14). During the transition stage, production of the AbrB protein decreases, thus allowing for the derepression of promoters regulated by AbrB (11). The modulation of AbrB synthesis depends upon the SpoOA protein (11,15).Although a large body of genetic evidence implicated the SpoOA protein in regulatory phenomena occurring at the onset of sporulation, the actual biochemical function of the SpoOA protein was unknown. In this communication we show that the SpoOA protein binds to a specific region of the abrB gene located downstream from the transcriptional start sites and functions as a repressor of transcription in vitro. MATERIALS AND METHODSDNase and Methylation Protection Assays. The 800-base-pair (bp) BamHI-HindIII fragment of pJM5134 (11) containing the abrB promoter and upstream region was labeled at the BamHI end by using either [a-32P] (12) with labeled DNA at 1-3 x 10-9 M final concentration and highly purified SpoOA protein (see below). The method used for determining which purine (both adenine and guanine) residues were protected from methylation by dimethyl sulfate due to binding of SpoOA was as follow...

Sporulation begins coincidentally with the expression of several stationary-phase-associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary-phase-associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96-amino-acid protein (molecular weight 10773) and contained a helix-turn-helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.