DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis

Abstract

ABSTRACT Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32 (Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag , and of the fla-che operon is strongly reduced in a degU32 (Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32 (Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che , thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the σ A -dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the P A promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.