Sequence organization and regulation of the Bacillus subtilis menBE operon

Menaquinone (MK) plays a central role in the respiratory chain of Bacillus subtilis. The biosynthesis of MK requires the formation of a naphthoquinone ring via a series of specific reactions branching from the shikimate pathway. "Early" MK-specific reactions catalyze the formation of o-succinylbenzoate (OSB) from isochorismate, and "late" reactions convert OSB to dihydroxynaphthoate, by utilizing an OSB-coenzyme A intermediate. We have cloned and sequenced the B. subtilis menE and menB genes encoding, respectively, OSB-coenzyme A synthase and dihydroxynaphthoate synthase. The MenB open reading frame encodes a potential polypeptide of 261 amino acid residues with a predicted size of 28.5 kDa, while the MenE open reading frame could encode a 24.4-kDa polypeptide of 220 amino acid residues. Probable promoter sequences were identified by high-resolution primer extension assays. Organization of these genes and regulatory regions was found to be menBp menB menEp menE. Expression of menE was dependent on both menEp and menBp, indicating an operonlike organization. A region of dyad symmetry capable of forming a stable RNA secondary structure was found between menB and menE. Culture cycle-dependent expression of menB and menE was measured by steady-state transcript accumulation. For both genes, maximal accumulation was found to occur within an hour after the end of exponential growth. The menBp and menEp promoters have sequences compatible with recognition by the major vegetative form of B. subtilis RNA polymerase, E sigma A. Both promoter regions also were found to contain homologies to a sequence motif previously identified in the menCDp region and in promoters for several B. subtilis tricarboxylic acid cycle genes.