Sequence organization and regulation of the Bacillus subtilis menBE operon

Abstract

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, EcA. 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.Menaquinone (MK; vitamin K2) is a lipophilic, nonprotein redox component in the electron transport chain of Bacillus subtilis which mediates electron flow between dehydrogenases and cytochromes (5). In addition to its role in respiration, MK is necessary for successful endospore formation and is involved in regulation of cytochrome formation (10, 28). The formation of MK appears to be regulated during growth of B. subtilis, with maximal levels being reached within 1 h (T1) after the end of exponential growth phase in batch culture (TO), followed by a rapid decline (10). The lack of MK in endospores of Bacillus cereus has been correlated with metabolic dormancy (8). Thus, study of the regulation of MK biosynthesis should provide insights into the factors governing modification of Bacillus respiratory chains in response to both the nutritional status of the growth medium and the cellular developmental sequence represented by sporulation.Much of the enzymology of MK biosynthesis has been carried out with Escherichia coli (1, 2). However, the pathway in B. subtilis appears to be similar if not identical (20,29 deficient. This has permitted cloning of men-specific sequences and assignment of gene functions to specific segments of DNA.The cloning of menCDBE has been previously reported, along with preliminary analysis o...

Genes coding for the benzoyl‐CoA pathway of anaerobic aromatic metabolism in the bacterium Thauera aromatica

“…The function of BadI has been deduced from the N-terminal amino acid sequence of the purified protein that catalyzed hydrolytic carbon ring opening of 2-oxocyclohexane-1-carboxyl-CoA producing pimelyl-CoA (Pelletier and Harwood, 1996). Furthermore, T. aromatica and R. palustris enzymes showed sequence similarities with dihydroxynaphthoic acid synthase from various organisms (Driscoll and Taber, 1992;Sharma et al, 1992). This synthase catalyzes the formal back reaction of the hydrolase, namely formation of the aromatic ring in naphthoate synthesis from the precursor o-succinylbenzoylCoA.…”

Transcriptional regulation of the Bacillus subtilis menp1 promoter

“…(3,20). At men locus, the locations of the menF, menD, open reading frame 3 (ORF3), menB, and menE genes are indicated (3,21,26 and 5% ␣-naphthol (Fluka) (0.2 ml) in a 2.5 N NaOH solution were added to 1 ml of culture supernatant or diluted supernatant containing between 0.01 and 0.25 mM acetoin. The A 540 was allowed to develop at room temperature for 60 min, at which time the color intensity was at its maximum.…”

“…At the oxidative metabolic stage, coordination of TCA cycle activity with the formation of respiratory chain components, especially MK, is essential for utilization of nonfermentable carbon sources. Most B. subtilis structural genes encoding biosynthesis of the naphthoquinone ring of MK have been isolated (3,21,26,33). The enzymology of MK biosynthesis is understood, and geneenzyme relationships have been established (3,21,23).…”

“…Most B. subtilis structural genes encoding biosynthesis of the naphthoquinone ring of MK have been isolated (3,21,26,33). The enzymology of MK biosynthesis is understood, and geneenzyme relationships have been established (3,21,23). In previous studies, B. subtilis genes encoding MK biosynthetic enzymes have been assigned to two major transcriptional units, the menFD and menBE operons (Fig.…”

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I

“…In several bacteria, vitamin K 2 (menaquinone) is used during fumarate reduction in anaerobic respiration (11). The genes encoding the enzymes involved in the conversion of chorismate to menaquinone have been cloned in Escherichia coli (12)(13)(14) and Bacillus subtilis (15,16). Although the route of phylloquinone biosynthesis has not been described in cyanobacteria, the pathway is likely to be similar to the pathway of menaquinone biosynthesis in other bacteria.…”