Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3-dihydroxybenzoate

Abstract

Bacillus subtilis has duplicate isochorismate synthase genes, menF and dhbC. Isochorismate synthase is involved in the biosynthesis of both the respiratory chain component menaquinone (MK) and the siderophore 2,3-dihydroxybenzoate (DHB). Several menF and dhbC deletion mutants were constructed to identify the contribution made by each gene product to MK and DHB biosynthesis. menF deletion mutants were able to produce wild-type levels of MK and DHB, suggesting that the dhbC gene product is able to compensate for the lack of MenF. However, a dhbC deletion mutant produced wild-type levels of MK but was DHB deficient, indicating that MenF is unable to compensate for the lack of DhbC. A menF dhbC double-deletion mutant was both MK and DHB deficient. Transcription analysis showed that expression of dhbC, but not of menF, is regulated by iron concentration. A dhbA::lacZ fusion strain was constructed to examine the effects of mutations to the iron box sequence within the dhb promoter region. These mutations abolished the iron-regulated transcription of the dhb genes, suggesting that a Fur-like repressor protein exists in B. subtilis.Isochorismate synthase is responsible for converting chorismate to isochorismate and is necessary for the biosynthesis of both the respiratory chain component menaquinone (MK) and the Bacillus subtilis siderophore 2,3-dihydroxybenzoate (DHB) (Fig. 1). Although it is known that the carbon source, growth phase, and oxygen tension have regulatory effects, the actual signals that induce expression of respiratory chain components are unknown (37). However, the production of DHB is known to be regulated by iron concentration (1,25,26). Therefore, isochorismate is required by cells under very different environmental conditions.B. subtilis has two distinct isochorismate synthases encoded by the menF (30) and dhbC (29) genes. MenF and DhbC are 47% identical at the DNA level and have 35% amino acid identity (29). menF is a promoter-proximal gene of the MK biosynthetic gene cluster located at 273Њ on the chromosome (30,38). MK is a lipophilic, nonprotein redox component mediating electron transfer between dehydrogenases and cytochromes (37). dhbC is the second gene of the DHB biosynthetic gene cluster located at 291Њ on the chromosome (29). Under conditions of iron deprivation, B. subtilis synthesizes DHB, a component of the specific transport system for the uptake of extracellular iron (1,25,26). This is not the only instance of gene duplication in B. subtilis. There are two thymidylate synthetases, TSaseA and TSaseB, that are encoded by the unlinked thyA and thyB genes, respectively (22). Both enzymes are functional in bacteria grown at 37ЊC or lower temperatures, but only TSaseA is active at 46ЊC (22). There are two different a-type terminal oxidases in B. subtilis that are encoded by separate loci (32). One of the oxidases is associated with a heme C-containing unit (32). The two membrane alkaline phosphatases of B. subtilis have substantial differences with respect to molecular weight, substrate sp...

A Plant Locus Essential for Phylloquinone (Vitamin K1) Biosynthesis Originated from a Fusion of Four Eubacterial Genes

“…A tendency to co-localize the menF and menD genes is extended to several eubacterial genomes, in line with the idea that conserved gene orders in operons of different bacteria often correspond to physical interactions of their gene products (29). These results suggest that the transcriptional linkage of men genes in an operon ensures a co-translational association of Men proteins promoting efficient assembly of a multienzymatic complex (27)(28)(29).…”

“…Thus, a selective driving force may have been acting over a long evolutionary distance to maintain this cluster of men genes as a fusion, possibly corresponding to a need of a multifunctional association of proteins required to channel metabolic intermediates of PhQ biosynthesis (23, 26 -32). This has been suggested similarly for the menaquinone pathway in the genomes of Escherichia coli and Bacillus subtilis, where co-localization of menF and menD genes in operons is essential for the proper channelling of isochorismate, the product of MenF, to the menaquinone pathway via MenD (27,28). A tendency to co-localize the menF and menD genes is extended to several eubacterial genomes, in line with the idea that conserved gene orders in operons of different bacteria often correspond to physical interactions of their gene products (29).…”

“…Therefore, it is conceivable that PHYLLO arose from the framework of a plastid operon as a result of selection forces adapting an analogous operon-directed association of enzymes in the eukaryotic genomes (23,(27)(28)(29). men genes are not fused in any of the eubacterial and plastid genomes sequenced so far.…”

The dhb Operon of Bacillus subtilisEncodes the Biosynthetic Template for the Catecholic Siderophore 2,3-Dihydroxybenzoate-Glycine-Threonine Trimeric Ester Bacillibactin

“…Transcription of the dhb operon was found to be controlled by a single A -dependent promoter that comprises a fur box-binding site, an iron regulatory element (26). According to the published data derived from B. subtilis genome strain 168, DhbF would end within a C domain.…”

Mapping of orthologous genes in the context of biological pathways: An application of integer programming

“…Test 2: Mapping of the menaquinone biosynthesis pathway. It is known that both B. subtilis and E. coli K12 have the menaquinone biosynthesis pathway (21,22). Currently, it is known that the menaquinone biosynthesis pathway in E. coli K12 includes the following genes: b3930 (menA), b2262 (menB), b2261 (menC), b2264 (menD), b2260 (menE), b2265 (menF), and b3833 (ubiE).…”