Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis

Abstract

NAD plays an important role in cellular metabolism, as it functions as a cofactor in numerous oxidation-reduction reactions. NAD is synthesized from quinolinic acid (Qa) via a pathway which is similar in all organisms which have been examined (Fig. 1). On the other hand, the pathway for Qa formation differs in different organisms (7). In Escherichia coli and Salmonella typhimurium, as well as in many other eubacteria, Qa is synthesized from L-aspartate and dihydroxyacetone phosphate by a Qa synthetase complex (7,33). This complex consists of two enzymes, L-aspartate oxidase and Qa synthetase A. Aspartate oxidase catalyzes oxidation of L-aspartate to iminoaspartate, which is subsequently condensed with dihydroxyacetone phosphate by Qa synthetase A to form Qa (Fig. 1). In E. coli, the latter two enzymes are encoded by the unlinked genes nadB and nadA4, respectively, both of which have been cloned and sequenced (6). Qa is then converted to nicotinic acid mononucleotide (NaMN) by Qa phosphoribosyltransferase, which is coded for by the nadC gene (23). NaMN then reacts with ATP in a reaction catalyzed by the nadD gene product to give nicotinic acid adenine dinucleotide, which is then amidated by the nadE gene product to form NAD. Cells can also convert exogenous nicotinic acid (Nic) to NaMN by Nic phosphoribosyltransferase. When either nadA, nadB, or nadC is inactivated, cells become dependent on Nic for growth.The NAD biosynthetic pathway in Bacillus subtilis has been thought to be the same as in E. coli (7), although the regulation of some of the B. subtilis enzymes, for example, Qa phosphoribosyltransferase, may be rather different than in E. coli (28). Nic-requiring auxotrophic mutants have been isolated in B. subtilis, and the mutations (termed nic) were mapped near the spoOB-pheBA region (2,10,13 The chromosomal DNA library was used to transform strain PS1589 to chloramphenicol resistance. Strain PS1589 carries the divIVB1 mutation, which is known to be closely linked to the site of nic mutations (16,26). Several different Cmr transformants were found to have the cat gene closely linked to divIVB1. Three-factor crosses showed that in one of the Cmr transformants, strain PS1591, the order of markers was divIVB-spoOB-cat.

The iscS Gene in Escherichia coli Is Required for the Biosynthesis of 4-Thiouridine, Thiamin, and NAD

“…This requirement has precedence, since in B. subtilis, an nifS gene is required for the biosynthesis of NAD (30). The proposed target of the nicotinate deficiency is quinolinate synthetase (NadA), one component of a system that condenses L-aspartate and dihydroxyacetone phosphate to give quinolinate (30).…”

Nitrogenase metalloclusters: structures, organization, and synthesis

“…Thus, it has been hypothesized that the nifS gene product activates sulfur by formation of an enzyme-bound persulfide which serves as the S donor in nitrogenase Fe-S cluster formation. Genes whose products have extraordinary primary sequence identity when compared to the nifS gene product have recently been identified in non-nitrogen-fixing organisms (50). Thus, it appears that the function of the nifS gene product in the mobilization of sulfur for nitrogenase metallocluster assembly could represent a universal path for Fe-S cluster formation.…”

bofC Encodes a Putative Forespore Regulator of the Bacillus Subtilis σk Checkpoint

“…The map shows (to scale) the 27 kb region of DNA beginning a t the spolVF locus and extending to the spoVB gene. This region of the chromosome has been cloned and partially sequenced and the map constructed from other work describing the characterization of the spolVF, spoOB, pheA, nifS, nadB and spoVB loci (Cutting et a/., 1991 b; Popham & Stragier, 1991;Sun & Setlow, 1993;Trach 81 Hoch, 1989). The physical map linking the nadB locus with DNA contained in pDG851 has been surmised from published work but not confirmed by Southern hybridization analysis.…”