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.

Characterization of l‐aspartate oxidase and quinolinate synthase from Bacillus subtilis

“…These results are in agreement with the data reported for NadA from E. coli and A. thaliana , which contain one highly oxygen‐sensitive [4Fe–4S] 2+ cluster per monomer of NadA [7,20,21]. The data are in keeping with the hypothesis proposed by Sun & Setlow [24], who suggested that NadA from B. subtilis may contain an [Fe–S] cluster, on the basis of the observation that, like E. coli , B. subtilis iscS − strains are auxotrophic for nicotinic acid and are unable to synthesize NAD de novo .…”

Characterization of l‐aspartate oxidase and quinolinate synthase from Bacillus subtilis

“…These results are in agreement with the data reported for NadA from E. coli and A. thaliana , which contain one highly oxygen‐sensitive [4Fe–4S] 2+ cluster per monomer of NadA [7,20,21]. The data are in keeping with the hypothesis proposed by Sun & Setlow [24], who suggested that NadA from B. subtilis may contain an [Fe–S] cluster, on the basis of the observation that, like E. coli , B. subtilis iscS − strains are auxotrophic for nicotinic acid and are unable to synthesize NAD de novo .…”

Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G

“…In conformity with the rule that most transcription occurs in the same direction as DNA replication (9, 77), all 35 genes in this interval are oriented in the same direction as DNA replication (left to right in Fig. 4a) with the exception of orfY and the nifS-orfi operon (69).…”

Regulation of the Expression of Genes Involved in NAD De Novo Biosynthesis in Corynebacterium glutamicum

“…In contrast to the E. coli iscS-deficient mutant strain, the C. glutamicum nadS-deficient mutant strain grew as well as the wild-type strain in nicotinate-supplemented minimal medium, suggesting that NadS is specifically involved in the de novo Fe-S cluster synthesis on the nascent NadA apoenzyme. Similar results are observed in the case of a B. subtilis mutant strain deficient in a cysteine desulfurase gene homologue, although the gene product has not yet been functionally characterized in detail (31). At present, whether or not the cysteine desulfurases specific for NAD de novo biosynthesis are additionally involved in Fe-S cluster repair under oxidative stress conditions remains to be determined.…”

Characterization of l‐aspartate oxidase and quinolinate synthase from Bacillus subtilis

“…These results are in agreement with the data reported for NadA from E. coli and A. thaliana , which contain one highly oxygen‐sensitive [4Fe–4S] 2+ cluster per monomer of NadA [7,20,21]. The data are in keeping with the hypothesis proposed by Sun & Setlow [24], who suggested that NadA from B. subtilis may contain an [Fe–S] cluster, on the basis of the observation that, like E. coli , B. subtilis iscS − strains are auxotrophic for nicotinic acid and are unable to synthesize NAD de novo .…”

Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G

“…In conformity with the rule that most transcription occurs in the same direction as DNA replication (9, 77), all 35 genes in this interval are oriented in the same direction as DNA replication (left to right in Fig. 4a) with the exception of orfY and the nifS-orfi operon (69).…”

Regulation of the Expression of Genes Involved in NAD De Novo Biosynthesis in Corynebacterium glutamicum

“…In contrast to the E. coli iscS-deficient mutant strain, the C. glutamicum nadS-deficient mutant strain grew as well as the wild-type strain in nicotinate-supplemented minimal medium, suggesting that NadS is specifically involved in the de novo Fe-S cluster synthesis on the nascent NadA apoenzyme. Similar results are observed in the case of a B. subtilis mutant strain deficient in a cysteine desulfurase gene homologue, although the gene product has not yet been functionally characterized in detail (31). At present, whether or not the cysteine desulfurases specific for NAD de novo biosynthesis are additionally involved in Fe-S cluster repair under oxidative stress conditions remains to be determined.…”

Characterization of l‐aspartate oxidase and quinolinate synthase from Bacillus subtilis

“…These results are in agreement with the data reported for NadA from E. coli and A. thaliana , which contain one highly oxygen‐sensitive [4Fe–4S] 2+ cluster per monomer of NadA [7,20,21]. The data are in keeping with the hypothesis proposed by Sun & Setlow [24], who suggested that NadA from B. subtilis may contain an [Fe–S] cluster, on the basis of the observation that, like E. coli , B. subtilis iscS − strains are auxotrophic for nicotinic acid and are unable to synthesize NAD de novo .…”

Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G

“…In conformity with the rule that most transcription occurs in the same direction as DNA replication (9, 77), all 35 genes in this interval are oriented in the same direction as DNA replication (left to right in Fig. 4a) with the exception of orfY and the nifS-orfi operon (69).…”

Regulation of the Expression of Genes Involved in NAD De Novo Biosynthesis in Corynebacterium glutamicum

“…In contrast to the E. coli iscS-deficient mutant strain, the C. glutamicum nadS-deficient mutant strain grew as well as the wild-type strain in nicotinate-supplemented minimal medium, suggesting that NadS is specifically involved in the de novo Fe-S cluster synthesis on the nascent NadA apoenzyme. Similar results are observed in the case of a B. subtilis mutant strain deficient in a cysteine desulfurase gene homologue, although the gene product has not yet been functionally characterized in detail (31). At present, whether or not the cysteine desulfurases specific for NAD de novo biosynthesis are additionally involved in Fe-S cluster repair under oxidative stress conditions remains to be determined.…”