NAD Metabolism Research Articles

Cryptococcus neoformansandCryptococcus gattiigenes preferentially expressed during rat macrophage infection

Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeast agents of cryptococcosis and facultative intracellular pathogens. The interaction of these yeasts with macrophages is essential for containing the infection. However, Cryptococcus spp. overcome this initial host defense barrier using a unique pathogenic strategy involving intracellular replication and cytoplasmic accumulation of polysaccharide-containing vesicles. Here, we employed representational difference analysis (RDA) to identify C. neoformans and C. gattii genes differentially expressed during intracellular growth in rat peritoneal macrophages. The upregulated transcripts of C. neoformans during macrophage interaction were related to ATP-binding cassette (ABC) transporters, intra-golgi transport, chaperone activity, ribosomal maintenance, NAD metabolism, histone methylation, stress response, and monosaccharide metabolism. In contrast, with C. gattii, upregulated genes were associated with cell growth, aerobic respiration, protein binding, microtubule nucleation, monosaccharides and nitrogen metabolism, inositol or phosphatidylinositol phosphatase activity, cellular signaling, and stress response. Our findings reveal new genes that may be necessary for the intracellular parasitism of C. neoformans and C. gattii.

Abstract 3673: Identifying the target of an orphan compound: CB30865 is a Nampt inhibitor

Abstract Background: Drug discovery based on cellular phenotypes is impeded by the challenge of identifying the molecular target. To alleviate this problem, Myriad Pharmaceuticals has developed a chemical proteomics process to identify cellular proteins that bind to small molecules. CB30865 is a potent and selective cytotoxic compound of previously unknown mechanism of action (1). Using our technology, we have unequivocally demonstrated that CB30865 cytotoxicity is due to inhibition of nicotinamide phosphoribosyltransferase (Nampt). Nampt catalyzes the first step in the synthesis of NAD from nicotinamide. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases. Materials and Methods: Direct target affinity purification (DTAP) was performed using analogs of CB30865 (MPI-0479883, MPI-0482594) coupled to beads. Purified proteins were identified by nano-flow reversed-phase liquid chromatography and tandem mass spectrometry using an LTQ-Orbitrap. In vitro Nampt activity was measured in a coupled biochemical assay based on the production of fluorescent resorufin. Nampt cellular activity was assayed by measuring levels of NAD and NAD-dependent polyADP-ribosylation. Results: As reported (1), 3-pyridyl, but not 2-pyridyl, analogs of CB30865 were potently cytotoxic. A 55 kD protein was purified efficiently using DTAP with MPI-0479883 (a 3-pyridyl CB30856 analog) but not MPI-0482594 (a 2-pyridyl analog). Preincubation of cellular lysates with uncoupled 3-pyridyl but not 2-pyridyl analogs potently blocked binding of the 55 kD protein. Based on LC-MS/MS analyses, the 55 kD protein was identified as Nampt. Immunoblotting of DTAPs using a Nampt specific antibody confirmed the MS identification. Furthermore, biochemical and cellular assays of Nampt activity demonstrated pyridyl-isomer specific enzyme inhibition. Finally, cellular Nampt inhibition and cytotoxicity were completely rescued by treating cells with 10 µM nicotinic acid as an alternate NAD source. Conclusions: The chemical proteomics DTAP approach is a powerful tool to identify molecular targets of orphan compounds. We demonstrate this here through identification of Nampt as the molecular target of CB30865 and further implicate NAD metabolism as a functional target for cancer. (1) Skelton LA, Ormerod MG, Titley J, Kimbell R, Brunton LA, Jackman AL. A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus. Br J Cancer. 1999 Apr;79(11-12):1692-701. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3673.

Metabolomics and Gene Discoveries Reveal Interrelationships between Glucose, Phosphate and NAD Metabolism

Early models that sought to explain how calorie restriction may alter NAD metabolism were formulated prior to the identification of all of the NAD metabolites and the enzymes that interconvert these metabolites. We have recently identified nicotinamide riboside and nicotinic acid riboside as metabolites and have identified roles for nicotinamide riboside kinase, uridine hydrolase, purine nucleoside phosphorylase, and two multifunctional 5′‐nucleotidases in yeast NAD metabolism. A metabolomic method has quantified the yeast NAD metabolome. Data from yeast cells whose lifespan is extended with vitamin provision or glucose restriction indicate that, while increasing net NAD synthesis is a mechanism for lifespan extension, it is not the mechanism that applies for glucose restriction, nor is a change in the NAD:nicotinamide or NAD:NADH ratio. Some of the same pathways that respond to glucose restriction are also regulated by phosphate restriction and the availability of vitamins. Thus, the calorie restricted metabolome appears to involve a large number of small changes that effect multiple targets rather than a small number of large changes that exclusively target sirtuins.

Genetic Differences between Five European Populations

Aims: We sought to examine the magnitude of the differences in SNP allele frequencies between five European populations (Scotland, Ireland, Sweden, Bulgaria and Portugal) and to identify the loci with the greatest differences. Methods: We performed a population-based genome-wide association analysis with Affymetrix 6.0 and 5.0 arrays. We used a 4 degrees of freedom χ² test to determine the magnitude of stratification for each SNP. We then examined the genes within the most stratified regions, using a highly conservative cutoff of p < 10^–45. Results: We found 40,593 SNPs which are genome-wide significantly (p ≤ 10^–8) stratified between these populations. The largest differences clustered in gene ontology categories for immunity and pigmentation. Some of the top loci span genes that have already been reported as highly stratified: genes for hair color and pigmentation (HERC2, EXOC2, IRF4), the LCT gene, genes involved in NAD metabolism, and in immunity (HLA and the Toll-like receptor genes TLR10, TLR1, TLR6). However, several genes have not previously been reported as stratified within European populations, indicating that they might also have provided selective advantages: several zinc finger genes, two genes involved in glutathione synthesis or function, and most intriguingly, FOXP2, implicated in speech development. Conclusion: Our analysis demonstrates that many SNPs show genome-wide significant differences within European populations and the magnitude of the differences correlate with the geographical distance. At least some of these differences are due to the selective advantage of polymorphisms within these loci.

Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5′-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD(+), which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD(+) metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5'-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5'-nucleotidase, and Sdt1, initially classified as a pyrimidine 5'-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD(+). Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD(+) metabolism.

Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation.

Poly-ADP-ribose polymerases (PARPs) use NAD(+) as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD(+) detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD(+) pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD(+) and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.

Microbial NAD metabolism: lessons from comparative genomics.

NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways.

Assimilation of Endogenous Nicotinamide Riboside Is Essential for Calorie Restriction-mediated Life Span Extension in Saccharomyces cerevisiae

NAD(+) (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD(+) metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD(+) metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD(+)-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD(+) metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD(+) precursors. Together, our studies provide a molecular basis for how NAD(+) homeostasis factors confer metabolic flexibility.

Role of ADP-Ribosyl Cyclase in the Pathogenesis of Neurological Disorders after Coronary Artery Bypass Surgery and Experimental Ischemia

The pathogenesis of neuronal dysfunction was evaluated from the viewpoint of cellular disturbances in NAD(+) metabolism and changes in activity of NAD(+)-utilizing enzymes (e.g., ADP-ribosyl cyclase/CD38). S-100B concentration and CD38 expression on peripheral blood lymphocytes were altered in patients after surgery for coronary heart disease with extracorporeal circulation. These changes in patients during the early postoperative period correlated with variations in CD38 expression on neuronal cells from postischemic rats with cognitive dysfunction.

Nampt (visfatin) associations with insulin metabolism are secondary to strong associations with BMI in children

Rationale: Nampt, also named visfatin, is an important enzyme in cellular NAD metabolism. Besides expression in adipose tissue, Nampt circulates in human serum and was hypothesized to be involved in the regulation of insulin secretion. There are only sparse data on Nampt association with obesity or insulin metabolism in humans applying valid assay systems. In this study we evaluated Nampt levels in normal lean children and adolescents compared to obese children and the relationship with metabolic and cardiovascular comorbidities. Results: In a cohort of n=138 healthy lean children, Nampt levels were not different between boys and girls matched for pubertal stage and there was also no dependency on pubertal development. Nampt levels were, however, significantly elevated in obese children compared to lean children (3.85±0.28, n=72 vs. 2.95±0.30ng/mL, n=69, P=0.029) and correlated with BMI SDS (r=0.35, P<0.0001) and other parameters of obesity in a case control study. We also identified significant associations with parameters of glucose (120min BG r=0.23, P=0.006) and insulin metabolism (Matsuda ISI r=-0.23, P=0.007) in univariate analyses that were, however, mainly attributed to underlying associations with BMI. Similarly, Nampt associations with endothelial function (RHI r=-0.25, P=0.003) or blood pressure (24h mean systolic BP: r=0.23, P=0.022) were abolished after correcting for BMI. The association with circulating endothelial progenitor cells was, however, robust after correction for BMI (rcorr=-0.26, Pcorr=0.01). Nampt levels significantly declined in response to a glucose load in an oral glucose tolerance test in obese children (n=28). Time after glucose challenge (b=-0.25, P=0.003) and insulin levels (b=-0.23 P=0.042) were significant predictors of Nampt in multiple regression analyses (r2=0.093, P=0.004), but not blood glucose. Conclusions: Nampt levels were strongly related with obesity in children in three independent groups of children and adolescents. The association with insulin resistance in obese subjects was secondary to the underlying association with obesity.

Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner

Tumor necrosis factor (TNF) synthesis is known to play a major part in numerous inflammatory disorders, and multiple transcriptional and post-transcriptional regulatory mechanisms have therefore evolved to dampen the production of this key proinflammatory cytokine. The high expression of nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the nicotinamide-dependent NAD biosynthetic pathway, in cells of the immune system has led us to examine the potential relationship between NAD metabolism and inflammation. We show here that intracellular NAD concentration promotes TNF synthesis by activated immune cells. Using a positive screen, we have identified Sirt6, a member of the sirtuin family, as the NAD-dependent enzyme able to regulate TNF production by acting at a post-transcriptional step. These studies reveal a previously undescribed relationship between metabolism and the inflammatory response and identify Sirt6 and the nicotinamide-dependent NAD biosynthetic pathway as novel candidates for immunointervention in an inflammatory setting.

Ectoenzymes and innate immunity: the role of human CD157 in leukocyte trafficking

CD157 is a glycosylphosphatidylinositol-anchored molecule encoded by a member of the CD38/ADP-ribosyl cyclase gene family, involved in the metabolism of NAD. Expressed mainly by cells of the myeloid lineage and by vascular endothelial cells, CD157 has a dual nature behaving both as an ectoenzyme and as a receptor. Although it lacks a cytoplasmic domain, and cannot transduce signals on its own, the molecule compensates for this structural limit by interacting with conventional receptors. Recent experimental evidence suggests that CD157 orchestrates critical functions of human neutrophils. Indeed, CD157-mediated signals promote cell polarization, regulate chemotaxis induced through the high affinity fMLP receptor and control transendothelial migration.

Nicotinamide Phosphoribosyltransferase (Nampt): A Link Between NAD Biology, Metabolism, and Diseases

New interest in NAD biology has recently been revived, and enzymes involved in NAD biosynthetic pathways have been identified and characterized in mammals. Among them, nicotinamide phosphoribosyltransferase (Nampt) has drawn much attention in several different fields, including NAD biology, metabolism, and immunomodulatory response. The research history of this protein is peculiar and controversial, and its physiological function has been a matter of debate. Nampt has both intra- and extracellular forms in mammals. Intracellular Nampt (iNampt) is an essential enzyme in the NAD biosynthetic pathway starting from nicotinamide. On the other hand, an extracellular form of this protein has been reported to act as a cytokine named PBEF, an insulin-mimetic hormone named visfatin, or an extracellular NAD biosynthetic enzyme named eNampt. This review article summarizes the research history and reported functions of this unique protein and discusses the pathophysiological significance of Nampt as an NAD biosynthetic enzyme vs. a potential inflammatory cytokine in diverse biological contexts.

The relationship between pyridine nucleotides and seed dormancy

To investigate the proposed role for NAD metabolism in regulating seed dormancy, NAD metabolites and associated enzyme activities were analysed in seed of Arabidopsis thaliana ecotypes ranging from Col-0, which has low seed dormancy, to Cvi, which is highly dormant. Seed poly(ADP-ribosyl)ation levels did not correlate well with the depth of seed dormancy but did correlate with the sensitivity of germination to the DNA damaging agent MMS. Cvi seed had relatively high NAD and low NADP levels compared with the less dormant ecotypes and the NAD : NADP ratios correlated well with dormancy. The activity of NAD kinase was relatively low, and NADP phosphatase was relatively high in dormant Cvi seed, indicating that these enzymes may be involved in controlling the NAD : NADP ratio. Dormant fresh Cvi and nondormant after-ripened Cvi seeds were used to investigate further. Measurement of reduced and oxidised pyridine nucleotides indicated that breaking of dormancy was associated with a reduction in NAD levels but not with an increase in NADP levels. It is proposed that poly(ADP-ribose) polymerase is involved in protecting the seed from genotoxic stress, whereas the level of NAD affects the depth of dormancy, perhaps by enhancing abscisic acid (ABA) synthesis.

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

NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low-GC Gram-positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe-4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe-4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species-specific between B. subtilis and Escherichia coli.

Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase/Visfatin Enzymatic Activity Identifies a New Inflammatory Pathway Linked to NAD

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Analysis of DBC1 and its homologs suggests a potential mechanism for regulation of Sirtuin domain deacetylases by NAD metabolites

Deleted in Breast Cancer-1 (DBC1) and its paralog CARP-1 are large multi-domain proteins, with a nuclear or perinuclear localization, and a role in promoting apoptosis upon processing by caspases. Recentstudies on human DBC1 show that it is a specific inhibitor of the sirtuin-type deacetylase, Sirt1, which deacetylates histones and p53. Using sensitive sequence profile searches and HMM-HMM comparisons weshow that the central conserved globular domain present in the DBC1 and it homologs from diverse eukaryotes is a catalytically inactive version of the Nudix hydrolase (MutT) domain. Given that Nudixdomains are known to bind nucleoside diphosphate sugars and NAD, we predict that this domain in DBC1 and its homologs binds NAD metabolites such as ADP-ribose. Hence, we propose that DBC1 and itshomologs are likely to regulate the activity of SIRT1 or related deacetylases by sensing the soluble products or substrates of the NAD-dependent deacetylation reaction. The complex domain architectures of the members of the DBC1 family, which include fusions to the RNA-binding S1-like domain, the DNA binding SAP domain and EF-hand domains, suggest that they are likely to function as integrators of distinct regulatory signals including chromatin protein modification, soluble compounds in NAD metabolism, apoptotic stimuli and RNA recognition.

PBEF1/NAmPRTase/Visfatin: A potential malignant astrocytoma/glioblastoma serum marker with prognostic value

Malignant astrocytomas comprise anaplastic astrocytoma (AA; grade III) and Glioblastoma (GBM; grade IV). GBM is the most malignant with a median survival of 10-12 months in patients. Using cDNA microarray based expression profiling of different grades of astrocytomas, we identified several fold increased levels of PBEF1 transcripts in GBM samples. Pre-B-cell colony enhancing factor 1 gene (PBEF1) encodes Nicotinamide phosphoribosyltransferase (NAmPRTase), which catalyses the rate limiting step in the salvage pathway of NAD metabolism in mammalian cells. Further validation using real time RT-qPCR on an independent set of tumor samples (n=91) and normal brain samples (n=9), GBM specific higher expression of PBEF1 was confirmed. Immunohistochemical staining for PBEF1 on a subset of the above samples largely reinforced our finding. We carried out ELISA analysis on serum samples of astrocytoma patients to determine whether this protein levels would correlate with the presence of tumor and tumor grade. PBEF1 serum levels were substantially elevated in many of the AA and GBM patients. Statistical analysis of these data indicates that in patients with astrocytoma, serum PBEF1 levels correlate with tumor grade and is highest in GBM. Immunohistochemical analysis of an independent set of 51 retrospective GBM cases with known survival data revealed that PBEF1 expression in the tumor tissue along with its co-expression with p53 was associated with poor survival. Thus, we have identified PBEF1 as a potential malignant astrocytoma serum marker and prognostic indicator among GBMs.

Transcriptional regulation of NAD metabolism in bacteria: NrtR family of Nudix-related regulators

A novel family of transcription factors responsible for regulation of various aspects of NAD synthesis in a broad range of bacteria was identified by comparative genomics approach. Regulators of this family (here termed NrtR for Nudix-related transcriptional regulators), currently annotated as ADP-ribose pyrophosphatases from the Nudix family, are composed of an N-terminal Nudix-like effector domain and a C-terminal DNA-binding HTH-like domain. NrtR regulons were reconstructed in diverse bacterial genomes by identification and comparative analysis of NrtR-binding sites upstream of genes involved in NAD biosynthetic pathways. The candidate NrtR-binding DNA motifs showed significant variability between microbial lineages, although the common consensus sequence could be traced for most of them. Bioinformatics predictions were experimentally validated by gel mobility shift assays for two NrtR family representatives. ADP-ribose, the product of glycohydrolytic cleavage of NAD, was found to suppress the in vitro binding of NrtR proteins to their DNA target sites. In addition to a major role in the direct regulation of NAD homeostasis, some members of NrtR family appear to have been recruited for the regulation of other metabolic pathways, including sugar pentoses utilization and biogenesis of phosphoribosyl pyrophosphate. This work and the accompanying study of NiaR regulon demonstrate significant variability of regulatory strategies for control of NAD metabolic pathway in bacteria.

Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulon

A comparative genomic approach was used to reconstruct transcriptional regulation of NAD biosynthesis in bacteria containing orthologs of Bacillus subtilis gene yrxA, a previously identified niacin-responsive repressor of NAD de novo synthesis. Members of YrxA family (re-named here NiaR) are broadly conserved in the Bacillus/Clostridium group and in the deeply branching Fusobacteria and Thermotogales lineages. We analyzed upstream regions of genes associated with NAD biosynthesis to identify candidate NiaR-binding DNA motifs and assess the NiaR regulon content in these species. Representatives of the two distinct types of candidate NiaR-binding sites, characteristic of the Firmicutes and Thermotogales, were verified by an electrophoretic mobility shift assay. In addition to transcriptional control of the nadABC genes, the NiaR regulon in some species extends to niacin salvage (the pncAB genes) and includes uncharacterized membrane proteins possibly involved in niacin transport. The involvement in niacin uptake proposed for one of these proteins (re-named NiaP), encoded by the B. subtilis gene yceI, was experimentally verified. In addition to bacteria, members of the NiaP family are conserved in multicellular eukaryotes, including human, pointing to possible NaiP involvement in niacin utilization in these organisms. Overall, the analysis of the NiaR and NrtR regulons (described in the accompanying paper) revealed mechanisms of transcriptional regulation of NAD metabolism in nearly a hundred diverse bacteria.