The comparative genomic approach was applied to characterize a novel NiaR regulon for transcriptional control of NAD metabolic genes in bacteria. The niacin-responsive transcription factor NiaR (YrxA) was originally identified and characterized as a nicotinic acid-responsive repressor of the de novo
NAD biosynthesis operon in B. subtilis
). NiaR orthologs were found in 30 out of 45 species from the Bacillus/Clostridium
group (Firmicutes), as well as in the deeply diverged groups, the Fusobacteria and Thermotogales (Supplementary Table S1). Two distinct types of DNA-binding sites of NiaR were identified, type I operator characteristic of the Firmicutes and Fusobacteria, and type II in the Thermotogales (A and C). Despite the apparent overall dissimilarity, both types of operators share palindromic signature sequences, TGT and ACA, albeit in opposite orientation (A and C). Comparison of closely related species showed high conservation of candidate NiaR-binding sites in the promoter regions of target genes (Supplementary Figure S3).
To validate the predicted NiaR-binding sites of both types, the NiaR proteins from two organisms, B. subtilis
NiaR) and T. maritima
NiaR), were cloned, purified, and assessed in EMSA assays for their ability to bind DNA fragments containing their target DNA sites. The results of these experiments () confirmed both the second type I operator site predicted in B. subtilis
and the predicted DNA sites of type II in T. maritima
. They also confirmed that, as previously shown for bs
), NA is a likely effector (corepressor) for the phylogenetically divergent tm
NiaR. Comparative 3D structural studies are underway to assess mechanistic details of the interactions with DNA and the effector in these two distant representatives of the NiaR family.
A combination of comparative genomic techniques, including positional gene clustering, phylogenetic footprinting of regulatory sites and metabolic reconstruction, helped us establish the details of the NiaR regulon organization in 35 bacterial species (A and ). In 18 genomes including species from the Firmicutes, Fusobacteria and Thermotogales groups, NiaR regulates de novo biosynthesis operon nadABC. Less frequent is the NiaR regulation of the niacin salvage genes pncB (in Lactobacillus plantarum) and/or pncA (in S. pyogenes, Streptococcus equi, and C. tetani) and the RNam salvage transporter pnuC (in S. pneumoniae and Streptococcus mutans). In addition, the NiaR regulon includes three previously uncharacterized gene families encoding membrane proteins of unknown function, to which we here tentatively assigned a role in niacin uptake. Of those, the most abundant NiaP family is present in 10 NiaR-containing species (Bacilli, Lactobacilli and Thermotogales) as well as in a number of species that do not contain the NiaR regulator. Among two other families, NiaX is present in twelve genomes (Streptococci and Clostridia), and NiaY is present in five genomes (Bacilli and Clostridia). The proposed involvement of these gene families in niacin uptake is supported by several lines of genomic evidence: (i) predicted coregulation with NAD biosynthesis and niacin salvage genes; (ii) clustering on the chromosome with the niaR genes; (iii) nonoverlapping (complementary) phyletic patterns of the genome occurrence; (iv) regulation by NiaR in species lacking the nadABC genes and fully dependent on niacin salvage for NAD synthesis; and (v) co-occurrence with the niacin salvage genes pncB-pncA ().
Although niacin uptake has been studied in many species, both bacterial, such as E. coli
) and E. faecalis
), and eukaryotic, such as yeasts (47
) and mammals (53
), the knowledge of the respective transport systems and mechanisms is quite limited. The only NA-specific permease was identified in yeast (47
), and the absence of its orthologs in other lineages suggests that other niacin transporters are yet to be identified.
The predicted niacin transporter NiaP, a member of the NiaR regulon in Firmicutes and Thermotogales, belongs to other NAD-related regulons in other bacterial species (B). Particularly, in two Actinobacteria and two Lactobacilli, NiaP is a member of the NrtR regulon, which is described in the accompanying paper (50
). Furthermore, an NiaP ortholog detected in a enterobacterium Proteus mirabilis
is presumably regulated by binding of the NadR repressor to its characteristic operator site (TGTTTAGTATACTAAACA) in the upstream region of the respective gene. The NiaP transporters belong to the MFS superfamily of transporters that catalyze uniport, solute:cation symport and solute:cation antiport of a great variety of metabolites (55
). NiaP is most similar to the aromatic acid/H+
symporters PcaK from Pseudomonas
spp. and BenK from Acinetobacter
spp. involved in 4-hydroxybenzoate and benzoate uptake, respectively (48
We selected a representative of the NiaP family encoded by the yceI gene in B. subtilis for the experimental testing of its proposed involvement in niacin uptake. Three types of growth experiments were performed to test for: (i) the growth acceleration of E. coli (nadA−) mutant strain in minimal medium supplemented with limiting concentrations of Nam, as a result of overexpression of the B. subtilis gene yceI; (ii) the increased sensitivity of E. coli (nadA+) to the growth suppression by a toxic analog of Nam (6ANam), as a result of overexpression of the B. subtilis gene yceI and (iii) the increased resistance of the B. subtilis (yceI−) mutant to growth suppression by toxic niacin analogs (6ANam and 6ANA). The results of these experiments ( and ) allowed us to conclude that bsNiaP is involved in niacin transport in B. subtilis, although there exist additional, unknown mechanism for niacin uptake. It is important to emphasize that although the performed experiments provided sufficient support for the predicted functional assignment of the NiaP family, additional studies are necessary to establish its actual mechanism, energy dependence and kinetic parameters, as well as to elucidate the mechanism of NiaP-independent niacin uptake.
High-scoring homologs of NiaP are widely distributed among Gram-positive and Gram-negative bacteria (Supplementary Table S1), and they are also present in eukaryotic genomes including nematodes, insects and mammals showing substantial (up to 30%) sequence identity with their bacterial counterparts (Supplementary Figure S4). Although the mechanism of niacin transport was characterized in human intestinal epithelial and liver cells, a gene encoding the respective transporter is yet unknown. At the same time, the human homolog of NiaP, a synaptic vesicle 2-related protein (SVOP) expressed in brain and endocrine cells, was extensively studied but its physiological and molecular function remained obscure (56
). It is tempting to speculate that human SVOP and its orthologs in other mutlicellular eukaryotes constitute a plausible candidate for a missing niacin transporter in these organisms. This hypothesis, however farfetched it may seem due to the evolutionary distance and well-anticipated differences between bacterial and eukaryotic membrane transport systems, is supported by a precedent in the field of vitamin transport. Indeed, the human homolog of the bacterial pantothenate (vitamin B5
) transporter of the Solute:Sodium Symporter family (e.g. PanF in E. coli
) was implicated in uptake of B5
(and possibly some other vitamins) in human cells (57
This work and the accompanying study (50
) demonstrated a significant variability of regulatory strategies for the transcriptional control of NAD metabolism in different bacterial species (see Supplementary Table S1 that summarizes the distribution of NAD metabolic and regulatory genes across genomes). This variability parallels substantial variations in the topology of NAD biosynthetic pathways [as illustrated in the ‘NAD and NADP biosynthesis’ subsystem provided by the SEED database (http://theseed.uchicago.edu/
) and briefly discussed in (58
)] and, more importantly, a variety of bacterial habitats and lifestyles. Although the NiaR regulatory mechanism bears some analogy with NadR regulation previously studied in Enterobacteria (e.g. suppression of the de novo
biosynthesis enhanced by interactions with a corepressor), fundamental differences between these two regulatory strategies are apparent. Whereas NadR blocks all biosynthetic routes to NAD as long as NAD concentration is above threshold (NAD operates is an effector of NadR), NiaR tends to switch the organism from the de novo
biosynthetic route to niacin salvage as long as niacin is available at the level sufficient to operate as an NiaR corepressor. On the other hand, niacin may also regulate its own uptake to maintain NAD homeostasis at the desired level as the genes of the downstream biosynthetic machinery (NadD and NadE) appear to be constitutively expressed, or at least not regulated by NiaR. Regulation of the niacin salvage enzymes (PncA and PncB) by NiaR is relatively rare, and happens only in species that lack a de novo
biosynthetic route. Both NadR and NiaR regulatory strategies are dramatically different from the NrtR regulatory network described in the accompanying paper (50
). The latter regulon includes the entire NAD biosynthetic machinery, including the downstream enzymes. The derepression appears to be triggered by the accumulation of NAD degradation products (e.g. ADP ribose that operates as NrtR antirepressor) likely interpreted by the cell as depletion of the NAD pool which thus needs to be replenished.
In summary, this study provided a comprehensive bioinformatic analysis of the NiaR regulon that constitutes a system of transcriptional regulation of NAD synthesis in several groups of Gram-positive bacteria. The key conjectures delivered by this analysis, two types of NiaR-binding DNA motifs and one of the three predicted families of niacin transporters, were experimentally validated providing additional support for these specific findings as well as for the reconstruction of the entire NiaR regulon. Despite the implications of this and the accompanying study (50
), the mechanisms and the specific genes involved in the regulation of NAD synthesis as well as in the uptake of its precursors in many groups of bacteria, remain to be elucidated.