Bacteria can trigger transcriptional and phenotypic programs to synchronize an adaptive response in reaction to environmental fluctuations or stresses (Edwards et al., 2013). This not only relies on the number of virulence factors it possesses, but also on the proper use of nutrients available in the human niches (Phillips et al., 1990; Titgemeyer and Hillen, 2002). A number of important vitamins and co-factors are required by bacteria to survive and grow successfully. Streptococcus pneumoniae, a major Gram-positive human pathogen and nasopharyngeal colonizer, encounters different environmental factors and has to fine-tune its gene expression accordingly (Bogaert et al., 2004; Kadioglu et al., 2008).

Niacin (nicotinic acid), a naturally occurring vitamin B complex, is a precursor of coenzymes NAD and NADP, and plays an important role in electron transfer during metabolic processes (Wei et al., 2014). Niacin has long been used for the treatment of lipid disorders and cardiovascular disease (Wei et al., 2014). It can regulate the activity of microbial two-component systems and, subsequently, modulate the genes and phenotypes that are controlled by these regulatory proteins (McPheat et al., 1983). Particularly, niacin has been reported to repress the expression of many genes including virulence factors in Bordetella pertussis, such as pertussis toxin, adenylate cyclase toxin, and filamentous hemagglutinin (Schneider and Parker, 1982; McPheat et al., 1983; Cotter and DiRita, 2000; Cummings et al., 2006). Furthermore, the two-component system BvgA/BvgS, which is known to have a role in the regulation of virulence and colonization, becomes inactive in B. pertussis when niacin is present in the medium (Miller et al., 1989). Similarly, the Escherichia coli EvgA/EvgS system that confers multidrug resistance and acid tolerance is regulated by niacin (Masuda and Church, 2002, 2003; Eguchi et al., 2003; Nishino et al., 2003). Both the BvgA/BvgS system of B. pertussis and the EvgA/EvgS system of E. coli are part of a family of proteins that utilize a multistep phosphor-relay to trigger their responsive pathways.

It has been proposed that in S. pneumoniae niacin enters the cell through NiaX and is converted to nicotinate (nicotinic acid)-mononucleotide by PncB (Johnson et al., 2015). Nicotinate mononucleotide is then converted to nicotinic acid adenine dinucleotide by NadD, whereafter NadE converts nicotinic acid adenine dinucleotide to nicotine adenine dinucleotide (NAD) (Johnson et al., 2015). Another important enzyme glyceraldehyde-3-phosphate dehydrogenase (GAP) is a highly conserved and a multifunctional protein with significant activity in several fundamental cell pathways (Sirover, 2011). Usually, the dehydrogenase reactions of metabolic pathways have been deemed the major sources of NADPH. Nevertheless, the importance of transhydrogenases, glucose dehydrogenases, and non-phosphorylating glyceraldehyde 3- phosphate dehydrogenase (GAPN), is becoming eminent, suggesting that the traditional view is over-simplistic (Sauer U. et al., 2004; Matsubara et al., 2011; Bräsen et al., 2014). As NAD is a vital cofactor used by all living organisms, all bacterial species make use of the pathways to reduce NAD⁺ to NADH (Jurtshuk, 1996). NAD⁺ is also used by bacteria as a substrate for dehydrogenases involved in breaking down aldehydes and alcohols (Nobelmann and Lengeler, 1996; Kotrbova-Kozak et al., 2007; Luong et al., 2015). Furthermore, several cellular processes in bacterial and mammalian cells also use NAD, for instance DNA ligation and repair, redox recycling in the pyruvate dehydrogenase pathway, and synthesis of acetyl-CoA for the tricarboxylic acid cycle (Ishino et al., 1986; Satoh and Lindahl, 1992; Wilkinson et al., 2001; Chalkiadaki and Guarente, 2012; Chiarugi et al., 2012; Patel et al., 2014).

YrxA (NiaR) was found to be a niacin-responsive repressor of NAD de novo synthesis in Bacillus subtilis and transcriptional regulation of NAD biosynthesis in bacteria having orthologs of B. subtilis yrxA was determined using a comparative genomic approach and expression studies (Rodionov et al., 2008a). NiaR family members are generally conserved in the Bacillus/Clostridium group and in the unrelated Fusobacteria and Thermotogales lineages (Rodionov et al., 2008a). The NiaR regulon is not limited to the transcriptional regulation of the nadABC but in some species it also covers niacin salvage (the pncAB genes) and contains uncharacterized membrane proteins putatively involved in niacin transport (Rodionov et al., 2008a). Moreover, members of the NiaP family (involved in niacin uptake) are not only conserved in bacteria but also in multicellular eukaryotes, including humans, suggesting the putative involvement of NiaP in niacin utilization in these organisms (Rodionov et al., 2008a).

This study explains the transcriptomic response of S. pneumoniae D39 to niacin and regulation of niaX, pnuC, and nadC genes. We established that the transcriptional regulator NiaR acts as a transcriptional repressor for niaX, pnuC, and nadC genes involved in niacin uptake and utilization. The putative operator site (5′-TACWRGTGTMTWKACASYTRWAW-3′ where R = A/G, K = G/T, S = G/C, Y = T/C, W = A/T and M = A/C) of NiaR in the promoter regions of niaX, pnuC, and nadC is predicted, and subsequently confirmed by mutating NiaR operator sites in the respective promoters.

NAD is an essential cofactor used by all living organisms. NAD synthesis is a tightly regulated intracellular process in bacteria (Huang et al., 2009). Bacteria acquire NAD in two main ways: through de novo synthesis and through the salvage pathway. Some bacteria do not have the ability to de novo synthesize NAD and must make use of the salvage pathway to import niacin or nicotinamide riboside through the substrate importers NiaX and PnuC, respectively. The de novo pathway synthesizes NAD from aspartic acid, whereas the salvage pathway brings intermediates many steps downstream into the NAD de novo synthesis pathway (Rodionov et al., 2008b). NiaX and PnuC are the two major importers in the NAD salvage pathway, where NiaX is responsible for niacin uptake, and PnuC transports nicotinamide riboside (Herbert et al., 2003; Sauer E. et al., 2004; Rodionov et al., 2008a, 2009). Our current study demonstrates the transcriptomic response of S. pneumoniae to niacin and reveals that a number of genes including pnuC, pncB, and nadC are differentially expressed under the tested conditions. We further demonstrate that a transcriptional regulator NiaR acts as a transcriptional repressor of niaX, pnuC, and nadC in the presence of niacin.

An extracellular protein capable of modifying nicotinamide mononucleotide to an importable form appears to help NiaX and PnuC for importing nicotinamide mononucleotide or there may be another import system in S. pneumoniae (Johnson et al., 2015). There is significant variability between PnuC homologs (Jaehme et al., 2014), and the PnuC homologs from Haemophilus influenzae and Salmonella typhimurium do not import nicotinamide mononucleotide, but can transform it to an importable form for PnuC (nicotinamide riboside) through NadN or AphA, respectively (Kemmer et al., 2001; Grose et al., 2005). The PnuC proteins from H. influenzae, S. typhimurium, and S. pneumoniae all possess the motif for nicotinamide mononucleotide binding. Nevertheless, PnuC homologs from many other organisms lack the consensus binding residues (Kemmer et al., 2001; Sauer E. et al., 2004; Grose et al., 2005). These observations indicate that different groups of NAD salvage substrate importers (annotated as PnuC) import nicotinamide riboside and/or nicotinamide mononucleotide, and that NiaX imports niacin and/or nicotinamide mononucleotide as preferred substrates. Moreover, the amino acids in Salmonella PnuC curtailing import of nicotinamide mononucleotide are not conserved in the pneumococci, suggesting that the pneumococcal PnuC may permit this substrate along with nicotinamide riboside. Although, both PnuC and NiaX in S. pneumoniae may have acquired the ability to import nicotinamide mononucleotide, an extra importer (that is yet to be characterized) may also be present (Johnson et al., 2015). The role of PnuC in pneumococcal pathogenesis has been studied and PnuC could be a potential viable small molecule therapeutic target to halt disease progression in the host (Johnson et al., 2015). The proposed NAD pathway in S. pneumoniae states that niacin and nicotinamide enter the cells through NiaX, and PnuC transports nicotinamide riboside to the inside of the cell, whereas the transporter for nicotinamide mononucleotide is unknown (Johnson et al., 2015). spd-1411 encodes a nicotinamidase (PncA) that converts nicotinamide into niacin, which is further converted into nicotinate mononucleotide by a nicotinic acid phosphoribosyltransferase (PncB) (Johnson et al., 2015). The nicotinate mononucleotide is then converted into NAD by NadD and NadE. Moreover, NadD (nicotinate/nicotinamide nucleotide adenylyltransferase) converts nicotinamide riboside and nicotinamide mononucleotide into NAD (Johnson et al., 2015). Nicotinamide riboside augmentation has been attributed to several advantageous functions in the host, including shielding against mitochondrial myopathy (Khan et al., 2014), hearing loss (Brown et al., 2014) and obesity (Cantó et al., 2012). These functions may not be due to increasing NAD synthesis (Frederick et al., 2015), but may be due to overall bioavailability. While nicotinamide riboside is required for pathogen and host, luckily pneumococcal PnuC and its homologous in other bacteria do not have sequence homology to any proteins in the animal kingdom. Hence, PnuC could be a potential therapeutic target in bacterial species shielding this pathway without mammalian significance as has been effectively shown with H. influenzae (Sauer E. et al., 2004).

NiaR orthologs have been found in 30 out of 45 species from the Bacillus/Clostridium group (Firmicutes), in addition to the diverged groups of the Fusobacteria and Thermotogales and for the Bacillus/Clostrida group another DNA binding site was proposed (Rodionov et al., 2008a). There are two different types of DNA-binding sites of NiaR i.e., type I operator found in Firmicutes and Fusobacteria, and type II in the Thermotogales. The niacin-responsive transcription factor NiaR (known as YrxA in B. subtilis) was first recognized as a nicotinic acid-responsive repressor of the de novo NAD biosynthesis operon (nadABC) in B. subtilis (Rossolillo et al., 2005). NiaR regulation of the niacin salvage genes pncB (in Lactobacillus plantarum), pncA (in Streptococcus pyogenes, Streptococcus equi, and Clostridium tetani), and/or the RNam salvage transporter pnuC (in S. pneumoniae and Streptococcus mutans) (Rodionov et al., 2008a) is less common. Moreover, the NiaR regulon contains membrane proteins that putatively have a role in niacin uptake. The most abundant NiaP family is found in ten NiaR-containing species (Bacilli, Lactobacilli and Thermotogales) in addition to several species that do not have the NiaR regulator (Rodionov et al., 2008a). Among Streptococci and Clostridia, NiaX is found in twelve genomes, and NiaY is found in five genomes (Bacilli and Clostridia). Several lines of genomic evidence support the putative involvement of these gene families in niacin uptake including the predicted co-regulation with NAD biosynthesis and niacin salvage genes, and co-occurrence with the niacin salvage genes pncB-pncA (Rodionov et al., 2008a). Our study demonstrates that niaX, pnuC, and nadC are the genes that have a putative NiaR operator site in their promoter regions and are repressed by NiaR in the presence of niacin. We have further confirmed the NiaR operator sites in the promoter regions of niaX, pnuC, and nadC by mutagenesis studies. There are some other genes that are differentially expressed under our tested conditions (fba, rex, gapN, pncB-nadE, gap, spd-1824, spd-1827, adhE, and adhB2). The change in the expression of these genes suggests that these genes may have a role in the transport and biosynthesis of niacin or they may be upregulated due to some indirect effect of niacin genes. These genes do not have a putative NiaR operator site in their promoter regions suggesting the role of another transcriptional regulator in the regulation of fba, rex, gapN, pncB-nadE, gap, spd-1824, spd-1827, adhE, and adhB2. Therefore, we propose that the study of the regulatory mode of the above-mentioned genes would shed light on this possibility.

Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work: MA, SS, and OK. Drafting the work or revising it critically for important intellectual content: MA, SS, and OK. Final approval of the version to be published: MA, SS, and OK. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: MA, SS, and OK.