• Organism: Streptomyces coelicolor A3(2)
  
  Experiment type(s): TF mutant, EMSA
  Grinberg I, Shteinberg T, Gorovitz B, Aharonowitz Y, Cohen G, Borovok I. (2006) The Streptomyces NrdR transcriptional regulator is a Zn ribbon/ATP cone protein that binds to the promoter regions of class Ia and class II ribonucleotide reductase operons. J , PMID: 16950922

• Organism: Streptomyces coelicolor A3(2)
  
  Experiment type(s): TF mutant, EMSA
  Grinberg I, Shteinberg T, Hassan AQ, Aharonowitz Y, Borovok I, Cohen G. (2009) Functional analysis of the Streptomyces coelicolor NrdR ATP-cone domain: role in nucleotide binding, oligomerization, and DNA interactions. J. Bacteriol., 191: 1169-79 , PMID: 19047342

• Organism: Mycobacterium tuberculosis
  
  Experiment type(s): TF mutant, qRT-PCR, microarray, EMSA, DNase I footprinting, target gene fusion
  Maciag A, Dainese E, Rodriguez GM, Milano A, Provvedi R, Pasca MR, Smith I, Palù G, Riccardi G, Manganelli R. (2007) Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J. Bacteriol., 189: 730-40 , PMID: 17098899

• Organism: Streptomyces coelicolor A3(2)
  
  Experiment type(s): TF mutant, S1 nuclease mapping, EMSA, DNase I footprinting
  Owen GA, Pascoe B, Kallifidas D, Paget MS. (2007) Zinc-responsive regulation of alternative ribosomal protein genes in Streptomyces coelicolor involves zur and sigmaR. J. Bacteriol., 189: 4078-86 , PMID: 17400736

• Organism: Streptomyces coelicolor A3(2)
  
  Experiment type(s): TF mutant, S1 nuclease mapping, EMSA, DNase I footprinting
  Shin JH, Oh SY, Kim SJ, Roe JH. (2007) The zinc-responsive regulator Zur controls a zinc uptake system and some ribosomal proteins in Streptomyces coelicolor A3(2). J. Bacteriol., 189: 4070-7 , PMID: 17416659

• Organism: Bacillus subtilis
  
  Experiment type(s): TF mutant, target gene fusion
  Nanamiya H, Akanuma G, Natori Y, Murayama R, Kosono S, Kudo T, Kobayashi K, Ogasawara N, Park SM, Ochi K, Kawamura F. (2004) Zinc is a key factor in controlling alternation of two types of L31 protein in the Bacillus subtilis ribosome. Mol. Microbiol., 52 , PMID: 15049826

• Organism: Escherichia coli K12
  
  Experiment type(s): TF mutant, target gene fusion, microarray, in vitro transcription, DNase I footprinting
  Giel JL, Rodionov D, Liu M, Blattner FR, Kiley PJ. (2006) IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol. Microbiol., 60: 1058-75 , PMID: 16677314

• Organism: Escherichia coli K12
  
  Experiment type(s): TF mutant, S1 nuclease mapping, in vitro transcription, EMSA, DNase I footprinting
  Yeo WS, Lee JH, Lee KC, Roe JH. (2006) IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins. Mol Microbiol., 61: 206-18 , PMID: 16824106

• Organism: Escherichia coli K12
  
  Experiment type(s): target gene fusion, TF mutant, site-directed mutagenesis of TFBSs, DNA-binding fluorescence assays
  Nesbit AD, Giel JL, Rose JC, Kiley PJ. (2009) Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe-S cluster ligation. J Mol Biol., 387: 28-41 , PMID: 19361432

• Organism: Escherichia coli K12
  
  Experiment type(s): TF mutant, qRT-PCR, EMSA
  Torrents E, Grinberg I, Gorovitz-Harris B, Lundström H, Borovok I, Aharonowitz Y, Sjöberg BM, Cohen G. (2007) NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. J. Bacteriol., 189: 5012-21 , PMID: 17496099

• Organism: Yersinia pestis
  
  Experiment type(s): TF mutant, microarray, qRT-PCR, EMSA, DNase I footprinting
  Li Y, Qiu Y, Gao H, Guo Z, Han Y, Song Y, Du Z, Wang X, Zhou D, Yang R. (2009) Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis. BMC Microbiol., 9: 128 , PMID: 19552825

• Organism(s): Thermotoga maritima MSB8; Bacillus subtilis
  
  Experiment type(s): EMSA
  Rodionov DA, Li X, Rodionova IA, Yang C, Sorci L, Dervyn E, Martynowski D, Zhang H, Gelfand MS, Osterman AL. (2008) Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulon. Nucleic Acids Res., 36: 2032-46. PMID: 18276644

The previously known facts

In Bacillus subtilis, the transcription factor NiaR encoded by the yrxA gene was shown to operate as a niacin-responsive transcriptional repressor of the NAD biosynthesis operon.

Comparative genomic analysis

Using the comparison of multiple genomes of bacteria from the Bacillus/Clostridium group that have an ortholog of NiaR, a candidate DNA-binding motif of NiaR was discovered. Further comparative genome analysis was used to predict additional NiaR binding sites and to reconstruct the NiaR regulon in every species. For the Thermotogales lineage, another taxonomic group possessing an NiaR ortholog, a distinct candidate NiaR-binding motif was identified. The reconstructed NiaR regulon was predicted to control various genes involved in the NAD biosynthesis and transport. The NiaR regulon reconstruction enables identification of the novel niacin transporter NiaP.t

Experimental validation

The predicted NiaR binding sites located upstream of two operons in Bacillus subtilis and Thermotoga maritima were experimentally verified by the electrophoretic mobility shift assays, and niacin was confirmed to serve as an effector of NiaR. The predicted NiaP transporter in B. subtilis was validated to be involved in niacin uptake.

• Organism(s): Synechocystis sp. PCC 6803; Shewanella oneidensis MR-1
  
  Experiment type(s): EMSA
  Rodionov DA, De Ingeniis J, Mancini C, Cimadamore F, Zhang H, Osterman AL, Raffaelli N. (2008) Transcriptional regulation of NAD metabolism in bacteria: NrtR family of Nudix-related regulators. Nucleic Acids Res., 36: 2047-59. PMID: 18276643

• Organism(s): Shewanella oneidensis MR-1
  
  Experiment type(s):In vitro transcription, DNase I footprinting
  Huang N, De Ingeniis J, Galeazzi L, Mancini C, Korostelev YD, Rakhmaninova AB, Gelfand MS, Rodionov DA, Raffaelli N, Zhang H. (2009) Structure and function of an ADP-ribose-dependent transcriptional regulator of NAD metabolism. Structure, 17: 939-51. PMID: 19604474

The previously known facts

Nothing was known about regulation which controls the NAD metabolism in bacteria.

Comparative genomic analysis

The comparative genome context analysis revealed a novel family of Nudix-related transcription factors, termed NrtR, that are responsible for the regulation of various aspects of NAD metabolism in diverse bacterial species. A genomic reconstruction of the NrtR regulons revealed a substantial variability in NrtR binding DNA motifs and regulon composition between various taxonomic groups. Although 18 derived NrtR motifs differ substantially in consensus sequence, most of them share a 21-bp palindrome symmetry and a conserved core with consensus GT-N7-AC. Among NrtR-regulated genes in different taxonomic groups are those involved in the de novo NAD biosynthesis and salvage pathways.

Experimental validation

A specific binding to the predicted DNA motifs was experimentally confirmed for the two diverse representative NrtR proteins from Synechocystis sp. and Shewanella oneidensis by the electrophoretic mobility shift assay. In a series of tested intermediates associated with NAD metabolism, 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. The cellular pool of ADP-ribose may reflect the extent of NAD consumption by various NAD glycohydrolases, and the increased level of this metabolite may be interpreted by a cell as a signal to replenish the cofactor pool by derepression of NAD biosynthetic genes and operons.