Published online 14 February 2008 Nucleic Acids Research 2008 Vol 36 No 6 2047ndash2059doi101093nargkn047

Transcriptional regulation of NAD metabolism inbacteria NrtR family of Nudix-related regulatorsDmitry A Rodionov12 Jessica De Ingeniis3 Chiara Mancini3

Flavio Cimadamore13 Hong Zhang4 Andrei L Osterman15 and Nadia Raffaelli3

1Burnham Institute for Medical Research La Jolla CA 92037 USA 2Institute for Information TransmissionProblems Russian Academy of Sciences 127994 Moscow Russia 3Istituto di Biotecnologie BiochimicheUniversita Politecnica delle Marche 60131 Ancona Italy 4Department of Biochemistry University of TexasSouthwestern Medical Center Dallas TX 75390 and 5Fellowship for Interpretation of Genomes Burr RidgeIL 60527 USA

Received December 5 2007 Revised January 8 2008 Accepted January 24 2008

ABSTRACT

A novel family of transcription factors responsiblefor regulation of various aspects of NAD synthesis ina broad range of bacteria was identified by com-parative genomics approach Regulators of thisfamily (here termed NrtR for Nudix-related tran-scriptional regulators) currently annotated as ADP-ribose pyrophosphatases from the Nudix family arecomposed of an N-terminal Nudix-like effectordomain and a C-terminal DNA-binding HTH-likedomain NrtR regulons were reconstructed indiverse bacterial genomes by identification andcomparative analysis of NrtR-binding sitesupstream of genes involved in NAD biosyntheticpathways The candidate NrtR-binding DNA motifsshowed significant variability between microbiallineages although the common consensussequence could be traced for most of themBioinformatics predictions were experimentally vali-dated by gel mobility shift assays for two NrtR familyrepresentatives ADP-ribose the product of glyco-hydrolytic cleavage of NAD was found to suppressthe in vitro binding of NrtR proteins to their DNAtarget sites In addition to a major role in the directregulation of NAD homeostasis some members ofNrtR family appear to have been recruited for theregulation of other metabolic pathways includingsugar pentoses utilization and biogenesis of phos-phoribosyl pyrophosphate This work and theaccompanying study of NiaR regulon demonstratesignificant variability of regulatory strategies forcontrol of NAD metabolic pathway in bacteria

INTRODUCTION

NAD cofactor in addition to its role in innumerableredox reactions is utilized in many metabolic andregulatory processes as a consumable co-substrate (1)Among NAD-consuming enzymes are histoneproteindeacetylase (2) bacterial DNA ligase (3) and a variety ofADP-ribosyltransferases (4) Maintaining homeostasis ofNAD cofactor pool via regulation of biosynthetic andrecycling pathways in a variety of growth conditionsappears to be of paramount importance Whereas mostbiochemical pathways related to NAD metabolism werestudied in detail [for reviews see (5ndash7)] our currentknowledge of respective regulatory mechanisms is ratherlimited Thus prior to this study only two types ofbacterial transcriptional regulators related to NADmetabolism have been identified in a limited set ofbacterial species (see subsequently) This prompted us tosearch for new candidate transcriptional factors andregulons associated with NAD metabolism in otherbacteria using the comparative genomics approach [asrecently reviewed in (8)]A schematic representation of the key pathways of NAD

biogenesis in bacteria including de novo biosynthesisfrom aspartate and various salvage pathways from theexogenous precursorsmdashnicotinamide (Nam) nicotinicacid (NA) and ribosyl nicotinamide (RNam)mdashis providedin Figure 1 and described in more details in the accom-panying paper (9) Different combinations of these meta-bolic routes result in a substantial diversity of the NADbiosynthetic machinery in various species Using a sub-system-based approach to comparative genome anal-ysis implemented in the SEED genomic platform (10)multiple versions of NAD metabolism were mapped inhundreds of completely sequenced bacterial genomes[as captured in the lsquoNAD regulationrsquo subsystem at

To whom correspondence should be addressed Tel +39 071 2204 682 Fax +39 071 2204 677 Email nraffaelliunivpmitCorrespondence may also be addressed to Dmitry A Rodionov Tel +1 858 646 31000 Fax +1 858 795 5249 Email rodionovburnhamorg

2008 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicenses

by-nc20uk) which permits unrestricted non-commercial use distribution and reproduction in any medium provided the original work is properly cited

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httptheseeduchicagoeduFIGsubsyscgi and brieflyoverviewed in (11)]The first transcriptional regulatory function for NAD

synthesis was originally linked to the nadR (nadI) gene ofSalmonella and Escherichia coli (12ndash14) prior to identifica-tion of the two mentioned enzymatic activities of thismultifunctional protein The repressor function of NadR(hence the name) is provided by an N-terminal helix-turn-helix (HTH) domain which is present only in enterobac-terial members of the NadR family The NadR dimer incomplex with theNAD co-repressor binds to a palindromic18-bp operator with consensus sequence TGTTTA-N6-TAAACA in the promoter region of genes involved in

de novo NAD biosynthesis and salvage pathways (1516)NadR provides an interesting example of a new transcrip-tional regulator emerging via fusion of a DNA-bindingdomain with a metabolic enzyme In contrast to otherknown examples of this evolutionary scenario [eg mem-bers of the ROK family (17)] the enzymatic domains ofNadR remain functionally active A recent comparativegenomic analysis of HTH-containing members of NadRfamily and corresponding regulons confirmed that theiroccurrence is restricted to a compact phylogenetic group ofEnterobacteria (18)

The second structurally and mechanistically distincttranscriptional regulator of NAD synthesis was recently

Figure 1 Overview of NAD biosynthesis and salvage pathways and a link with other metabolic pathways via ADP-ribose NrtR-controlled steps areindicated by a red asterisk Metabolic enzymes and uptake transporters are shown by solid and dashed lines respectively and colored by a metabolicpathway De novo NAD biosynthesis pathway utilizes L-aspartate oxydase (the product of nadB gene) quinolinate synthase (nadA) and quinolinatephosphoribosyltransferase (nadC) Universal NaMN to NAD pathway utilizes nicotinate mononucleotide adenylyltransferase (nadD) and NADsynthetase (nadE) In Nam salvage pathways NaMN is synthesized from NA and Nam precursors that are taken up by niacin transporter (niaP)Salvage I pathway involves nicotinamide deaminase (pncA) and nicotinate phosphoribosyltransferase (pncB) Salvage II pathway uses nicotinamidephosphoribosyltransferase (nadV) and nicotinamide mononucleotide adenylyltransferase (nadMAT) (3141) In the third salvage pathway NAD issynthesized from the exogenous RNam precursor delivered by the RNam transporter (pnuC) via consecutive reactions catalyzed by two separatedomains of NadR nicotinamide mononucleotide adenylyltransferase (nadRAT) and nicotinamide riboside kinase (nadRK)(4748) Endogenous Namand ADP-ribose are generated by enzymes hydrolyzing the N-glycosidic bond of NAD Enzymes linked to NAD metabolism via ADP-ribose areribose phosphate pyrophosphokinase (prs) ribose phosphate isomerase (rpi) ribulose phosphate epimerase (rpe) transketolase (tkt) transaldolase(tal) as well as xylose (xylAB) and arabinose (araBAD) utilization enzymes Asp aspartate Trp tryptophan IA iminoaspartate Qa quinolinicacid NaMN nicotinate mononucleotide NaAD nicotinate adenine dinucleotide NA nicotinic acid Nam nicotinamide RNam ribosylnicotinamide NMN nicotinamide mononucleotide ADPR ADP-ribose PRPP phosphoribosyl pyrophosphate Rib-P ribose-5-phosphate Xyl-Pxylulose-5-phosphate Ara L-arabinose Xyl D-xylose

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discovered and characterized in Bacillus subtilis (19) andstudied in more details in the accompanying paper (9) TheB subtilis niacin-responsive DNA-binding regulator YrxA(tentatively re-named to NiaR) represses transcription ofthe de novo biosynthesis operon nadABC and the niacintransporter niaP (formerly yceI) by direct binding tooperator sites in the promoter regions of target genes(919) The comparative genomic reconstruction of ortho-logous NiaR regulons confirmed conservation of thisregulon in bacteria from the BacillusClostridium groupand in the deep-branched group of Thermotogales (9)However the mechanism of transcriptional regulation ofthe NAD metabolism in many other bacterial lineages stillremained unknown

In this study we expanded the use of comparativegenomic techniques toward a prediction of a noveltranscription regulator of NAD metabolism and de novoreconstruction of respective regulons in many diversebacterial lineages beyond those containing NadR or NiaRregulators The analysis of conserved NAD biosyntheticoperons led to a tentative identification of a previouslyuncharacterized family of Nudix-related transcriptionalregulators (termed here NrtR) that are composed of anN-terminal domain homologous to ADPR pyrophospha-tase of the Nudix family and a C-terminal HTH-likedomain In the proposed mechanism of transcriptionalregulation the Nudix domain is responsible for a specificbinding of an effector molecule interfering with the abilityof HTH-domain to bind to operator sites in the promoterregions of regulated genes NrtR-binding sites werepredicted in a variety of bacterial genomes allowingin silico reconstruction of NrtR regulons that primarilyinclude genes involved in NAD metabolism Two selectedrepresentatives of the NrtR family from diverse bacteriaSynechocystis sp (Slr1690) and Shewanella oneidensis(SO1979) were experimentally characterized using gel-shift mobility assays This analysis validated NrtR cognatebinding sites deduced for these species and showed thatADP-ribose (ADPR) one of the intermediates of NADdegradation can operate as an effector molecule weaken-ing the NrtRndashDNA complex The detailed bioinformaticanalysis revealed a number of remarkable features of theNrtR family such as (i) conservation over a largephylogenetic distance along with (ii) significant variationsin DNA-binding motifs between species and (iii) functionaldiversity of regulated biochemical processes that inaddition to NAD metabolic pathways include utilizationof sugar pentoses and biogenesis of phosphoribosylpyrophosphate (PRPP) a central precursor for thesynthesis of nucleotides and amino acids

MATERIALS AND METHODS

Bioinformatics tools and resources

Functional annotations of genes involved in NADmetabolism and related pathways in 400 bacterialgenomes were from the collection of metabolic subsys-tems in The SEED comparative genomic database(httptheseeduchicagoedu) (10) Complete and partialbacterial genomes were downloaded from GenBank (20)

Preliminary sequence data were also obtained fromthe web sites of The Institute for Genomic Research(httpwwwtigrorg) the Welcome Trust Sanger Institute(httpwwwsangeracuk) and the DOE Joint GenomeInstitute (httpjgidoegov) Multiple sequence align-ments of protein sequences were produced by the Clustalseries of program (21) The PHYLIP package was used forconstruction of maximum likelihood phylogenetic treefor the NrtR protein family including bootstrappingwith 100 replicates and drawing of a consensus tree (22)The Protein Families database (Pfam) (httpwwwsangeracukSoftwarePfam) and the Clusters ofOrthologous Groups (COG) database (23) were used toidentify conserved functional domains Three-dimensionalprotein structures were obtained from the Protein DataBank (wwwrcsborgpdb) Structural similarity searcheswere performed using the SSM server (httpwwwebiacukmsd-srvssmssmstarthtml) (24) Functional cou-pling of genes via clustering on the chromosome wereanalyzed using The SEED toolsTo identify candidate regulatory motifs we started from

sets of potentially coregulated genes (using functional andgenome context considerations) An iterative motif detec-tion procedure implemented in the program SignalX wasused to identify common regulatory DNA motifs in a setof upstream gene fragments and to construct the motifrecognition profiles as previously described (25) For eachgroup of NrtR proteins on the phylogenetic tree we useda separate training set of the upstream regions ofcandidate target genes to construct the NrtR binding siteprofile The constructed group-specific recognition ruleswere used to scan a subset of genomes that encode anNrtR regulator from the corresponding group Positionalnucleotide weights in the recognition profile and Z-scoresof candidate sites were calculated as the sum of therespective positional nucleotide weights [as previouslydescribed in (26)] Genome scanning for additionalcandidate sites recognized by specific regulatory motifswas performed using GenomeExplorer software (27) Thethreshold for the site search was defined as the lowestscore observed in the training set Sequence logos for thederived group-specific DNA binding motifs were drawnusing WebLogo package v26 (httpweblogoberkeleyedu) (28)

Cloning and expression of Synechocystis sp andShewanella oneidensis nrtR genes

NrtR genes were amplified from genomic DNA ofSynechocystis sp strain PCC 6803 and S oneidensisMR-1 with the set of primers shown in SupplementaryData Table S3 and designed to incorporate a BamHI orNdeI restriction site at the 50 end and a Bpu1102I or aBamHI site at the 30 end of each gene respectivelyPolymerase chain reaction (PCR) conditions were asfollows 5min at 948C 1min at 948C 1min at 588C3min at 728C for 25 cycles Reactions were performed inthe presence of 03 pmolml of each primer 15mMMgCl2 02 nmolml deoxynucleoside triphosphatesand 2 units of 004Uml Taq polymerase (FinnzymesEspoo Finland) PCR fragments were purified digested

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with the respective enzymes and inserted into thecorresponding restriction sites of the expression vectorpET15b Escherichia coli TOP10F0 was used for plasmidpropagation and the nucleotide sequences of the insertswere confirmed by DNA sequencing For protein expres-sion the constructs were used to transform E coli BL21(DE3) Cells were grown at 378C in LB mediumsupplemented with ampicillin (100mgml) After reachingan OD600 of 03 cultures were shifted at 258C andexpression was induced with 1mM isopropyl b-D-1-thiogalactopyranoside at an OD600 of 06 After 12 hinduction cells were harvested by centrifugation at 5000gfor 10min and stored at 208C

Purification of recombinant NrtR

Cell pellets were resuspended in one-twentieth of theoriginal culture volume of 10mM potassium phosphatebuffer pH 75 03M NaCl 7mM b-mercaptoethanol(buffer A) containing 1mM phenylmethanesulfonylfluor-ide (PMSF) and 002mgml each of leupeptin antipainchymostatin and pepstatin After disruption by FrenchPress at 1000 psi suspensions were centrifuged at40 000g for 20min Supernatants were diluted 10-foldwith buffer A containing 1mM PMSF and subjected toNi+2-chelating chromatography For syNrtR purificationthe cell free extract deriving from 40ml culture was loadedonto 500ml Ni-NTA agarose (Qiagen) column equili-brated with buffer A After washing with 100mMimidazole in buffer A elution was performed with350mM imidazole in the same buffer For soNrtRpurification the cell-free extract deriving from 40mlculture was applied to a 1-ml HisTrap HP column(Amersham Biosciences) equilibrated with buffer AThe column was washed with 30mM imidazole in bufferA and elution was performed with an imidazole gradientfrom 30mM to 130mM in buffer A Fractions containingthe target proteins were collected concentrated byultrafiltration using YM-10 membranes (CentriconAmicon Bedford MA) and subjected to gel filtrationchromatography on a Superose 12 HR 1030 column(Amersham Biosciences) For elution a buffer containing50mM TrisHCl pH 80 03M NaCl and 1mMdithiothreitol (DTT) was used Samples were analyzedby Tricine Sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDSndashPAGE) (29) Protein concentrationwas determined as described by Bradford (30) usingbovine serum albumin as the standard

Assay of enzymatic activity

The HPLC-based assay described in (31) was used tomeasure the Nudix hydrolase activity of both NrtRproteins The reaction mixture contained 50mMHEPES pH 80 5mM MgCl2 or 5mM MnCl2 1mMDTT and 1mM nucleoside diphosphate derivatives as thesubstrate and varying amounts of pure recombinantproteins After incubation at 378C for 30min the reactionwas stopped with HClO4

Electrophoretic mobility shift assay

Regions containing the predicted regulatory sites wereamplified by PCR from genomic DNA by using the primerpairs shown in Supplementary Table S3 One of theprimers was 50-biotinylated by Sigma-Aldrich Corp(St Louis MO USA) By using Synechocystis sp DNAas the template primers nadMVfw and nadMVrevamplified a 55-bp fragment in the upstream region of theoperon sequence primers nadAfw and nadArev generateda 71-bp fragment consisting of 68 bp of upstream and 3 bpof 50 nadA sequence primers nadEfw and nadErevproduced a 60-bp fragment including 3 bp of 30 nrtRsequence all of the intergenic region between nrtR andnadE (49 bp) and 8 bp of 50nadE sequence By using theS oneidensis DNA as the template primers prsfw andprsrev generated a 144-bp fragment including the twopredicted DNA sites in the intergenic region between nrtRand prs sequences PCR products were purified by usingthe High Pure PCR Product Purification kit (RocheBasel Switzerland) and spectrophotometricallyquantitated

For the elecrophoretic mobility shift assay the biotin-labeled DNA (1 nM) was incubated with the indicatedamount of purified NrtR in 20 ml of binding buffercontaining 10mM Tris pH 75 50mM KCl 1mMDTT 25 glycerol 5mM MgCl2 005 NP-40 and050mgml of bovine serum albumin After 20minincubation at 258C the reaction mixtures were electro-phoresed on a 5 native polyacrylamide gel in 05TrisndashboratendashEDTA for 60min at 120V at 48C The gel waselectrophoretically transferred (30min at 380mA) onto anylon membrane (Pierce Rockford Ill) and fixed by UVcross-linking Biotin-labeled DNA was detected withthe LightShift Chemiluminescent EMSA kit (PierceRockford Ill) as recommended by the manufacturerSpecificity of the NrtRndashDNA interaction was establishedby including a 200-fold molar excess of non-biotinylatedtarget DNA (specific competitor) or 1 mg poly(dI-dC)(non-specific competitor) in binding reaction mixturesThe effect of potential effectors on NrtR-DNA bindingwas tested by pre-incubation of the proteins with NADmetabolites at the indicated concentrations for 10min at258C before the biotinylated DNA was added

RESULTS

We began this study by performing a bioinformatics surveyof candidate genes for transcriptional regulators of NADmetabolism in those bacterial species that do not containorthologs of known regulators NadR or NiaR Weconsidered a conserved clustering on the chromosomewith known genes of NAD biogenesis as primary evidencefor implication of such candidates (32) Further prioritiza-tion of candidate genes was performed based on theirdomain structure analysis for a presence of putativeDNA-binding motifs as well as on additional evidence offunctional coupling such as occurrence profiles andpresence of shared regulatory sites (833) as metabolictranscriptional regulators are often auto regulated

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Reconstruction of NAD metabolic and regulatorynetworks in 400 bacterial species with completelysequenced genomes was performed using a subsystems-based approach (10) implemented in the SEED genomicplatform (see lsquoNAD regulationrsquo subsystem at httptheseeduchicagoeduFIGsubsyscgi) A genome contextanalysis evidenced a strong tendency of genes involved inNAD biogenesis (including regulatory gene niaR) to formconserved operon-like clusters (Supplementary DataTable S1) Among other genes with similar chromosomalclustering patterns of particular interest was a family ofNudix hydrolase homologs Members of this family wereconsidered primary candidates for the NAD regulatoryrole and annotated as possible NrtR based on thefollowing key observations

(i) presence of a C-terminal domain with winged HTH-fold characteristic of many prokaryotic transcrip-tion factors (34)

(ii) homology of the NrtR N-terminal domain tomembers of the Nudix hydrolase family related toNAD metabolism such as ADPR pyrophosphataseinvolved in NAD recycling

(iii) lack of conservation within the Nudix hydrolasesignature sequence suggesting that many NrtRmembers could have lost a catalytic activity whilepotentially retaining an ability to specifically bindrelevant metabolites (eg ADPR)

Chromosomal co-localization of nrtR genes

In most cases (42 out of 62 analyzed) nrtR genes arepositionally linked to various NAD metabolism genes(Supplementary Table S1) The tendency of nrtR genes tocluster on the chromosome with NAD biosynthesis genesis illustrated in Figure 2 For instance nrtR genes occur inclusters with nadABC in Actinobacteria nadD-nadE andpncB in Cyanobacteria prs-nadV in g-proteobacterianadR-pnuC in Streptococcus and niaP in Bifidobacterium

In Streptomyces species the nrtR gene occurs next to agene coding for a putative ADP-ribosyl-glycohydrolase(draG) which catalyzes the removal of the ADPR groupcovalently linked to target proteins (Figure 1) ProteinADP-ribosylation was proven to be an indispensableregulatory mechanism in Streptomyces species (35) eventhough its role remains to be elucidated

Additional types of conserved chromosomal clusterswere observed pointing to possible functional coupling ofNrtR with other metabolic pathways For example ninecases of nrtR chromosomal clustering with genes involvedin utilization of pentoses (L-arabinose and D-xylose) weredetected in some g-proteobacteria and in BacteroidetesNotably the pentose utilization pathways have connec-tions with NAD metabolism via the shared intermediateribose 5-phosphate (Rib-P) In fact glycohydroliticdegradation of NAD generates ADPR which is furtherconverted to Rib-P by Nudix hydrolases Rib-P can berecycled to generate NAD via PRPP formation(Figure 1) Rib-P is also produced by the pentosesutilization routes after they merged to the pentosephosphate pathway The result is a combination of

pentoses utilization NAD degradation and recycling ina compact subnetwork (Figure 1)

Phylogenetic distribution and domain compositionof NrtR proteins

We constructed the maximum likelihood phylogenetic treefor 62 NrtR protein family representatives selected fromdiverse bacterial species (Figure 3) The distribution ofNrtR orthologs largely coincides with known taxono-mic groups with several exceptions eg in the case oftwo proteins from g-proteobacteria Pseudoalteromonasatlantica and Saccharophagus degradans whose clusteringwith the Bacteroidetes group is a likely result of lateralgene transfer NrtR proteins from Actinobacteria and

Figure 2 Genomic organization of nrtR-containing loci involved inNAD metabolism (A) pentose utilization (B) and other pathways (C)Genes encoding the predicted NrtR regulator are shown by red arrowsthe color code and the abbreviations for other genes correspond tothose used in Figure 1 Red circles indicate the predicted NrtR-bindingsites

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Firmicutes are split on the tree into two separate groupswhich may reflect the actual functional divergence eg bythe set of co-regulated genes or by the consensus DNA-binding motif (see next section) Moreover some of thesespecies contain two NrtR paralogs For instance twoNrtR paralogs in Streptomyces species (groups 2a and 2b)likely resulted from a recent duplication event In anotherspecies of Actinobacteria Kineococcus radiodurans thesituation is different two NrtR paralogs are located onthe most divergent branches of the tree (groups 1aand 2b)A common feature of the NrtR family is the invariant

presence of the N-terminal Nudix domain (PF00293 orCOG1051) fused with a characteristic C-terminal domain(PB002540) which is similar to C-terminal part ofproteins from uncharacterized COG4111 familyHowever COG4111 and NrtR proteins have extremelydivergent N-terminal domains (see Discussion section forfurther details on COG4111) The schematic representa-tion of the NrtR domain arrangement compared with thatof some of the known members of the Nudix hydrolasefamily (where the Nudix domain is combined with other

domains) and the COG4111 protein family is shown inSupplementary Fig S1 A multiple alignment of selectedNrtR proteins including two proteins with known 3Dstructure is provided in Supplementary Fig S2 TheNudix domain is typical of a family of hydrolases found innearly all known species in all three domains of lifeTypical substrates of Nudix hydrolases are nucleosidediphosphates with large variation of residues (x) attachedto the phosphate moiety (hence the name nudix) Theseenzymes hydrolyze a pyrophosphate bond in a wide rangeof organic pyrophosphates including nucleoside di- andtriphosphates dinucleoside polyphosphates and nucleo-tide sugars (such as ADPR) with varying degrees ofsubstrate specificity The number of Nudix-like genes inprokaryotic genomes is a subject of significant variationreaching up to 30 copies depending on metaboliccomplexity and adaptability of species [reviewed in (36)]Among various Nudix hydrolases those with establishedsubstrate preference for ADPR (ie ADPR pyrophospha-tases catalyzing ADPR hydrolysis to AMP and Rib-P)show the most significant similarity with the Nudixdomain of NrtR proteins

Figure 3 Maximum likelihood phylogenetic tree and DNA recognition motifs for the NrtR family of transcriptional regulators NrtR proteinsrecognizing the same DNA motif are grouped (the group names are given) and the corresponding motif sequence logos are shown on the left andright sides Species content of the NrtR groups as well as the content of corresponding regulons and the NrtR-regulated pathways are summarized inTable 1 Genome context of nrtR genes is shown by squares with colors corresponding to the color code of functional roles in Figure 1 NrtRproteins possessing intact Nudix signature are in red Proteins from B thetaiotaomicron and E faecalis with solved 3D structures are highlightedProteins studied in this work are boxed The numbers indicate the number of bootstrap replications out of 100 that support each node on the tree

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A detailed analysis of conserved sequence motifs withinthe NrtR family indicated that in contrast to functionalNudix hydrolases many members of this family couldhave lost a catalytic activity This conjecture is based onthe apparent lack of conservation within a signaturesequence GX5EX7REUXEEXGU (where U is a hydro-phobic residue and X is any residue) which is strictlyconserved in all active Nudix hydrolases identified so far(37) In most members of the NrtR family at least one ormore of the conserved signature residues are replaced in amore or less random fashion (Supplementary Fig S2)Previously published results of biochemical characteriza-tion of two divergent members of the NrtR familyfrom cyanobacteria NuhA from Synechococcus sp PCC7002 and Slr1690 from Synechocystis sp providedadditional insights for interpretation of functional motifsin this family Whereas the NuhA protein with an intactNudix signature displayed a high ADPR pyrophosphatase

activity (38) the hydrolytic activity of its homolog Slr1690with a severely perturbed signature was barely detectable(kcat 14 104s1) (39) Restoring the canonical Nudixsignature by a directed mutagenesis of the slr1690 gene ledto a 600-fold increase of the catalytic rate (39) These factssuggest that the presence of an intact Nudix signature inNrtR proteins correlate with their catalytic activity

Structural analysis of NrtR proteins

A comparative structural analysis of C-terminal domainsin NrtR proteins provided the key evidence for their rolein the regulation of transcription This analysis wasenabled by the availability of 3D structures determinedat the Midwest Center for Structural Genomics (httpwwwmcsganlgov) for the two NrtR family membersfrom Bacteroides thetaiotaomicron (BT0354 PDB code2FB1) and Enterococcus faecalis (EF2700 PDB code2FML) (Figure 4) It is important to note that while the

A

B

Figure 4 Crystal structures and ligand-binding sites of E faecalis EF2700 (A) and B thetaiotaomicron BT0354 (B) Structures of EF2700 (PDBaccession number 2FML) and BT0354 (2FB1) were solved at the Midwest Center for Structural Genomics The C-terminal wHTH domains areshown in grey and the N-terminal Nudix domains of each monomer are shown in cyan and green respectively The co-crystallized glycerol andphosphate molecules are indicated In the insets are the close-ups of the Nudix domain active sites with a modeled ADPR molecule in EF2700 and aRib-P molecule in BT0354 respectively based on the superposition with the Synechocystis sp NadMndashADPR complex structure In the bindingpocket of BT0354 the Rib-P moiety (R5P) of ADPR is shown as sticks while the rest of the molecule is show as thin lines Protein residues that arepredicted to interact with the bound ligand are also shown as sticks Dotted lines represent potential hydrogen bonds Residues Y27B and Y5B ofEF2700 and BT0354 respectively come from the second subunit of the dimer

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results of similarity searches for NrtR C-terminal domainbased solely on sequence comparison were rather incon-clusive a structure-based search by the SSM server (24)revealed a substantial similarity with winged helix-turn-helix (wHTH) domains A three-stranded wHTH fold(a1-b1-a2-a3-b2-b3) of the C-terminal domain(Supplementary Fig S2) is typical for DNA-bindingdomains present in many families of prokaryotic tran-scription factors (34)The 3D structures of BT0354 and EF2700 show that

both NrtR proteins form dimers with clear domainswapping The Nudix domain of both proteins is verysimilar to the ADPR pyrophosphatase domain of thebifunctional enzyme NadM from Synechocystis sp whosestructure has been recently solved in complex with ADPR(Huang N and Zhang H unpublished data) Inparticular root mean square deviations of 14 A and15 A have been calculated for 115 and 108 superimposedCa atoms between NadM and EF2700 and NadM andBT0354 respectively The residues demonstrated to bedirectly involved in ADPR binding in the NadM structureare well conserved in EF2700 (Figure 4A) Most of theresidues interacting with ADPR are also conserved in theBT0354 active site however only the ribose-phosphatemoiety of ADPR fits well into the binding pocket(Figure 4B) These structural considerations allowed usto suggest ADPR and Rib-P primary candidates aspossible NrtR effector molecules

Prediction of NrtR-binding sites and reconstructionof NrtR regulons in bacterial genomes

To identify possible DNA motifs specifically recognizedby NrtR in various taxonomic groups we created trainingsets by combining the upstream regions of NAD meta-bolic genes from complete bacterial genomes containingnrtR genes Based on the phylogenetic analysis the NrtRfamily was divided into several taxonomic groups andsome of them were further split to distinct branches(Figure 3) They were used to compile group-specifictraining sets For nrtR genes that occur in conservedchromosomal clusters with genes other than thoseinvolved in NAD metabolism the respective trainingsets included upstream regions of these gene clusters(eg nrtR-draG locus in Streptomyces and ara or xyl lociin Bacteroidetes)By applying the motif-detection program SignalX with

the inverted repeat option (40) we have identifiedcandidate NrtR recognition sites conserved in each ofthe compiled training sets and constructed the correspond-ing NrtR profiles and sequence logos (Figure 3) Althoughthe derived motifs are substantially different in consensussequence and information content (depending on thenumber and diversity of species within groups and thenumber of candidate target genes) most of them share a21-bp palindrome symmetry and a conserved corewith consensus GT-N7-AC The most divergent NrtRconsensus sequences were detected for two groups of theg-proteobacteria (Vibrio and Pseudomonas) and one groupof the Firmicutes (Streptococcus) For the Deinococcus

and Enterococcus species we were unable to identify NrtRrecognition profiles and regulons

The constructed NrtR-binding site recognition profileswere used to detect new candidate members of the NrtRregulons in the genomes containing nrtR genes Table 1gives a list of genes and operons predicted to be undercontrol of NrtR The detailed information about thesequence position and score of each predicted NrtR siteas well as the genomic identification numbers of down-stream genes are provided in Supplementary Table S2The key features of the reconstructed NrtR regulons areoutlined in details by taxonomic groups in SupplementaryText S1

Functional gene content of the reconstructed NrtRregulons varies significantly between different taxonomicgroups of bacteria (Table 1 and Figure 3) The NrtR-regulated pathways include the universal NAD biosynth-esis and salvage I pathways in Cyanobacteria andg-proteobacteria Pirellula and Chloroflexi the de novoNAD biosynthesis pathway in Actinobacteria niacinuptake and salvage I and III pathways in Lactobacillales(Firmicutes) and the pentose utilization pathways inBacteroidetes (see color square code in Figure 3)Taxonomic distribution of NrtR regulators is comple-menting to the distribution of two other transcriptionalregulators of NAD metabolism NadR (18) and NiaR [seethe accompanying paper (9)] with the exception of somespecies of Firmicutes where both NrtR and NiaR appearto regulate non-overlapping aspects of NAD metabolism(Supplementary Table S1) For instance in Clostridiumactobutylicum NrtR regulates the nicotinamide salvagepathway (pncAB) and the universal NAD synthesis(nadDE) whereas NiaR controls the de novo NADsynthesis (nadBCA) The Lactobacillales species representanother example of the large variability in the content ofNAD regulons In Lactobacillus casei NiaR and NrtRregulate the niaP and pncAB genes respectively whereasin L plantarum they control the pncB and niaP genesrespectively (9)

The position of the candidate NrtR-binding sites in theregulatory gene regions either overlapping the predictedpromoter elements or lying between the promoter and thetranslation start site of the downstream gene stronglysuggests that these regulators might act as repressors oftranscription (Supplementary Figure S3) They areexpected to bind to target genes via the wHTH C-terminaldomain and a postulated interaction of the NudixN-terminal domain with an effector molecule isanticipated to weaken the NrtRndashDNA complex leadingto derepression of target genes

While many aspects of the proposed mechanism are yetto be investigated in this study we chose to perform anexperimental validation of selected NrtR family membersin order to provide minimal required support for thesuggested novel functional annotations For these valida-tion experiments we have chosen two representatives ofthe NrtR family Slr1690 from Synechocystis sp andSO1979 from S oneidensis The candidate NrtR-bindingsites in Synechocystis sp precede the nadA nadE andnadM-nadV genes whereas the predicted NrtR regulon inS oneidensis contains a single target operon prs-nadV

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(Table 1) The choice of these two species was dictated bythe following considerations (i) unambiguous associationof the chosen NrtR groups with NAD metabolism (ii) adivergent nature of these groups at the level of taxonomicplacement of respective organisms as well as at the level ofprotein sequences (position on the NrtR tree andconsensus DNA motifs) (iii) availability of biochemicaldata for the Slr1690 protein (39) from the best studiedmodel cyanobacterium Synechocystis sp including thedetailed analysis of the NAD biosynthetic machinery (41)and (iv) S oneidensis being an important model organismwith a rapidly growing body of physiological and genomicdata (42)

Experimental characterization of two NrtR familyrepresentatives

To experimentally test the ability of NrtR to specificallybind to the predicted DNA sites and to assess possibleeffectors slr1690 from the Synechocystis sp (furtherreferred to as syNrtR) and SO1979 from S oneidensis(soNrtR) were cloned and overexpressed in E coli BothHis6-tagged recombinant proteins were purified to homo-geneity by Ni+2-chelating chromatography followed bygel filtration as described in the Materials and Methodssection SDS-PAGE of pure syNrtR and soNrtR proteinsrevealed molecular masses of about 32 and 29 kDa

Table 1 Operon structure for nrtR genes and predicted NrtR sites in bacteria

Organism NrtR operonregulon structurea NrtR-regulatedpathwayb

NrtR DNA-bindingsequence logo profilec

Synechocystis sp PCC 6803 nrtR-nadE nadM-nadV nadA NAD(dus2) CyanobacteriaNostoc Anabaena spp Crocosphaera watsonii

Thermosynechococcus elongatus pncB-nadD-nrtR-nadE nadA NAD(dus1) CyanobacteriaTrichodesmium erythraeum pncB-nadD-nrtR NAD(us1) CyanobacteriaCorynebacterium glutamicum Cefficiens nrtR-nadA-nadC-sufS NAD(d) Actinobacteria-1aKineococcus radiotolerans Leifsonia xyli nrtR-nadA-nadB-nadC-sufS NAD(d) Actinobacteria-1aBifidobacterium longum nrtR-nadA-nadB-nadC-sufS-niaP NAD(dt) Actinobacteria-1aBifidobacterium adolescentis nrtR-niaP pncB NAD(ts1) Actinobacteria-1aCorynebacterium diphtheriae nrtRltgtnadA-nadB-nadC NAD(d) Actinobacteria-1bMycobacterium species Nocardia farcinica nrtRltgtnadA-nadB-nadC NAD(d) Actinobacteria-1bThermobifida fusca nrtRltgtnadA NAD(d) Actinobacteria-1bStreptomyces species - (1) nrtR1-draG-SCO1765 ADPR Actinobacteria-2aStreptomyces species - (2) nrtR2 nrtX-nrtY Actinobacteria-2bKineococcus radiotolerans - (2) Frankia alni nrtR2ltgtnrtX-nrtY Actinobacteria-2bLactobacillus casei Ldelbrueckii nrtR-pncA-pncB NAD(s1) Firmicutes-1aLactobacillus plantarum Lmesenteroides nrtR-niaP NAD(t) Firmicutes-1aStreptococcus mitis Sgordonii Ssanguinis nadR-pnuC-nrtR NAD(s3) Firmicutes-1bClostridium acetobutylicum nrtR-pncA-pncB-nadD-nadE NAD(us1) Firmicutes-2aClostridium thermocellum nrtR pncA-pncB NAD(s1) Firmicutes-2bShewanella oneidensis Sputrefaciens nrtR-prs-nadV NAD(s2p) Gammaproteo-1aAcinetobacter sp ADP1 prs-nadV-nrtR NAD(s2p) Gammaproteo-1aProteus mirabilis nrtRltgtnrtX-nrtY Gammaproteo-1aHahella chejuensis nrtRltgtpnuC-nadR-nadM-pncB nrtX-nrtY NAD(s1s2s3) Gammaproteo-1aChromobacterium violaceum nrtR-prs-nadV NAD(s2p) Gammaproteo-1aPseudomonas aeruginosa Pfluorescens nrtR-nadDltgtpncA-pncB-nadE NAD(us1) Gammaproteo-1bPseudomonas entomophila nrtRltgtpncA-pncB NAD(s1) Gammaproteo-1bVibrio cholerae nrtRltgtpncB NAD(s1) Gammaproteo-1cVibrio parahaemolyticus Vvulnificus pncA-nrtRltgtpncB NAD(s1) Gammaproteo-1cCytophaga hutchinsonii nrtR-prs-nadV nadE NAD(us2p) CytophagaRoseiflexus sp RS-1 nrtR-pncA pncB nadC-nadA-nadB NAD(bs1) ChloroflexiBlastopirellula marina pncB-nrtR-pncA nadE NAD(us1) PirellulaBacteroides fragilis B thetaiotaomicron - (1) nrtR-xylB-xylA-xylT Xyl Bacteroidetes-1aB thetaiotaomicron - (2) galM-araT-nrtR2-araD-araB-araA Ara Bacteroidetes-1bRobiginitalea biformata nrtRltgtabf-araB-araD-araA-araT Ara Bacteroidetes-1bFlavobacterium sp MED217 - (2) nrtR2ltgtabf-abn-xyn-xyl araB-araD-araT-araA Xyl Ara PP Bacteroidetes-1bFlavobacterium johnsoniae UW101 - (2) nrtR2ltgtabf-araB-araD-araA other genes Ara Bacteroidetes-1bSaccharophagus degradans nrtRltgtxylB-xylA other genes Xyl PP Bacteroidetes-1cPseudoalteromonas atlantica nrtR-abf Ara Bacteroidetes-1cFlavobacterium sp MED217 - (1) nrtR1ltgtxylB-xylA Xyl Bacteroidetes-1c

aGenes forming one putative operon (with spacer lt100 bp) are separated by dashes Different loci are separated by semicolons The direction oftranscription in divergons (two oppositely directed transcriptional units) is shown by angel brackets Predicted NrtR-binding sites are denoted by lsquorsquoFunctions of NrtR-regulated genes are described in the legend to Figure 1 SufS is a homologue of IscS cysteine desulfurase involved in the in vivomaturation of Fe-S clusters possibly necessary to assemble NadAB complex Other predicted NrtR-regulated genes of unknown function are denotednrtX and nrtYbBiochemical pathways predicted to be regulated by NrtR are abbreviated according to Figure 1 Functional roles involved in NAD synthesis dde novo biosynthesis u universal NAD synthesis s1 s2 and s3 stand for salvage pathways I II and III p PRPP synthesis t niacin transport Othermetabolic pathways are pentose-phosphate pathway (PP) xylose (Xyl) arabinose (Ara) utilization and reversible protein ADP-ribosylation (ADPR)cGenomes are grouped according to the predicted DNA-binding profiles (see the phylogenetic tree of NrtR proteins on Figure 3) and in most casesthe NrtR groups coincide with the taxonomic groups of organisms

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respectively in agreement with the expected size(Supplementary Figure S4)As expected no appreciable ADPR pyrophosphatase

activity could be detected for syNrtR and soNrtR proteinsin the presence of Mg2+ or Mn2+ This is consistent withobserved alterations in their Nudix signatures(Supplementary Figure S2) as well as with the previousreport of extremely low enzymatic activity of Slr1690protein several orders of magnitude lower than thatmeasured for other ADPR pyrophosphatases inSynechocystis sp (39) Likewise both NrtR proteinswere unable to hydrolyze other nucleoside diphosphatecompounds including (20)phospho-ADP-ribose (pADPR)NAD flavine adenine dinucleotide diadenosine poly-phosphates (ApnA n=2 4) and GDP-mannoseWe used electophoretic mobility shift assay (EMSA) to

test the specific DNA-binding of the purified syNrtR andsoNrtR proteins to their predicted operator sites derivedfrom the upstream regions of nadA and nadE genes thenadM-nadV operon from Synechocystis sp and the prs-nadV operon from S oneidensis A substantial shift of theDNA band was observed in all cases upon incubation ofthe target DNA fragments with respective proteins(Figure 5) A typical protein concentration dependenceof DNA-binding is illustrated in Figure 5A showing theincreasing intensity of the shifted DNA band (corre-sponding to a predicted nadM-nadV target site) in thepresence of increasing amounts of the syNrtR protein Theband shift was suppressed in the presence of 200-foldexcess unlabeled DNA fragments but not in the presenceof negative control DNA poly(dCdI) confirming aspecific nature of the NrtRndashDNA interactions(Figure 5A) A similar specific binding in the presence ofcompeting DNA was observed between syNrtR and itscognate DNA-sites from the upstream regions of nadEand nadA genes (Figure 5B) Two distinctly shiftedproteinndashDNA complexes were observed in the case ofsoNrtR interaction with the prs-nadV DNA targetconfirming a functional competence of both predictedtandem NrtR-binding sites (Figure 5C)Of the tested intermediary metabolites associated with

NAD biosynthetic salvage and recycling pathways(Figure 1) Nam NA quinolinic acid NMN NaMNNAD PRPP Rib-P ADP and AMP at 100 mM concen-tration did not interfere with complex formation betweensoNrtR and the prs-nadV tandem DNA-site This isillustrated for some of these compounds by the results ofEMSA analysis (Figure 6A) In the same experiment100mM of ADPR and pADPR significantly suppressedsoNrtR-DNA binding (Figure 6A) and a similar effectwas observed with 100 mM of nicotinate adenine dinucleo-tide (NaAD) nicotinate adenine dinucleotide phosphate(NaADP) NADH NADPH and NADP (data notshown) As shown in the lower panel of Figure 6Athese metabolites exerted a similar effect on soNrtR-DNAbinding complex formation even at 10 mM with ADPRbeing the most effective of all ADPR was also shown tosuppress specific DNA-binding of syNrtR-DNA asillustrated in Figure 6B for the nadM-nadV target whereasNAD and Rib-P had no effect under the same conditions

DISCUSSION

Identification of a novel family of transcriptional factorsfor the NAD metabolism allowed us to fill a substantialgap in the knowledge of transcriptional regulation of thekey metabolic pathways in bacteria Prior to this studytranscriptional regulation of NAD biosynthesis waselucidated only in the classic model systems E coliSalmonella and B subtilis (1519) Of the two knownfamilies of transcriptional regulators of NAD biosynth-esis NadR appears to be operational within only a limitedgroup of Enterobacteria (18) whereas the distribution ofthe NiaR (YrxA) family is mostly restricted to speciesfrom the BacillusClostridium and Thermotogales group(9) This limited knowledge is in marked contrast with

prs nadV

nadVnadM

nadAnadE

A

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8

1 2 3 4 5 6

C

DNA-protein complex

free DNA

DNA-protein complexes

free DNA

free DNA

DNA-protein complexB

Figure 5 EMSA demonstrating specific NrtR binding to DNADNA fragments used in the assays are defined by their genomicpositions and are shown as dark circles in the top of each panel(A) EMSA with nadM-nadV DNA fragment (07 ng) in the absence(lane 1) and in the presence (lanes 2ndash5) of increasing syNrtR proteinconcentrations (025 05 10 and 50 nM respectively) The specificityof interaction of syNrtR (2 nM) with DNA fragment (lane 7) was testedby competition with 1mg polydCdI (lane 8) and 140 ng unlabeled DNAfragment (lane 9) Lane 6 contains 07 ng of the biotylinated DNAfragment only (B) EMSA with nadE and nadA DNA fragments inthe absence (lanes 1 and 5) and in the presence of syNrtR (2 nM)(lanes 2 and 6) The specificity of interaction was tested with polydCdI(lanes 3 and 7) and unlabeled DNA fragment (lanes 4 and 8)(C) EMSA with prs-nadV DNA fragment in the absence (lane 1) and inthe presence of 2 nM soNrtR (lanes 2 and 5) and 5 nM soNrtR (lane 6)Competition assays were performed with 2 nM soNrtR in the presenceof polydCdI (lane 3) and unlabeled DNA (lane 4)

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a comprehensive understanding of NAD biosyntheticmachinery that was reliably reconstructed by comparativegenomic analysis in nearly all bacteria with completelysequenced genomes (11)

In this study we used a combination of comparativegenomic techniques including analysis of genome contextand regulatory DNA motifs (8) to predict the novelregulator NrtR and reconstruct respective regulons inseveral divergent groups of bacteria The NrtR proteinfamily of transcriptional regulators described here is thefirst known example of a functional fusion between aNudix domain and a DNA-binding domain The com-parative analysis of predicted NrtR-binding motifsrevealed their significant variability between taxonomicgroups that were largely in agreement with a phylogenetictree of the NrtR protein family (Figure 3) The derivedmotifs for 12 NrtR groups have a relatively conserved core(TG-N7-CA) whereas the other six groups appear to haveadopted dissimilar NrtR-binding sites This degree ofvariability of NrtR DNA-binding motifs is higher thanwhat is usually observed for most other broadly conservedbacterial transcription factors such as BirA ArgR andNrdR (843ndash45)

The predicted composition of NrtR regulons in differentbacterial genomes appears to be the subject of substantial

variations that to some extent reflect various combinationsof pathways involved in NAD biosynthesis (Figure 1 andTable 1) NrtR regulation appears to play a central role inthe transcriptional control of all aspects of NADbiosynthesis salvage and recycling in Cyanobacteria [fora comparative genomic reconstruction of NAD metabo-lism in this bacterial lineage see (41)] In contrast theNrtR regulon in S oneidensis includes only one targetoperon prs-nadV Although the functional associationbetween these two genes is straightforward (Figure 1) aninferred co-regulation of the PRPPndashsynthesizing enzymePrs with a rather local Nam salvage pathway via NadV isquite unexpected However unlike most bacteria having asingle copy of the prs gene S oneidensis and several otherspecies with prs-nadV operons contain two prs paralogsonly one of which is predicted to be regulated by NrtRA functional significance of having separate machinery forPRPP production committed to Nam to NMN conversionis yet to be elucidatedWe chose representatives of the two NrtR groups

discussed earlier to experimentally assess their specificDNA-binding properties and to test possible smallmolecule effectors Both recombinant purified proteinsfrom Synechocystis sp and from S oneidensis were shownto form a complex with their cognate target DNAsequences (Figure 5) Among various tested metabolitesassociated with NAD metabolism ADPR was the mosteffective in suppressing in vitro binding of both regulatorsto their target sequences (Figure 6) At the same timeenzymatic characterization of both proteins revealed noappreciable catalytic (ADPR pyrophosphatase) activity inagreement with their substantially perturbed Nudixsignature sequences as well as with the previous reportabout the Synechocystis sp protein (39) Although thein vitro EMSA data alone were insufficient for unambig-uous identification of the actual physiological NrtReffector molecule(s) ADPR appears to be the mostlikely candidate for this role As discussed earlier thiseffector specificity could be anticipated from the sequenceand structure similarity between the NrtR Nudix domainand the ADPR pyrophosphatase domain of the bifunc-tional enzyme NadM (Figure 4) Notwithstanding thesecaveats the obtained experimental data are generallysupportive of a role of NrtRndasheffector interactions in theproposed mechanism of de-repression of NrtR-regulatedgenes They also allowed us to exclude common NADprecursors (including NA the known NiaR co-repressor)as well as NAD (the known effector of the NadRregulator) from the list of possible NrtR effectorsThe proposed physiological role of ADPR as an anti-

repressor of NAD synthesis is based on the assumptionthat the cell may interpret the accumulation of ADPR as asignal to replenish the NAD cofactor pool This assump-tion is reasonable as the only source of ADPR in the cell isconsumption of NAD by direct enzymatic hydrolysisBinding of ADPR to an NrtR Nudix domain wouldpromote dissociation of NrtRndashDNA complexes leadingto de-repression of transcription of NAD biosyntheticgenes ADPR itself may contribute to NAD production byproviding a Rib-P precursor for PRPP synthesis upon itsNudix-mediated hydrolytic cleavage This provides an

100mM

10mM

A

025 05 10 025 05 10 025 05 10 025 05 10

1mM NAD 1mM ADPRNrtR[nM]

DNA-proteincomplex

free DNA

B

1 ADPR pADPR NAD NMN Nam PRPP Na NaMN ADP

DNA-proteincomplexes

free DNA

1 pADPR ADPR NaAD NaADP NADH NADPH NADP

DNA-proteincomplexes

free DNA

1mM Rib-P

Figure 6 Effect of NAD metabolites on NrtRndashDNA binding(A) Electrophoretic mobility of prs-nadV DNA fragment incubatedwith purified soNrtR (2 nM) in the absence (lane 1) and in the presenceof 100 and 10 mM of the indicated compounds (B) Electrophoreticmobility of nadM-nadV DNA fragment incubated with increasingconcentrations of purified syNrtR in the absence and in the presence of1mM NAD 1mM ADPR and 1mM Rib-P

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additional rationale for the observed regulatory connec-tions between the NAD biosynthetic machinery and prsgene Intriguingly some members of the NrtR family thathave an intact Nudix signature may directly contribute toADPR hydrolysis in addition to their role in regulation oftranscriptionThe most remarkable aspect of the already-mentioned

functional plasticity of the NrtR family is its apparentadaptation to the regulation of metabolic pathways thatare not immediately related to NAD metabolism Amongthe examples analyzed in this study are pentoses utiliza-tion and the pentose phosphate pathway in Bacteroidetesand in two species of g-proteobacteria (Table 1) Indeedthese pathways share the intermediate Rib-P with NADdegradation and recycling (Figure 1) It is tempting tospeculate that in these species NrtR regulons may utilizeRib-P (rather than ADPR) as an effector molecule as alsosuggested by ligand modeling in the active site of NrtRfrom B thetaiotaomicron (Figure 4) Further experimentsare required to test this hypothesisThe data analyzed in this study allowed us to suggest a

likely scenario for the evolution of the NrtR family thatincludes a fusion of an ADPR-preferring Nudix hydrolasewith a DNA-binding domain giving rise to a transcrip-tional regulator An ancestral version of NrtR regulatorcould retain ADPR pyrophosphatase activity Later inspeciation NrtR proteins could lose the hydrolase activitythat was redundant in the presence of other active Nudixhydrolase(s) Regardless of the actual effector specificitywhich is yet to be explored across the NrtR family thisevolutionary scenario is another example of a noveltranscription factor emerging via a fusion between theenzymatic and DNA-binding domains This appears to bequite an efficient strategy as may be deduced from a largeand growing number of transcription factors lsquodesignedrsquousing similar principles Among other prominent examplesin bacteria are biotin repressor BirA a fusion offunctional biotin-ligase and DNA-binding HTH domain(43) xylose repressor XylR and other regulators of theROK family composed of a sugar kinase-like domainfused with HTH domain (17) transcriptional repressorPutA that also carries out the enzymatic steps in prolinecatabolism (46) and NadR a NAD salvage enzyme andthe transcriptional regulator of NAD synthesis inEnterobacteria (154748)In this work and the accompanying study (9) we

characterized the NrtR and NiaR families of transcrip-tional factors implicated in the control of NAD home-ostasis Although these findings substantially expandedour knowledge in this important but relatively unexploredarea mechanism of transcriptional regulation of NADmetabolism remains unknown in many other bacteriallineages including a- b- d- and e-proteobacteria Notablyanother family of putative transcriptional regulators ofNAD metabolism (COG4111 here named NadQ) wasidentified in 15 species of a-proteobacteria and in somepathogenic b-proteobacteria (Supplementary Table S1)Members of this family share similar C-terminal HTHdomains with NrtR proteins but have highly divergentN-terminal domains (as illustrated in SupplementaryFigure S1) NadQ encoding genes also have a strong

tendency to cluster on the chromosome with genesinvolved in NAD metabolism The genomic identificationof NadQ-binding sites and functional characterization ofNadQ regulon are currently underway Altogether thesefindings demonstrate the significant variability of regula-tory strategies for control of NAD metabolic pathways inbacteria

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENTS

We want to thank Dr Giulio Magni and Dr SilverioRuggieri (Universita Politecnica delle Marche AnconaItaly) for discussions and advice in the field of NADmetabolic biochemistry Dr Mikhail Gelfand (IITPMoscow Russia) for useful discussions and commentsduring the preparation of this article and Dr AndreiMironov (Moscow State University Russia) for softwarefor the analysis of regulons We are grateful to theMidwest Center for Structural Genomics team for sharingthe results of structural analysis that were instrumental forour functional prediction of the novel family of transcrip-tional regulators We thank Ross Overbeek VeronikaVonstein Gordon Push and other members of The SEEDdevelopment team at Fellowship for Interpretation ofGenomes (FIG) for their help with the use of The SEEDgenomic resource This work was partially supported bygrants from Ministero dellrsquoIstruzione dellrsquoUniversita edella Ricerca (MIUR) lsquoCharacterization of key enzymesof NAD(P) biosynthesis in bacteria and their regulatio-nessential steps to find targets of new drugsrsquo (PRIN 2005to NR) National Institute of Health (NIH) lsquoGenomicsof Coenzyme Metabolism in Bacterial Pathogensrsquo (1-R01-AI066244-01A2 to AO) and Department of Energy(DOE) lsquoIntegrated Genome-Based Studies of ShewanellaEcophysiologyrsquo (DE-FG02-07ER64384 to AO) Fundingto pay the Open Access publication charges for this articlewas provided by National Institute of Health researchgrant 1-R01-AI066244-01A2

Conflict of interest statement None declared

REFERENCES

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2 SchmidtMT SmithBC JacksonMD and DenuJM (2004)Coenzyme specificity of Sir2 protein deacetylases implications forphysiological regulation J Biol Chem 279 40122ndash40129

3 WilkinsonA DayJ and BowaterR (2001) Bacterial DNA ligasesMol Microbiol 40 1241ndash1248

4 DomenighiniM and RappuoliR (1996) Three conservedconsensus sequences identify the NAD-binding site ofADP-ribosylating enzymes expressed by eukaryotes bacteria andT-even bacteriophages Mol Microbiol 21 667ndash674

5 BegleyTP KinslandC MehlRA OstermanA andDorresteinP (2001) The biosynthesis of nicotinamide adeninedinucleotides in bacteria Vitam Horm 61 103ndash119

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6 MagniG AmiciA EmanuelliM RaffaelliN and RuggieriS(1999) Enzymology of NAD+ synthesis Adv Enzymol RelatAreas Mol Biol 73 135ndash182 xi

7 GerlachG and ReidlJ (2006) NAD+ utilization inPasteurellaceae simplification of a complex pathway J Bacteriol188 6719ndash6727

8 RodionovDA (2007) Comparative genomic reconstruction oftranscriptional regulatory networks in bacteria Chem Rev 1073467ndash3497

9 RodionovDA LiX RodionovaIA YangC SorciLDervynE MartynowskiD ZhangH GelfandMS andOstermanAL (2008) Transcriptional regulation of NAD metabo-lism in bacteria Genomic reconstruction of NiaR (YrxA) regulonNucleic Acids Res X XXX

10 OverbeekR BegleyT ButlerRM ChoudhuriJVChuangHY CohoonM de Crecy-LagardV DiazN DiszTEdwardsR et al (2005) The subsystems approach to genomeannotation and its use in the project to annotate 1000 genomesNucleic Acids Res 33 5691ndash5702

11 OstermanAL and BegleyTP (2007) A subsystems-basedapproach to the identification of drug targets in bacterial pathogensProg Drug Res 64 131 133ndash170

12 HolleyEA SpectorMP and FosterJW (1985) Regulation ofNAD biosynthesis in Salmonella typhimurium expression of nad-lacgene fusions and identification of a nad regulatory locus J GenMicrobiol 131 2759ndash2770

13 TritzGJ and ChandlerJL (1973) Recognition of a gene involvedin the regulation of nicotinamide adenine dinucleotide biosynthesisJ Bacteriol 114 128ndash136

14 ZhuN and RothJR (1991) The nadI region of Salmonellatyphimurium encodes a bifunctional regulatory protein J Bacteriol173 1302ndash1310

15 GroseJH BergthorssonU and RothJR (2005) Regulation ofNAD synthesis by the trifunctional NadR protein of Salmonellaenterica J Bacteriol 187 2774ndash2782

16 PenfoundT and FosterJW (1999) NAD-dependent DNA-bindingactivity of the bifunctional NadR regulator of Salmonellatyphimurium J Bacteriol 181 648ndash655

17 TitgemeyerF ReizerJ ReizerA and SaierMHJr (1994)Evolutionary relationships between sugar kinases and transcrip-tional repressors in bacteria Microbiology 140(Pt 9) 2349ndash2354

18 GerasimovaAV and GelfandMS (2005) Evolution of the NadRregulon in Enterobacteriaceae J Bioinform Comput Biol 31007ndash1019

19 RossolilloP MarinoniI GalliE ColosimoA andAlbertiniAM (2005) YrxA is the transcriptional regulator thatrepresses de novo NAD biosynthesis in Bacillus subtilisJ Bacteriol 187 7155ndash7160

20 BensonDA Karsch-MizrachiI LipmanDJ OstellJ andWheelerDL (2007) GenBank Nucleic Acids Res 35 D21ndashD25

21 ChennaR SugawaraH KoikeT LopezR GibsonTJHigginsDG and ThompsonJD (2003) Multiple sequencealignment with the Clustal series of programs Nucleic Acids Res31 3497ndash3500

22 FelsensteinJ (1996) Inferring phylogenies from protein sequencesby parsimony distance and likelihood methods Methods Enzymol266 418ndash427

23 TatusovRL GalperinMY NataleDA and KooninEV (2000)The COG database a tool for genome-scale analysis of proteinfunctions and evolution Nucleic Acids Res 28 33ndash36

24 KrissinelE and HenrickK (2004) Secondary-structure matching(SSM) a new tool for fast protein structure alignment in threedimensions Acta Crystallogr D Biol Crystallogr 60 2256ndash2268

25 GelfandMS NovichkovPS NovichkovaES and MironovAA(2000) Comparative analysis of regulatory patterns in bacterialgenomes Brief Bioinform 1 357ndash371

26 MironovAA KooninEV RoytbergMA and GelfandMS(1999) Computer analysis of transcription regulatory patterns incompletely sequenced bacterial genomes Nucleic Acids Res 272981ndash2989

27 MironovAA VinokurovaNP and GelrsquofandMS (2000)[Software for analyzing bacterial genomes] Mol Biol 34 253ndash262

28 CrooksGE HonG ChandoniaJM and BrennerSE (2004)WebLogo a sequence logo generator Genome Res 14 1188ndash1190

29 SchaggerH and von JagowG (1987) Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa Anal Biochem 166368ndash379

30 BradfordMM (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding Anal Biochem 72 248ndash254

31 RaffaelliN LorenziT AmiciA EmanuelliM RuggieriS andMagniG (1999) Synechocystis sp slr0787 protein is a novelbifunctional enzyme endowed with both nicotinamidemononucleotide adenylyltransferase and lsquoNudixrsquo hydrolaseactivities FEBS Lett 444 222ndash226

32 HuynenM SnelB LatheW3rd and BorkP (2000) Predictingprotein function by genomic context quantitative evaluation andqualitative inferences Genome Res 10 1204ndash1210

33 OstermanA and OverbeekR (2003) Missing genes in metabolicpathways a comparative genomics approach Curr Opin ChemBiol 7 238ndash251

34 AravindL AnantharamanV BalajiS BabuMM and IyerLM(2005) The many faces of the helix-turn-helix domaintranscription regulation and beyond FEMS Microbiol Rev 29231ndash262

35 ShimaJ PenyigeA and OchiK (1996) Changes in patterns ofADP-ribosylated proteins during differentiation of Streptomycescoelicolor A3(2) and its development mutants J Bacteriol 1783785ndash3790

36 McLennanAG (2006) The Nudix hydrolase superfamily Cell MolLife Sci 63 123ndash143

37 BessmanMJ FrickDN and OrsquoHandleySF (1996) The MutTproteins or lsquoNudixrsquo hydrolases a family of versatile widelydistributed lsquohousecleaningrsquo enzymes J Biol Chem 27125059ndash25062

38 OkudaK NishiyamaY MoritaEH and HayashiH (2004)Identification and characterization of NuhA a novel Nudixhydrolase specific for ADP-ribose in the cyanobacteriumSynechococcus sp PCC 7002 Biochim Biophys Acta 1699245ndash252

39 OkudaK HayashiH and NishiyamaY (2005) Systematic char-acterization of the ADP-ribose pyrophosphatase family in theCyanobacterium Synechocystis sp strain PCC 6803 J Bacteriol187 4984ndash4991

40 GelfandMS KooninEV and MironovAA (2000) Prediction oftranscription regulatory sites in Archaea by a comparative genomicapproach Nucleic Acids Res 28 695ndash705

41 GerdesSY KurnasovOV ShatalinK PolanuyerBSloutskyR VonsteinV OverbeekR and OstermanAL (2006)Comparative genomics of NAD biosynthesis in cyanobacteriaJ Bacteriol 188 3012ndash3023

42 GralnickJA and HauHH (2007) Ecology and biotechnology ofthe genus Shewanella Annu Rev Microbiol 61 237ndash258

43 RodionovDA MironovAA and GelfandMS (2002)Conservation of the biotin regulon and the BirA regulatory signal inEubacteria and Archaea Genome Res 12 1507ndash1516

44 MakarovaKS MironovAA and GelfandMS (2001)Conservation of the binding site for the arginine repressor in allbacterial lineages Genome Biol 2 Research0013

45 RodionovDA and GelfandMS (2005) Identification of abacterial regulatory system for ribonucleotide reductases byphylogenetic profiling Trends Genet 21 385ndash389

46 Ostrovsky de SpicerP and MaloyS (1993) PutA proteina membrane-associated flavin dehydrogenase acts as a redox-dependent transcriptional regulator Proc Natl Acad Sci USA 904295ndash4298

47 KurnasovOV PolanuyerBM AnantaS SloutskyR TamAGerdesSY and OstermanAL (2002) Ribosylnicotinamide kinasedomain of NadR protein identification and implications in NADbiosynthesis J Bacteriol 184 6906ndash6917

48 RaffaelliN LorenziT MarianiPL EmanuelliM AmiciARuggieriS and MagniG (1999) The Escherichia coliNadR regulator is endowed with nicotinamide mononucleotideadenylyltransferase activity J Bacteriol 181 5509ndash5511

Nucleic Acids Research 2008 Vol 36 No 6 2059

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