An Indispensable Gene for NAD Biosynthesis in Salmonellatyphimurium
KELLY T. HUGHES, DUBRAVKA LADIKA, JOHN R. ROTH, AND BALDOMERO M. OLIVERA*
Department ofBiology, University of Utah, Salt Lake City, Utah 84112
Received 11 March 1983/Accepted 2 May 1983
We have located the nadD locus between lip and leuS at 14 min on theSalmonella typhimurium chromosome, and we have shown it to be the structuralgene for nicotinic acid mononucleotide adenylyltransferase. This is the firstindispensable gene of pyridine nucleotide metabolism that has been identified.Mutants altered at this locus, isolated by their 6-aminonicotinamide resistancephenotype, accumulate abnormally large pools of nicotinic acid mononucleotidein vivo; many exhibit a temperature-sensitive lethal phenotype. Enzyme assaysreveal markedly lower transferase activity in mutant extracts than in nadD+extracts. The partial dominance of nadD mutants when placed in a nadD+lnadDdiploid suggests that nicotinic acid mononucleotide adenylyltransferase is amultimeric enzyme.
NAD and NADP and their reduced forms,NADH and NADPH, are the major electronacceptors and donors in cellular metabolism.The pathways for the biosynthesis of these keycofactors are complex. In bacteria such as Sal-monella typhimurium and Escherichia coli,there is both a de novo pathway, starting withaspartate and dihydroxyacetone phosphate, anda complex series of salvage pathways. Thus, S.typhimurium can use nicotinamide, nicotinicacid, nicotinamide ribonucleoside, and even nic-otinamide mononucleotide as exogenous precur-sors for the synthesis of NAD. The presentlydefined pathways for NAD biosynthesis in S.typhimurium are shown in Fig. 1.There is a preference hierarchy for the differ-
ent pathways of NAD biosynthesis in bacteria;when an exogenous source of the pyridine ring isavailable, endogenous synthesis is suppressed(6). However, little is known about the regula-tory interactions between these pathways. All ofthe known genes of pyridine nucleotide metabo-lism map at widely separated locations in boththe S. typhimurium and E. coli chromosomes (2,14).
Inspection of the biosynthetic pathways dia-grammed in Fig. 1 shows that all of the presentlyidentified mutations are in genes in the branchesof the pathway which can be bypassed (for areview, see reference 10). Thus, a lesion in thenadA, nadB, or nadC gene causes auxotrophy,but NAD and NADP can still be synthesizedfrom exogenous precursors. Mutations in thepncA and pncB genes are viable since de novosynthesis, as well as nucleoside salvage, can stilloccur. Even double mutants in the nad and pnc
genes survive if given nicotinamide ribonucleo-side or nicotinamide mononucleotide as a pyri-dine nucleotide source.These branches of the pathway which can be
bypassed converge to the key metabolite, nico-tinic acid mononucleotide (NaMN). NaMN canbe formed by three different enzymatic reac-tions: in the de novo pathway from quinolinate,in the Preiss-Handler salvage pathway from nic-otinic acid, and in the nucleoside salvage path-way by deamidation of nicotinamide mononucle-otide (10). In bacteria, there are no knownalternatives for the metabolic steps betweenNaMN and NADP. Mutants blocked in thesesteps cannot be recovered as auxotrophs sincethe required metabolites are not taken up bycells. One of the initial aims of our study ofpyridine nucleotide metabolism is to obtain mu-tants in this essential branch of the biosyntheticpathway for NAD and NADP. In this report, wepresent evidence that the recently describednadD locus in S. typhimurium (11) is the struc-tural gene for the first essential enzyme ofpyridine nucleotide biosynthesis, NaMN ade-nylyltransferase.
MATERIALS AND METHODS
Bacterial strains. All strains used in this study arelisted with their sources in Table 1. All strains werederived from S. typhimurium LT2.
Media. The E medium of Vogel and Bonner (18),supplemented with 0.2% glucose, was used as minimalmedium. Difco nutrient broth (NB; 8 g/liter), withNaCI (5 g/liter) added, was used as rich medium. Difcoagar was added to a final concentration of 1.5% forsolid medium. The following additives were includedin media as needed (final concentrations): amino acids
FIG. 1. NAD metabolism in S. and E. coli. Theenzyme designations are: 1, L-aspartate oxidase; 2,quinolinic acid synthetase; 3, quinolinic acid phos-phoribosyltransferase, 4, NaMN adenylyltransferase;5, NAD synthetase; 6, nicotinamide mononucleotidedeamidase; 7, nicotinamide mononucleotide glycohy-drolase; 8, nicotinamide deamidase; 9, nicotinic acidphosphoribosyltransferase. The relationship of knowngenetic markers is given. Abbreviations: Na, nicotinicacid; Nm, nicotinamide; NMN, nicotinamide mononu-cleotide; NmR, nicotinamide ribonucleoside; Qa, qu-molinic acid.
(approximately 0.3 mM), adenine and uracil (0.4 mM),lipoic acid (5 ng/ml), tetracycline hydrochloride (25 pLg/ml in rich medium or 10 ,ug/ml in minimal medium),kanamycin sulfate (50 ,ug/ml in rich medium or 125 ,ug/ml in minimal medium), 5',5',5'-trifluoro-DL-leucine(250 ,ug/ml), nicotinic acid and nicotinamide (2 ,ug/ml),6-aminonicotinamide (50 g/ml), 6-aminonicotinic acid(50 ,ug/ml), and quinolinic acid (5 mM), which wasrecrystallized in cold 40%o acetic acid before use.Plates for the assay of leucine excretion were preparedas suggested by J. Calvo (personal communication). A10-mI portion of E-glucose agar was poured into petridishes and allowed to solidify. Another 10-ml portionof the same agar was cooled to 48°C, and the leu-447mutant was added to a concentration of 10i cells perml, miXed, poured on top, and allowed to solidify. Athird 10-ml layer of E-glucose agar cooled to 48°C wasthen poured on top.
Transductional methods. The high-frequency gener-alized transducing bacteriophage P22 mutant (HT105/1) (15) int-201 (1) was used for all transductionalcrosses. Selective plates were spread with 108 cellsand 108 to 109 phage. Transductants were purified andmade phage free by streaking nonselectively on greenindicator plates (5). Phage-free clones were thenchecked for phage sensitivity by cross-streaking withP22 H5 (clear-plaque mutant) phage.
Conjugational methods. F- recipients were grownovernight in nutrient broth before mating. F' donorswere grown overnight in selective minimal media.Plate matings were performed as described by Miller(12). Transconjugants were purified by two successivesingle-colony isolations on selective plates. F' merodi-ploids were cured of their episome with acridineorange by the method of Miller (12).Construction and characterization of tandem nadD
merodiploids. Merodiploids for the nadD region wereconstructed by using TnlO insertions as regions forhomologous recombination (8). TnlO insertions thatmapped near the nadD gene were isolated, and prox-imity was estimated by cotransductional frequency oflinkage to the lip gene. Two TnlO insertions (zbe-1023and zbe-1028) were isolated and were found to be onopposite sides of nadD by three-factor crosses (datanot shown). Hfr strains were constructed with theseTnlO insertions as sites for integration via homologousrecombination with TnlO on several F plasmids (7).Hfr matings determined the TnlO insertions to be inthe A orientation (7) on the chromosome (data notshown). These TnlO insertions near nadD were usedto generate tandem duplication merodiploids (nadD+'lnadD'-) as diagrammed in Fig. 4. The nadD/nadDmerodiploids for complementation tests were alsoconstructed as diagrammed in Fig. 4, except that thenadD189(Ts) allele was used as donor.
Putative merodiploid strains were grown overnightin NB liquid medium to full density (2 x 109 cells perml). These strains were then diluted 106-fold and wereplated for single colonies on NB plates. After over-night incubation, single colonies were replica plated toNB and NB + tetracycline plates. The accumulationof tetracycline-sensitive clones and the presence ofeither nadD+ or nadD mutants (thermosensitive le-thal, 6-aminonicotinamide resistant) were used as indi-cations of merodiploidy.
In the cases in which segregrants of nadD/nadDmerodiploids had identical phenotypes, specific alleleswere determined by recombinational tests. For exam-ple, each segregant from a nadD1891nadDIS7 diploidwould harbor only one of the two alleles. The ability ofphage grown on a segregant to recombine with a straincarrying the nadD157 allele and not with a straincarrying the nadD189 allele indicated that the segre-gant harbored oaly the nadD189 allele. The ability ofnadD mutant diploids to segregate two different alleleswas used to demonstrate that heterozygosity of theduplicated region had been achieved.
solation of frameshift mutations in the E. coli nadDgene present on episome F'254. Strain TT7513[nadD(Ts)/(F'nadD+)] was grown overnight in selec-tive minimal medium and plated onto selective mini-mal agar containing 6-aminonicotinic'acid. A drop ofICR-191 (1 mg/ml) was added to the center of eachplate. Two classes of 6-aminonicotinic acid-resistantmutants were expected. First, those lacking the pncBgene product would be resistant because conversion of6-aminonicotinic acid to 6-amino NAD would beblocked; this class would be analog resistant andwould grow at 42°C. The second mutant class wouldinclude those lacking the F'254 E. coli nadD+ geneproduct. These would be temperature sensitive andanalog resistant owing to the 6-aminonicotinic acidresistance phenotype characteristic of the chromo-sonmal nadD(Ts) haploid, which is recessive to theF'254 nadD+ allele (see below). The 6-aminonicotinicacid-resistant-colonies that arose around the ICR-191spot were picked and screened for temperature sensi-tivity at 42°C. They were then screened for ICR-191revertibility to growth at 42°C with concomitant recov-ery of analog sensitivity. An identifying characteristicof frameshift mutations is their revertibility by theframeshift mutagen ICR-191 (13). The nadD frameshiftmutants isolated were then screened for the inability to
complement the nadD157 allele when the episomecarrying the mutation was introduced into a straincarrying this allele.
Preparation of [t4CINaAD. Radiolabeled [carboxyl-"C]nicotinic acid adenine dinucleotide ([carboxyl-"C]NaAD) was prepared by the nicotinamide adeninedinucleotidase-catalyzed exchange reaction between[carboxyl-14C]nicotinic acid and unlabeled NAD. Thereaction mixture contained 74 mg ofNAD, 0.3 ,mol of[carboxyl-14Cjnicotinic acid (New England NuclearCorp.; specific activity, 50.4 mCi/mmol), 0.1 mmols ofTris-hydrocholoride (pH 8.0), and 23 mg of nicotin-amide adenine dinucleotidase (Sigma Chemical Co.) ina reaction volume of 1 ml. The reaction was incubatedat 37°C with shaking and was monitored by spotting 1,Il onto GFC-filter disks and then washing in 7:3 (vol/vol) acetone-ethanol-'(this washes off nicotinic acidwhile retaining NaAD). The reaction was terminatedwhen the production of [14C]NaAD leveled off (2 to 3h). The entire reaction mixture was spread on What-man 3MM filter paper and was chromatographed withcitrate-ethanol used as the developing solvent (19).The chromatogram was dried, and the NaAD bandwas cut into 1.5-cm strips. These were desalted bysuspension in methanol for 15 min and then dried. The[14C]NaAD was concentrated to one end of the stripby ascending chromatography with distilled water asthe solvent. When the solvent front reached the top,the strips were centrifuged at 5,000 rpm for 20 min,and the eluate was collected.
Preparation of [14CJNaMN. Radiolabeled [carboxyl-14C]NaMN was prepared from [14C]NaAD (above) bythe action of snake venom phosphodiesterase (Sigma).Before using a commercial preparation of the phos-phodiesterase, contaminating 5'-nucleotidase was in-activated (17). The reaction mixture for the productionof [14C]NaMN was 10 ,umol of Tris-hydrochloride (pH8.6), 0.1 ,mol of MgSO4, 1 ,ug of snake venomphosphodiesterase, and 30 pmol of [14C]NaAD in atotal volume of 1 ml. The reaction was monitored onpolyethyleneimine-impregnated cellulose by thin-layerchromatography (solvent: 100 ml of sodium phosphatebuffer, pH 6.8, 60 g of NH4SO4, and 2 ml of n-propanol). When thin-layer chromatography indicatedthat the reaction was complete ([14C]NaMN produc-tion leveled off), the reaction mixture was spread on3MM chromatography paper and chromatographedwith a citrate-ethanol buffer system. The chromato-gram was dried, and the NaMN band was cut out anddesalted by suspension in methanol for 15 min. It wasthen cut into 1.5-cm strips, and the ['4C]NaMN wasisolated as described for [14C]NaAD above.
Preparation of crude extracts. Overnight cultures ofstrains to be assayed were grown in NB or (formerodiploids) NB t 15 ,ug of tetracycline per ml.These cultures were used to inoculate 100 ml ofNB orNB + tetracycline to a cell density of approximately10 Klett units. The cultures were grown to 100 Klettunits (ca. 6 x 10' cells per ml), harvested by centrifu-gation, washed once with E medium and a second timein 0.2 M Tris-hydrochloride buffer (pH 7.5), andsuspended in 2.5 ml of 0.2 M Tris (pH 7.5). The cellswere lysed by use of a French press. The extract wascentrifuged at 15,000 rpm for 25 to 30 min to removedebris. Samples of extract (200 RI.l) were quick-frozenin dry ice-methanol and were kept at -70°C untilneeded for NAD synthetase assays. All NaMN ade-
nylyltransferase assays were done with freshly pre-pared extracts. All NAD synthetase assays reportedwere done with extracts frozen at -70°C for 1 week.No significant NAD synthetase or NaMN adenylyl-transferase activity was lost after the extracts hadbeen frozen for 2 weeks at -70°C. Protein concentra-tions were determined by a Coomassie dye-bindingassay (3) with commercial reagents (Bio-Rad Labora-tories). Bovine serum albunmin (Sigma) was used as astandard.Assay for NaMN adenylyltransferase. NaMN adenyl-
yltransferase was assayed essentially as described byDahmen et al. (9), except that the NaMN concentra-tion used was 80 ,uM. After termination, the reactionmixture was spread onto 3MM chromatography paperand was chromatographed with the citrate-ethanolbuffer system. The chromatogram was cut into 1.5-cmstrips, which were counted with a Beckman LS200liquid scintillation counter.
Assay for NAD synthetase. NAD synthetase wasassayed as described by Spencer and Priess (16).However, unlabeled NaAD was added to a final con-centration of 10-5 M. Upon completion, the reactionmixture was spread onto DEAE chromatography pa-per and was chromatographed with 0.25 M ammoniumbicarbonate. The chromatogram was cut into 1.5-cmstrips and counted as described above.
Analysis of pyrldine nucleotide pools in vivo. Culturesof wild-type S. typhimurium (Nad+) or nadD mutantswere grown in E medium containing 0.2% glucose toan absorbance at 650 nm of 0.4. Cultures were thenlabeled with [3H]nicotinic acid (to a final concentrationof 0.05 mCi/ml, 0.8 Ci/mmol), and the cells wereharvested after 15 min at 37°C or, in the case of thetemperature-sensitive mutants, 1 h at 42°C. The cul-tures were harvested by centrifugation in an RC2BSorvall centrifuge, and the cell pellet was washedtwice with E medium containing 0.2% glucose, 10 ,gof nicotinic acid per ml, and 10 ,ug of nicotinamide perml. The cell pellet was suspended in 0.4 ml of 0.3 MHCl and kept overnight at 0°C.Chromatography was performed by using either
DEAE-paper (Whatman DE81) with 0.25 MNH4HCO3 as the developing system, or the citrateethanol system of Witholt (19). Before chromatogra-phy on DEAE-paper, the cell extract was neutralizedwith 1 M Tris-hydrochloride (pH 8). A commercialpreparation of bacterial alkaline phosphatase (Wor-thington Diagnostics; BAF-F; 3 ILI) was added, and themixture was incubated for 15 min at 37°C. A second 3-,ul sample of alkaline phosphatase was then added, anda second incubation for 15 min at 37°C was carried out.The phosphatase-treated extract (in which NADP isconverted to NAD and NaMN is converted to nicotin-ic acid riboside) was then chromatographed on DEAE-paper, and the radioactivity was analyzed as previous-ly described. This is a rapid method for assessing thefraction of'NaMN present.
Aisay'for leucine excretion. Excretion of leucine wasdetermined qualitatively. Colonies were transferredwith sterile toothpicks to the surface of the leucineexcretion assay plates described above. After incuba-tion for 36 to 48 h, leucine excretion was detected asgrowth of the leu-447 mutant forming a halo of growthbeneath the transferred colonies. Both leucine excre-tion and resistance to trifluoroleucine were used toscreen for the presence of the leuS marker (4).
FIG. 2. Analysis of nicotinic acid metabolites in anS. typhimurium nadD strain shifted to the nonpermis-sive temperature. S. typhimurium TR6418, which car-
ries the nadDI59(Ts) allele, was grown at 30°C to an
absorbance at 650 nm of 0.4. The culture was thenshifted to 42°C and incubated for 1 h at 42°C in thepresence of [3H]nicotinic acid. The cells were thenharvested, and the internal pools of pyridine nucleo-tides were analyzed as described in the text. Thechromatography system used is the citrate ethanolsystem described by Witholt (19). Marker positionsare indicated by the bars. NADP migrates near theorigin, overlapping the NAD peak in this chromato-graphic system.
RESULTS
Isolation and characterization of mutants defec-tive in NaMN adenylyltransferase activity. Theisolation of 6-aminonicotinamide-resistant mu-tants of S. typhimurium, including mutants thatwere also thermosensitive lethals, has been de-scribed (10). All thermosensitive lethal mnutantsand some which are simply 6-aminonicotinamideresistant were found to be closely linked to thelip locus of S. typhimurium. This previouslyunmapped locus involved in pyridine nucleotidemetabolism was designated the nadD locus. Wehave further characterized nadD strains by la-beling pyridine nucleotide metabolites in vivo.To identify the metabolic defect in these nadD
strains, the mutant cells were grown in mediumcontaining [3H]nicotinic acid, and the intracellu-lar pyridine pools were analyzed (Fig. 2). In awild-type strain, all intracellular radioactivity isfound in fractions near the origin in the chro-matographic system used, consistent with theNAD and NADP pools being the only signifi-cantly labeled metabolites. However, in thenadD mutant, a major fraction of the intracellu-lar radioactivity was present as NaMN (Fig. 2).A variety of biochemical criteria established thatthe material accumulating was NaMN. The ra-
dioactive material comigrated with the authenticnucleotide (NaMN) in two separate chromatog-
raphy systems. After treatment with alkalinephosphatase, the radioactivity comigrated withauthentic nicotinic acid ribonucleoside. In theparticular nadD mutant shown in Fig. 2, therewas more NaMN than NAD; in nadD+ strains,the NaMN pool was below detectable limits. Inaddition to a large pool ofNaMN, this nadD(Ts)mutant also accumulates a substantial nicotinicacid pool under these conditions.Although the largest pools ofNaMN are found
in the thermosensitive-lethal nadD mutants, asignificant pool of NaMN accumulates even inthe non-temperature-sensitive nadD190 mutant,which has a pool of NaMN which is 20% of thetotal pyridine nucleotide pool. No NaMN isdetectable in the wild-type strain (results notshown). These results are consistent with adefect in NaMN adenylyltransferase (also calledNaAD pyrophosphorylase). The fact that manynadD mutants are temperature-sensitive lethalsis also consistent with this assignment in that theenzyme catalyzing the conversion of NaMN toNaAD would be an indispensable enzyme forNAD biosynthesis, since none of the subsequentintermediates in the pathway is taken up andused as a nutritional supplement.Enzyme assays. The accumulation of NaMN
suggests that a defect in NaMN adenylyltrans-ferase in these mutants causes the analog resist-ance and temperature sensitivity. Crude extractsof all nadD mutants were assayed for NaMNadenylyltransferase activities (Table 2). AllnadD temperature-sensitive strains have lessthan 2% of the NaMN adenylyltransferase activ-ity of the wild-type strain, even when grown andassayed at the permissive temperature. ThenadDI90 mutant, which does not show a tem-perature-sensitive phenotype, had less than 25%of wild-type activity. As a control, NAD synthe-tase activity was also assayed. There was nosignificant difference in the NAD synthetaseactivities between the mutants and the wildtype. These assays are strong evidence that thenadD gene codes for NaMN adenylyltransfer-ase.
Genetic mapping of the nadD gene. Our previ-ous studies have shown that the nadD locus istightly linked to the lip gene (located at 14 min)by P22-mediated transductional crosses (11).Three-factor crosses were used to locate theprecise position of the nadD gene. The nadDgene is located between lip and the leuS locus(Table 3). A detailed map of the region is shownin Fig. 3. This map includes P22 cotransductionfrequencies between genetic markers in thenadD region. The locations of the two TnJOinsertions used to generate nadD tandem diploidstrains (discussed below) are also included.Complementation and recombination studies.
a All strains were grown in NB medium at 30°C before assay.b The value of 1 represents a specific activity of 868 and 672 pmol/min per mg of protein, respectively, for the
transferase and synthetase.
constructed by homologous recombination ofTnlO insertions as described by Chumley andRoth (8) (Fig. 4). Homologous recombinationgenerated a small duplication of the nadD regionwith a TnlO insertion betwen the two copies.The duplication is maintained by selection oftetracycline resistance; in the absence of tetra-cycline, tetracycline-sensitive clones arise viahomologous recombination, causing deletion ofthe TnlO and retention of only one of the twonadD alleles. In the first set of complementationexperiments, one nadD(Ts) allele (nadDl89)was Used in the donor strain, and dipfoids of thisallele and all other nadD mutant alleles wereconstructed to test for complementation.
All of the tandem duplication complementa-tion diploids listed in Table 4 are able to growand show analog resistance at 30°C, as do theparental haploid strains. At the nonpermissivetemperature (42°C); which is lethal for all tem-perature-sensitive haploids, the diploids are un-able to grow, except for the diploid which in-cludes the nadDI90 (temperature resistant)
TABLE 3. Three-factor crosses: position Qf nadDwith respect to lip and leuSa
Resulting No. with RelativeCos Selected characters indicated frequencyCosmarker (no. (% of
aDonor was strain TR6419 (lip-2+ nadD157+leuS2); recipient was strain TT7261 (lip-2 nadD157leuS2+).
allelle (Table 4). This demonstrates that allnadD(Ts) alleles tested belong to a single com-plementation group.
All of the nadD(Ts)InadD(Ts) merodiploids inTable 4A (lines b through g) recombine to giveoccasional haploid nadD+ (tetracycline-sensi-tive) segregants, except for nadD1891nadD158and nadD1891nadDJ89. All nadD(Ts) alleleswere tested in standard transductional tests forthe ability to recombine with each other to yieldnadD+ recombinants (data not shown). ThenadD(Ts) alleles were found to belong to threerecombinational groups: (i) nadD157, nadD187,and nadD188; (ii) nadD158 and nadD189; and(iii) nadD159. Since all these belong to a singlecomplementation group, these results suggestthat the nadD(Ts), 6-aminonicotinamide-resist-ant mutants affect three local regions within asingle complementation group.
purEzbe-1 023::Tn 10lip A nadD
zbe-1 028::TnlOleuS A nag
- 5 - -83-0
37~~~8*-981 .4 ~~61
I 47-- *-
57--
(<0.2)(
(<0.2)(<0.2)
FIG. 3. P22-mediated cotransduction frequenciesof the nadD region of the S. typhimurium genetic map.A scaled portion of the map from 12 to 15 n-in isillustrated here. Included in the map are the locationsof the two TnlO insertions used to generate tandemduplications of the nadD region (see the text). ThenadD157 allele was used in all mapping transductions.The orientations of the TnlO insertions were deter-mined as described by Chumley et al. (7).
FIG. 4. Generation of tandem duplications of thenadD region. Unequal recombination between TnlOelements yields lip+ nadD+ recombinants containingtwo copies of the nadD region. The TnlO insertions inthe donor and recipient strains have the same orienta-tion as determined by TnlO-directed Hfr formation.When selection for tetracycline resistance is removed,tetracycline-sensitive clones arise in which homolo-gous recombination between the duplicated region hasoccurred. This results in the loss of TnlO and one ofthe two nadD alleles. Recombination event I results ina tetracycline-sensitive nadD+ haploid segregant; re-combination event II results in a tetracycline-sensitivenadD haploid segregant.
A second set of complementation experimentswas carried out with an E. coli episome (F'254)which is known to carry the homologous regionof the E. coli chromosome (from 7 to 14 min)(see Fig. 5) (2). This E. coli episome corrects thetemperature sensitivity phenotype when intro-duced into a nadD(Ts) mutant and restoressensitivity to 6-aminonicotinamide. This epi-some therefore presumably carries an E. colinadD+ gene (Table 4B, lines b through g). To testfor complementation, a frameshift mutation wasintroduced into the E. coli nadD allele by usingICR-191 mutagenesis of a merodiploid straincarrying nadDJ57 in the chromosome and the E.coli F'254 nadD+ episome (13). Such a mutant(nadD) episome no longer complemented thenadD157 allele of S. typhimurium (Table 4B, linej), resulting in 6-aminonicotinamide resistance
(the episomal nadD mutation is induced to re-vert by ICR-191, so it is not a large deletionmutation). The mutant episome was tested forits ability to complement all other mutant Sal-monella nadD alleles. As shown in Table 4B,lines j through p, the mutant E. coli nadDframeshift mutant allele failed to complementany of' the Salmonella nadD mutant alleles,consistent with all mutations belonging to asingle complementation group.Dominance studies. Merodiploids containing
tandem duplications with both the nadD+ andnadD mutant alleles were constructed as shownin Fig. 4. The nadD+InadD merodiploids werecharacterized, and as expected, the nadD+ al-lele was found to be dominant to the tempera-ture-sensitive mutant alleles at 42°C (i.e., themerodiploids were viable at 42°C). However, thesecond phenotype, 6-aminonicotinamide resist-ance, was partially dominant at the permissivetemperature for all nadD(Ts) and nontempera-ture-sensitive alleles tested. Wild-type nadD+strains do not grow on 6-aminonicotinamide-containing plates. The nadD mutants form colo-nies that are visible after approximately 12 h at
FIG. 5. Chromosome of S. typhimurium with rele-vant markers. Included in the figure is the E. coliepisome F'254, which harbors the nadD region on theE. coli chromosome (2).
30°C; the nadD+InadD(Ts) merodiploid formscolonies that are detectable at 24 h, and thenadD+InadD190 (temperature resistant) merodi-ploid forms colonies that appear at 36 h at thesame temperature. At 42°C, no nadD+lnadD(Ts)merodiploid grew on 6-aminonicotinamide-con-taining plates. Thus, nadD+ is dominant overnadD(Ts) at 42°C but is codominant with respectto 6-aminonicotinamide resistance at permissivetemperatures. In contrast, the E. coli episomeF'254, which complemented all mutant nadDalleles, was dominant to Salmnonella nadD al-leles for both temperature sensitivity and analogresistance. These results suggest that the Salmo-nella nadD product is a multimeric enzyme andthat the nadD(Ts) mutants produce no function-al product at 42°C. At 30°C, the mutant proteinmay be able to form active but abnormal mul-timers with the wild-type Salmonella proteinand allow 6-aminonicotinamide resistance. TheE. coli product may be unable to form mixedmultimers with the Salmonella nadD product.
This leads to dominance of the E. coli nadD+allele for both the temperature sensitivity and 6-aminonicotinamide resistance phenotypes.Enzyme assays were also performed on a
merodiploid containing the nadD+lnadD(Ts) al-leles grown at various temperatures. If thenadD+/nadD(Ts) merodiploid was grown andassayed at 30°C, enzyme activity was signifi-cantly lower than with the wild type. However,if the diploid was pregrown at 42°C, the enzy-matic activity detected was essentially wild type(Table 5). Under our assay conditions, all mero-diploids showed significant enzyme activity, butthe specific activity was decreased in nadD+InadD(Ts) cells grown at 30°C as compared to thesame strains grown at 42°C, consistent with thecodominant phenotype.
DISCUSSIONIn a previous report, we described three new
classes of mutants that are resistant to 6-amino-nicotinamide (11). One of these classes, whichwe designated nadD, mapped at a previouslyundefined locus of pyridine nucleotide metabo-lism and yielded a high proportion of mutantswhich were both temperature-sensitive lethaland 6-aminonicotinamide resistant; in fact, alltemperature-sensitive lethals obtained by thisselection map at the nadD locus. In the presentstudy, the nadD gene and the encoded producthave been more precisely defined, both geneti-cally and biochemically. Additional mappingdata demonstrate that nadD maps between thelip and leuS loci at 14 min on the Salmonellachromosome.We have also presented several lines of evi-
dence in this report that the nadD gene codes foran enzyme in the essential branch of the NADbiosynthetic pathway, NaMN adenylyltransfer-ase (EC 2.7.7.18), an enzyme catalyzing themetabolic reaction: NaMN + ATP -* NaAD +
PPi.First, in vivo studies show that mutants in the
nadD locus accumulate NaMN, which would beexpected if the transferase is the defective en-
TABLE 5. NaMN adenylyltransferase assays of nadD diploidsActivity at:
a Strain LT2 was grown in NB medium before assay. The nadD diploid strains were grown in NB plus 15 Fig oftetracycline per ml to maintain the duplication. All assays were performed at 30°C.
b Specific activity expressed in picomoles per minute per milligram of protein.
zyme. Second, enzymatic assays of mutant ex-tracts show reduced NaMN adenylyltransferaseactivity. Indeed, all mutants which were both 6-aminonicotinamide resistant and temperature-sensitive lethal had no detectable activity forthis enzyme either at the permissive or nonper-missive temperature. The one mutant studiedthat was 6-aminonicotinamide resistant but nottemperature-sensitive lethal had 25% of the ac-tivity of the wild-type strain. Finally, merodi-ploids with both the nadD+ and nadD(Ts) allelesare viable at 42°C (nadD+ is dominant) andpossess enzymatic activity.The nadD locus comprises a single comple-
mentation group. As expected, nadD+ is domi-nant over nadD(Ts) with respect to viability at42°C. An unexpected observation is that nadD+InadD(Ts) merodiploids were codominant withrespect to 6-aminonicotinamide resistance at thepermissive temperature. In contrast, the merodi-ploids at the nonpermissive temperature were 6-aminonicotinamide sensitive. The most straight-,forward explanation for this result is that NaMNadenylyltransferase is a multimeric enzyme. Atthe nonpermissive temperature, protein codedfor by the temperature-sensitive allele of thenadD gene cannot be assembled. Thus, all trans-ferase activity found is wild type, leaving the celltemperature resistant and 6-aminonicotinamidesensitive. At the permissive temperature,mixed-subunit enzyme is made, and the mixed-multimer protein causes the intermediate pheno-type observed. The results with nadD+InadD(Ts) diploids constructed by introducingthe E. coli F'254 episome are consistent with theabove hypothesis if it is assumed that mutantSalmonella subunits do not assemble into mul-timers with heterologous, wild-type E. coli sub-units so that wild-type E. coli multimers confer a6-aminonicotinamide sensitivity phenotype onthe interspecies merodiploid at all temperatures.The identification of the nadD locus as a
structural gene for NaMN adenylyltransferaseshould permit a detailed examination of howexpression of the essential branch of the pyri-dine nucleotide biosynthetic pathway is regulat-ed.
ACKNOWLEDGMENTSThis work was supported by Public Health Service grant no.
GM25654 from the National Institutes of Health. K.T.H. wassupported by predoctoral training grant T32-GM-07537 fromthe National Institutes of Health.
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