Activity of the Nicotinamide Mononucleotide Transport Is Regulated in Salmonella typhimurium NING ZHU , BALDOMERO M. OLIVERA , AND JOHN R. ROTH * Department of Biology , University of Utah , Salt Lake City , Utah 84112 System Transport of nicotinamide mononucleotide ( NMN ) requires two functions , NadI ( T ) and PnuC .
The PnuC protein is membrane associated , as judged by isolation of active TnphoA gene fusions and demonstration that the fusion protein is membrane associated .
The PnuC function appears to be the major component of the transport system , since mutant alleles of the pnuC gene permit NMN transport in the absence of NadI ( T ) function .
We present evidence that the activity of the NMN transport system varies in response to internal pyridine levels ( presumably NAD ) .
This control mechanism requires NadI ( T ) function , which is provided by a bifunctional protein encoded by the nadI gene ( called nadR by Foster and co-workers [ J. W. Foster , Y. K. Park , T. Fenger , and M. P. Spector , J. Bacteriol .
The nadl protein regulates transcription of the nadA and nadB biosynthetic genes and modulates activity of the NMN permease ; both regulatory activities respond to the internal pyridine nucleotide level .
provided by the pnuC gene product and that this transport activity is regulated by the nadI ( T + ) function , probably in response to internal NAD levels .
We propose that the bifunctional NadI protein senses internal NAD and controls both transcription of synthetic genes and the activity of the NMN transport system .
Salmonella typhimurium can derive pyridines from exog-enous nicotinamide mononucleotide ( NMN ) by several routes .
The first route , which does not require NMN transport , involves cleavage of NMN by a periplasmic glycohydrolase to yield nicotinamide ( NM ) , which can be taken up and converted to NAD by the sequential action of the pncA , pncB , nadD , and nadE gene products ( 2 , 15 , 17 , 18 , 22 , 23 ) ( Fig. 1 ) .
The second route requires transport of intact NMN ( 10 , 17 , 30 , 31 , 46 , 51 ) .
Internal NMN can be deaminated to yield nicotinic acid mononucleotide ( NAMN ) , which is converted to NAD by the sequential action of the nadD and nadE gene products ( 18 , 22 , 23 ) .
Some internal NMN may also be cleaved by an internal glycohydrolase to yield NM ( Fig. 1 ) .
Two classes of mutations , pnuA and pnuC , have been described which fail to use exogenous NMN as a pyridine source .
It has been inferred that these mutants fail to transport NMN ( 17 , 30 , 46 , 51 ) .
The pnuC gene maps at min 17 of the Salmonella chromosome ( 46 , 51 ) and is located promoter distal to the nadA gene in a regulated operon ( 51 ) .
The pnuC gene is expressed by both the regulated main promoter of this operon and a weaker constitutive promoter located within the operon ( 51 ) .
The pnuA mutations map at min 99 , immediately adjacent to nadI mutations which eliminate a repressor ; this repressor mediates transcriptional control of the nadB gene and the nadA-pnuC operon ( 10 , 16 , 20 , ' 50 ) .
Genetic studies and DNA sequencing of the nadI-pnuA region indicate that ` both types of mutations affect a single gene that encodes a bifunctional protein , acting both as a repressor ( NadI function ) and as a component of the NMN transport system ( PnuA function ) ( 17a , 52 ) .
We will designate this gene nadI and refer to the R ( repression ) and T ( transport ) functions .
Mutations causing only a defect in transport will be designated nadI ( R + T - ) ; mutations eliminating both transport and repression will be designated nadI ( R-T - ) .
Mutants defective only in repression will be designated nadI ( R-T + ) .
( Foster and co-workers [ 17a ] refer to this gene as nadR .
) In this report , we present evidence that NMN transport is MATERIALS AND METHODS Bacterial strains .
All strains used in this study are derived from S. typhimurium LT2 and are listed in Table 1 .
Mu dA refers to a conditionally transposition-defective derivative ( 24 ) of the original Mu dl ( Lac Ap ) phage of Casadaban ` and Cohen which forms operon fusions ( 5 ) .
Mu dJ refers to a transposition-defective mini-Mu phage , Mu dl-1734 ( Lac Kmr ) , constructed by Castilho et al. ( 6 ) .
This phage lacks transposition functions and carries kanamycin resistance .
Mu dP refers to a transposable P22 prophage flanked by ends of the Mu prophage ' ( 49 ) .
TnJOd ( Tc ) refers to a small transposition-defective derivative of TnJO ( TnJO Dell6 Dell7 Tetr ) constructed by Way et al. ( 48 ) .
TnJOd ( Cm ) refers to a transposition-defective derivative of transposon TnJO constructed by Elliott and Roth ( 14 ) .
The E medium of Vogel and Bonner ( 47 ) , supplemented with 0.2 % glucose , was used as the minimal-medium .
Difco nutrient broth ( NB ; 8 g/liter with 5 g of NaCl per liter ) was used as the rich-medium .
Difco agar was added at a final concentration of 1.5 % for solid medium .
Nutrients to feed auxotrophs were included in minimal media at final concentrations described ` by Davis et al. ( 12 ) ; exceptions are indicated in the ' text .
Antibiotics were added to media at the following final concentrations : ampicillin ( sodium salt ) , 30 , ug/ml in NB and 15 , ug/ml in E medium ; tetracycline hydrochloride and chloramphenicol , 20 , ug/ml in NB and 10 , ug/ml in E medium ; and kanamycin sulfate , 50 , ug/ml in NB and 125 , ug/ml in E medium .
All antibiotics were obtained from Sigma Chemical Co. .
Media containing ampicillin were always prepared fresh before use .
5-Bromo-4-chloro-3-in-dolyl-p-D-galactoside ( X-Gal ) dissolved in N,N-dimethyl formamide ( 20 mg/ml ) was added to media at a final concentration of 25 , ug/ml .
5-Bromo-4-chloro-3-indolylphosphate , toluidine salt ( X-P ) , also dissolved in N,N-dimethyl formam-ide ( 20 mg/ml ) was added to media at a final concentration of 50 pug/ml .
All transductional crosses were mediated by a mutant of phage P22 ( HT105/1 int-201 ) which performs generalized transduction with high frequency .
The int mutation of this phage was added by Roberts ( 39a ) to the P22 HT105/1 phage of Schmieger ( 45 ) .
To select for the inheritance of the Kmr marker of Mu dJ and the Cmr marker of TnJOd ( Cm ) , the mixture of recipient cells and donor phages was spread on NB plates and incubated overnight before replica printing to selective plates .
In all other crosses , selective plates were spread directly with 2 x 108 cells and 108 to 109 phages .
Transductants were purified , and phage-free clones were isolated by being streaked nonselectively onto green indicator plates ( 7 ) .
Phage-free clones were identified on green indicator plates and then tested for phage sensitivity by cross-streaking with P22 H5 phage , a clear-plaque mutant of P22 .
Hydroxylamine mutagenesis of localized regions of the Salmonella chromosome was done by treating P22 transducing phage as described by Davis et al. ( 12 ) .
By selecting for inheritance of fragments carrying a particular region , one can obtain strains mutagenized for a single small region of the chromosome .
The general method is that of Hong and Ames ( 21 ) .
Diethyl sulfate mutagenesis of cells was done as described by Roth ( 41 ) with some modifications .
Minimal medium ( 5 ml ) with no added carbon source was equilibrated with 0.2 ml of diethyl sulfate at 37 °C for 1 h before addition of 0.1 ml of an overnight culture .
After mutagenesis for 30 min at 37 °C , an aliquot was removed , diluted 1:50 into fresh me-dium without mutagen , and grown to saturation .
Mutants can be isolated either by direct selection or by screening among single colonies derived from the culture .
Insertion mutagenesis with the Mu dJ and Mu dK transposons was done by the cis complementation method described previously ( 25 ) .
The nonspecific acid phosphatase of S. typhimurium ( 29 ) is sufficiently active to make all strains score positive for the chromogenic phosphatase substrate , X-P .
Therefore , all TnphoA insertion mutagenesis ( 32 ) is done in strains carrying a phoN or a phoP mutation that eliminates this activity ( 53 ) .
To introduce the TnphoA element for transposition mutagenesis , a donor strain ( TT15088 ) was constructed which carries the TnphoA element near a properly oriented locked-in P22 prophage ( Mu d-P22 ) ( 49 ) ; this strain also carries a plasmid which produces P22 tail protein .
When the P22 prophage is induced , the resulting lysate has a very high frequency of transducing particles that include TnphoA .
This lysate is a suitable donor of TnphoA for mutant isolation ; the lysate is used to transduce a recipient strain that carries a phoN or phoP mutation .
Selection is made for Kmr , and transductant colonies are screened for interesting insertion mutations ( 2a ) .
PhoA + insertion mutants were initially identified as blue colonies on media containing X-P ; they were then assayed for alkaline phosphatase activity .
Selection for revertants of NMN transport mutants .
A pnuA or pnuC mutant strain was grown overnight in NB , and cells were washed once with E medium lacking glucose .
A 0.1-ml aliquot of washed cell suspension was plated on medium containing 10-4 M NMN .
Revertants able to transport NMN grow up as colonies after incubation at 37 °C for 2 days .
All enzymatic activity assays were de scribed previously : the NADH-ferricyanide oxidoreductase assay is that of Jaworowski et al. ( 28 ) ; the glutamate dehydrogenase assay is that of Coulton and Kapoor ( 11 ) ; P-lac-tamase activity was assayed as described by Sargent ( 42 ) and Sawai et al. ( 43 ) .
The units of,3-lactamase activity are reported as nanomoles of substrate hydrolyzed per minute by enzymes from 1 ml of original cell culture , based on the calculation described by Sawai et al. ( 43 ) .
The P-galactosi-dase activity assay is that of Miller ( 35 ) , and the,-galactosi-dase activity reported is nanomoles per minute per optical density unit ( at 650 nm ) of cells .
The alkaline phosphatase assay is that of Brickman and Beckwith ( 4 ) and Manoil ( 31b ) , and the units of phosphatase were calculated as described by Brickman and Beckwith ( 4 ) .
Cell fractionation was done by the method of Manoil and Beckwith ( 33 ) .
The modifications of the method are that cells were resuspended in 1/6 volume of cold spheroplast buffer containing 40 % sucrose before osmotic shock , and lysed spheroplasts were centrifuged for 60 min at 40,000 rpm in a 45 Ti rotor to separate the membrane and cytoplasmic fractions .
Each fraction was assayed for glutamate dehydrogenase , NADH-ferricyanide oxidoreductase , and 3-lactamase .
These enzymes are known to be associated with cytoplasmic , membrane , and periplasmic fractions , respectively ( 11 , 28 , 42 , 43 ) .
Preparation of carbonyl - ' 4C-NMN .
Carbonyl - ' 4C-NMN was made from carbonyl-4C-NAD by a nucleotide pyro-phosphatase reaction ( 31a ) .
The reaction mixture contains 47.5 , uM NAD , 9.5 , uCi of carbonyl - 4C-NAD per ml ( 1 p.Ci / 20 , Il ) , 4.75 mM MgCl2 , 9.5 mM Tris hydrochloride ( pH 7.4 ) , and 0.2 U of nucleotide pyrophosphatase ( type II [ from Crotalus sp. ] ; Sigma ) .
The reactions were carried out at 37 °C for 60 min and monitored by spotting 2 , u1 of reaction mix onto a thin-layer chromatograph plate ( plastic polyethylene-imine-cellulose F ; EM Science ) with 2 , ul of 10 mM NAD-O0 mM NMN solution spotted at the same place as markers 131 nadC nadE nadA n nadD I ( 4 nadB coon CH2 m c COOHl Ha '' NADP cs - / 1 ( 2 ) i N , Ad NaR-ADt-R \ NaAD ( 5 ) N * Ad ( 6 ) R ( 1 ) OuII o.c a2copos H2M COOH R-N-A NAD A -0 T-10cH2 Asp QA IA DHAP NH2 ; , NN -0 -- 0-CH NO 0P03 NA NM NMN ( 9 ) ( 12 ) pnuE NAD FIG. 1 .
The NAD metabolic pathway of S. typhimurium .
The enzymes included are ( 1 ) L-aspartate ( Asp ) oxidase , ( 2 ) quinolinic acid ( QA ) synthetase , ( 3 ) quinolinic acid phosphoribosyltransferase , ( 4 ) nicotinic acid mononucleotide ( NaMN ) adenyltransferase , ( 5 ) NAD synthetase , ( 6 ) NAD kinase , ( 7 ) DNA ligase ( lig ) , ( 8 ) NMN deamidase , ( 9 ) NMN glycohydrolase , ( 10 ) NM deamidase , ( 11 ) NA phosphoribosyltransferase , and ( 12 ) NAD pyrophosphatase .
DHAP , Dihydroxyacetone phosphate ; IA , iminoaspartate ; PRPP , 5-phosphoribosyl-1-pyrophosphate .
Genetic loci corresponding to enzymatic steps are indicated above the reaction arrows .
b Nomenclature is as described by Demerec et al. ( 13 ) , Chumley et al. ( 8 ) , and Schmid and Roth ( 44 ) .
The thin-layer chromatography plates were developed by 1 M LiCl .
After drying , the NAD and NMN spots were located under UV light and cut out to ` count radioactivity .
Greater than 96 % of the NAD was converted to NMN .
Strains were grown overnight in E medium containing 10-6 M nicotinic acid ( NA ) , diluted 10-fold in the same medium , and grown to mid-log phase ( 140 Klett units ) .
Cells were harvested by centrifugation and washed twice in E salt solution before being resuspended into a half volume of E medium containing glucose ( 0.2 % ) .
Cells were preincubated by shaking for 20 min at 37 °C in a water bath .
The assay was started by addition of 14C-NMN to a final concentration of 10 , uM ( 0.57 ' .
One milliliter of sample ' was removed at ` each time point , and cells were harvested by filtration through Millipore filters ( 0.45-ptm pore size ) .
Filters were washed three times ` with 1.5 ml of E salt solution and then dried before determination of radioactivity .
The effect of NA on NMN uptake was tested by adding NA to a final concentration of 2 x 10-4 M ; NA was added 20 min after addition of 14C-NMN .
The effect of chloramphenicol was tested by adding the antibiotic ( 20 mg/ml in 95 % ethanol ) to a final concentration of 20 , ug/ml .
Chloramphenicol was added with 14C-NMN when the assay was started .
Assay of protein synthesis .
The method was modified from that of Imamoto ( 27 ) .
Cells to be assayed were prepared as for NMN uptake assays ( see above ) .
The assay was ' started by adding 14C-leucine ( 251 mCi/mmol ) and nonradioactive NMN to cell ` suspensions to final concentrations of 4 nCi/ml and 10 FM , respectively .
A 0.5-ml sample was removed at each time point and mixed with 0.5 ml of 10 % trichloroacetic acid ( 200 , ug of leucine per ml ) .
Protein precipitates ' were recovered by filtration on Millipore filters ( 0.45 - , um pore size ) and washed three times with 1.5 ml of deionized H20 .
Filter disks were dried before radioactivity was measured .
The effect of chloramphenicol on protein synthesis was tested by adding the antibiotic ( 20 mg/ml in 95 % ethanol ) to a final concentration of 20 jig/ml ; chloramphenicol was added at the initiation of the reaction .
An equal volume of 95 % ethanol was added to controls .
0x v ) 7 / ' - NadI ( R + T + ) PnuC + E z w-NadI ( Re T - ) PnuC * -LCB- NadI ( R + T - ) PnuC + / N r l. adI ( R + T + ) PnuC ¬ 100 .
NMN uptake in a pnuA + pnuC + strain ( TT15335 ) ( O ) , a nadl ( R + T - ) pnuC * strain ( TT15877 ) ( U ) .
RESULTS Both nadI ( R + T - ) and pnuC mutants are deficient in NMN transport .
Mutants defective in NMN transport were originally isolated in parent strains that required exogenous pyridine ( due to a nadB mutation ) and could not utilize free NM ( due to a pncA mutation ) ( 17 , 30,46 , 51 ) .
These parental strains can use NMN as a pyridine source and apparently transport it intact before converting it to NAMN and ultimately to NAD ( Fig. 1 ) .
Mutants defective in NMN utilization fell into two classes , nadI ( T - ) ( originally called pnuA ) and pnuC mutants .
Both mutant types possessed NMN deamidase activity and were inferred to be defective in transport of NMN ( 30 , 46 ) .
To clarify the roles played by the nadl and pnuC gene products , we measured the NMN uptake of nadI ( R + T - ) and pnuC mutants ( TT13124 and TT14946 ) and a wild-type strain ( TT15335 ) ( Fig. 2 ) .
In agreement with previous conclusions ( 30 , 46 ) , no NMN uptake could be detected in either the nadI ( R + T - ) mutant or the pnuC mutant .
The NMN uptake by a nadA : : Mu dJ insertion mutant ( TT15336 ) was normal and could not be distinguished from that of a nadB : : Mu dJ insertion strain , TT15335 ( data not shown ) .
Thus , the polar effect of the nadA insertion on the pnuC gene ( in the same operon ) does not limit NMN transport ; apparently , the pnuC gene expression provided by the internal promoter , downstream of the Mu dJ insertion , is sufficient to provide transport activity ( 51 ) .
The pnuC * mutant in Fig. 2 will be described later .
Neither NadI ( T + ) nor PnuC function is essential for expression of-the other .
Since two distinct mutations eliminate NMN transport , we tested the possibility that the NMN transport deficiency of one mutant type could be due to a regulatory effect on expression of a single transport function encoded by the other .
Operon and gene fusions to the nadI and pnuC genes were isolated by insertion of Mu dJ and Mu dK elements into a nadB pncA mutant strain .
All mutants unable to use NMN map at either the nadI or the pnuC locus ; no other class of mutants was found .
Since Mu dJ and Mu dK insertions generate lac operon fusions and protein fusions , respectively , the transcription and translation of the nadI and pnuC genes can be monitored by assaying the levels of ' - galactosidase from these strains [ e.g. , TT13121 for nadl : : Mu dJ ( R-T - ) , TT13124 forpnuC : : Mu dJ , TT13521 for nadI : : Mu dK ( R-T - ) , and TT15527 forpnuC : : Mu dK ] .
These NMN-mutants were scored for their Lac phenotype , and the effect of various mutations in the other gene was scored .
The expression of nadI : : Mu dJ and nadI : : Mu dK operon and protein fusions is constitutive and is not affected either by the concentration of exogenous NA or NMN or by the presence of pnuC mutations , including the pnuC * mutation ( data not shown ) .
[ The pnuC * mutation allows NMN transport without , a nadI ( T + ) function and will be described below .
] Because the nadA-pnuC operon is regulated by the nadI repressor , assaying the influence of nadI mutations on pnuC gene expres ` sion is complicated .
We have tested all three phenotypic classes of nadl mutations : R + T - , R-T , and R-T - .
Both R-T + and R-T-mutations cause derepression of the pnuC gene ; this finding confirms the previous conclusion that the pnuC gene is expressed by transcription from the nadA promoter and is therefore regulated by the NadI repre ` ssor ( 46 , 51 ) .
` However , nadI ( R ' T - ) mutations , which ` eliminate NMN transport , have no effect on the expression of the pnuC gene at a transcriptional or translational level from either the nadA promoter or the pnuC promoter ( 51 ; other data not shown ) .
Spector et al. had previously shown that nadI ( T - ) mutations have no effect on transcription of the pnuC gene ( 46 ) .
On the basis of all o these results , we conclude that the NMN transport defect of nadI and pnuC mutants is not due to a failure to express the other gene .
An alteration of PnuC function permits NMN transport without NadI ( T + ) function .
To investigate the relationship between the NadI ( T + ) and PnuC functions , revertants of a nadI ( R-T - ) deletion mutant ( TT15478 ) selected ; these were revertants can use 1o-4 M NMN pyridine as a source despite the lack of the NadI protein .
The revertants were selected in a strain that carries both a nadB and a pncA insertion mutation ; both parental mutations are retained by the revertants .
The revertant frequency was about 10-8 .
All 16 revertants tested map at the pnuC locus and are phenotypically NadA + .
The revertants were designated pnuC * ( e.g. , TT15480 ) .
The pnuC * mutants can be distinguished from pnuB mutants reported previously ( 31 ) by position and map by the fact that the pnuC * able 10-4 mutants are to grow on M but not 10-M NMN as a pyridine source , while pnuB mutants are selected for their ability to use the lower concentration of NMN .
Suppression of the nadI defect by pnuC * is not due to increased expression of the pnuC gene .
We conclude this because known constitutive mutations for the nadA-pnuC operon ( nadAc ) do not suppress the transport defect of nadI ( T - ) mutants .
That is , a strain ( TT15539 ) of genotype nadAc nadB pncA nadI ( R-T - ) is unable to grow on 10-4 M NMN .
The ability of pnuC * mutations to suppress the transport defect of nadI ( T - ) mutations is not allele specific .
Even a deletion mutant lacking the entire nadI locus is able to transport NMN following introduction of a pnuC * mutation ( Table 2 , lines 1 and 2 ; Fig. 2 ) .
Similarly , nadI point mutants with either a simple transport defect ( R + T - ) or lacking both transport and repressor functions ( R-T - ) are corrected for transport by all pnuC * mutations ( Table 2 , lines 3 to 6 ) .
A pnuC * mutation alone causes no transport defect .
This was tested in strains with a normal nadI ( R + T + ) region ( Table 2 , lines 7 and 8 ) and in strains with a nadI regulatory mutation ( R-T + ) causing constitutive expression of nadA-pnuC and nadB ( Table 2 , lines 9 and 10 ) .
Superrepressing nadI ( Rs ) mutations all cause an NMN transport defect that is not corrected by pnuC * .
This is apparently due to reduced expression of the pnuC * mutant gene , caused by the superrepressing nadI ( Rs ) allele .
The repression effect of nadl ( Rs ) can be circumvented by a mutation causing constitutive expression of the nadA-pnuC operon ( nadAc ) ; under these conditions , the pnuC * mutation can provide NMN transport ( Table 2 , lines 11 to 13 ) .
This situation is expected since nadI ( R5 ) mutations ( superrepressor ) cause a failure to express the nadA-pnuC operon from its main promoter .
When the pnuC * mutant gene is not expressed at a high level , there is apparently not enough pnuC * gene product for NMN transport .
The need for high levels of the pnuC * product is confirmed by the effect of a nadA219 : : Mu dJ insertion mutation .
NMN uptake in a pnuA + pnuC + strain ( TT15335 ) ( O ) , a nadl ( R + T - ) pnuC * strain ( TT15877 ) ( U ) .
Phenotypes of mutants on lo-4 M NMN medium Growth on 10-4 M NMN Relevant genotype Line Strain DEL085 ( serB-nadI ) 1 2 TT15478 TT15480 TT15872 TT15877 TT15871 TT15618 TT15869 FT15874 TT15870 TT15875 TT15873 TT15878 TT15611 + DEL1085 ( serB-nadI ) pnuC * 127 nadI3J2 ( T-R + ) nadI312 ( T-R + ) pnuC * 127 nadI275 ( T-R - ) nadI275 ( T-R - ) pnuC * 127 nadI + ( T + R + ) nadI + ( T + R + ) pnuC * 127 nadI260 ( T + R - ) nadI260 ( T + R - ) pnuC * 127 nadPSll ( T-RS ) nadPSll ( T-RS ) pnuC * 127 nadPSll ( T-RS ) nadAc532 pnuC * 127 nadA219 : : Mu dJ nadA219 : : Mu dJ pnuC * 127 nadA219 : : Mu dJ pnuC * 127 DEL794 ( nadl ) 3 4 5 6 7 8 9 10 11 12 13 + + + + + + Although this insertion is located upstream of the pnuC gene within the same operon , it does not reduce pnuC gene expression sufficiently to prevent NMN transport in an otherwise normal strain ( nadI pnuC + ) .
This is due to the constitutive internal promoter within the nadA-pnuC operon downstream of the nadA219 insertion ; this promoter can provide sufficient pnuC + function to allow nadI ( T + ) - depen-dent transport .
However , in a pnuC * strain , a nadA insertion prevents NMN transport with or without nadI ( T + ) function ( Table 2 , lines 14 to 16 ) .
The internal promoter is apparently not sufficient to permit a pnuC * allele to provide transport .
Presumably , NMN transport by the PnuC * protein with or without nadI ( T + ) is less efficient than transport by the wild-type protein aided by the nadI ( T ) function .
The pnuC * mutations are dominant .
Since the pnuC gene must be expressed at a high level to permit pnuC * to correct a nadI ( T - ) defect , one would predict that the pnuC * mutation might be dominant .
A dominance test was done in a strain carrying a tandem duplication of the chromosome segment from pyrC ( 11 min ) to purE ( 23 min ) and a TnlO element at the duplication join point ( TT15521 ) ( 51 ) .
This strain carries a nadI ( R-T - ) deletion ( outside the duplicated region ) and is diploid for the pnuC + allele .
A pnuC * mutation was introduced into one copy of the duplication by using the linked marker , aroG : : Mu dJ .
The resulting pnuC + I pnuC * heterozygote ( TT15522 ) was able to grow on 1O-4 M NMN .
The genotype of this heterozygous strain ( pnuC + I pnuC * ) was verified by scoring the phenotypes of haploid segregants .
Evidence that the pnuC gene product is membrane associated .
We have isolated a TnphoA insertion mutant of the pnuC gene ; this mutant expresses alkaline phosphatase activity .
The TnphoA element , constructed by Manoil and Beckwith ( 32 ) , has the gene for alkaline phosphatase ( phoA ) near one end of the transposable sequence ; this phoA gene lacks a translation initiation site and the signal sequence required for its secretion to the periplasm ( 32 , 34 ) .
Alkaline phosphatase is active only if the protein can be transported outside of the cytoplasmic membrane .
To produce active phosphatase , the TnphoA element must insert in the proper reading frame into a structure gene that can provide both a translational initiation site and a functional signal sequence .
This phoA fusion technique has been used extensively to study the topology of membrane proteins and the process of protein transport ( 1 , 3 , 9 , 19 , 33 ) .
An insertion mutation which expresses alkaline phosphatase indicates that the target gene has a signal sequence and that its product is probably located in either the membrane or the periplasm .
The pnuCJ49 : : TnphoA mutation appears to be a simple insertion within the pnuC gene ; it does not affect the promoter-proximal nadA gene .
Furthermore , the expressed alkaline phosphatase is regulated in response to pyridine levels as are lac operon fusions to the pnuC gene .
Data for regulation of phoA are in Table 3 ; all of the strains described carry a phoN mutation which eliminates the Salmonella nonspecific acid phosphatase .
The first two strains in Table 3 carry the nonpolar nadA542 mutation to block the synthetic pathway .
Alkaline phosphatase is repressed in response to an increased level of exogenous nicotinic acid ( Table 3 , line 1 ) .
Alkaline phosphatase is constitutively expressed when a nadl ( R-T - ) mutation is introduced ( line 2 ) .
A TnlOd ( Tc ) insertion in nadA exerts a polar effect , reducing expression of the pnuC : : TnphoA fusion ( line 3 ) .
The cellular location of the hybrid PnuC-PhoA protein produced by the pnuC149 : : TnphoA fusion gene was determined by cell fractionation as described in Materials and Methods .
Each fraction was assayed for alkaline phosphatase and for the marker proteins,-lactamase ( periplasmic ) , NADH-ferricyanide oxidoreductase ( membrane associated ) , and glutamate dehydrogenase ( cytoplasmic ) .
Most alkaline phosphatase is associated with membranes , suggesting that the phoA sequences have been fused to a protein that is anchored in the membrane ( Table 4 ) .
This finding suggests that the pnuC gene encodes a membrane-associated protein .
A model for the role of the NadI protein in regulation of transcription of de novo pathway and NMN transport .
To explain the effects of nadl mutations on both transport and transcription , we propose that the NadI protein is allosteric , alternating between two distinct regulatory states ( Fig. 3 ) .
When NAD levels are low , the protein assumes a conformation that stimulates transport of NMN and allows increased expression of biosynthetic genes ( Fig. 3a ) .
When NAD levels are high , the protein represses transcription of the nadB and nadA genes and ceases to stimulate NMN transport ( Fig. 3b ) .
The role of NadI ( T + ) in modulating activity of the transort system is a functional parallel with its role as a transcriptional repressor ( 52 ) .
Depending on the supply of NAD , two routes of pyridine acquisition ( synthesis and transport ) are either restricted or increased .
The model makes several testable predictions .
Most important of these is that the activity of the transport system should vary in response to internal pyridine levels .
We have tested this prediction .
Regulation of NMN transport function .
Transport of NMN was assayed for a strain ( TT15335 ) in which the pyridine moiety of NMN is assimilated only by the NadI ( T + ) - PnuC route ( any NM generated by a periplasmic glycohydrolase can not be assimilated ) .
Following addition of a high concentration of NA , transport of NMN stops ( Fig. 4a ) .
This cessation of NMN transport is a consequence of internal NAD accumulation ( not competition between NMN and NA for a common transport system ) , since NA has no regulatory effect in a nadE ( NAD synthetase ) mutant which is blocked in the last step of NAD synthesis ( Fig. 4b ) .
The nadE mutation used is a temperature-sensitive mutation known to stop NAD synthesis and accumulate nicotinic acid adenine dinucleotide ( NaAD ) at high-temperatures ( 23 ) ; the assay presented was performed at 42 °C .
The cessation of NMN transport following NA addition is not due to repression of the nadA-pnuC operon by the NadI function , since the NA effect still occurs in strains which carry a nadAc or nadI ( R-T + ) mutation , both of which cause constitutively high expression of the nadA-pnuC operon ( Fig. 4c and d ) .
The regulatory effect of NA on NMN transport does not require protein synthesis .
When protein synthesis is blocked by chloramphenicol , NA still shows its negative effect on NMN transport ( Fig. 4e and f ) .
The variation in transport activity seems to depend on the NadI ( T + ) function .
Transport is eliminated by nadI ( R + T - ) mutations ( Fig. 2 ) .
Furthermore , regulation of transport is lost in pnuC * mutants which are insensitive to NadI ( T + ) .
NMN transport in the pnuC * nadl ( R-T - ) and pnuC * nadI ( R + T - ) mutants ( Fig .
Sb and c ) is comparable to that o a wild-type strain under starvation conditions ( Fig. 5a ) but has become insensitive to NA addition .
Since pnuC * mutants have simultaneously become independent of nadI ( T + ) and have lost regulation of NMN transport by NA , we conclude that the regulation of transport is via the NadI ( T + ) function .
Strains with both a pnuC * mutation and a nadI ( T + ) allele have a hyperactive NMN transport system .
This activity is reduced to the slower PnuC * rate when NA is added ( Fig. 5d and e ) .
We conclude that the regulatory effect of NA on NMN transport is due to internal accumulation of NAD which causes the NadI ( T + ) function to stop activating the PnuC transport system .
The transport system is fully shut off within about 20 min of NA addition .
According to the model ( Fig. 3 ) , this time is required for NA to be converted to NAD , for the increased NAD to be sensed by the NadI protein , and for the pnuC transport system to return to its inactive state .
+ + 14 15 16 TT13008 TT15556 TT16346 TABLE 3 .
Effects of nadA insertion and nadl mutation on expression of the pnuC149 : : TnphoA fusion Alkaline phosphatase activity ( U ) in medium with indicated NA concn 10-6 M 2 x 10-4 M 13 1 13 13 1 Line Strain Relevant genotype 1 TT15593 TT15599 FT15598 pnuC149 : : TnphoA 2 pnuC149 : : TnphoA nadI242 : : TnlO nadA379 : : TnlOd ( Tc ) pnuC149 : : TnphoA 3 TABLE 4 .
Cellular location of PnuC : : PhoA fusion protein Enzyme activitya In whole-cell Recovery after Distribution of recovered activity in indicated fraction extractb fractionation Periplasm Membrane Cytoplasm P-Lactamase 18 16 ( 89 ) 15 ( 94 ) 1 ( 6 ) 0 ( 0 ) NADH-ferricyanide oxidoreductase 43 37 ( 86 ) 2 ( 5.5 ) 33 ( 89 ) 2 ( 5.5 ) Glutamate dehydrogenase 9.8 9.3 ( 95 ) 0.5 ( 5 ) 0.1 ( 1 ) 8.7 ( 94 ) PnuC : : PhoA alkaline phosphatase 4.3 3.6 ( 84 ) 0.8 ( 22 ) 2.5 ( 70 ) 0.3 ( 8 ) a Normalized as activity units in the amount of extract or fraction prepared from 1 ml of starting cell culture .
The optical density at 650 nm of the starting cell culture in this experiment was 0.553 .
Numbers in parentheses are percentages .
Data are from one fractionation experiment in which all activities were assayed .
Enzyme assayed ( a. ) L NAD Pool ow NINNIN na ( lIB - fnd adA p-Fpriu1C I ( b. ) 4 , High NAD l'ool 1 ) ; ; ¶ iiadll : 10 1 \ # 1 ) [ tl ( m : : , FIG. 3 .
Model for coordinate control of NMN uptake and transcription of the NAD de novo biosynthetic genes .
( a ) When the internal NAD level is low , NadI bifunctional protein is in a conformation that is able to bind with the membrane protein , PnuC , to form functional NMN transport systems but is not able to bind with the nadA and nadB promoters .
The nadA and nadB genes are expressed .
( b ) When the internal NAD level is high , NadI protein binds NAD , stabilizing a conformation of the protein that is able to bind to operator sites in DNA but unable to activate transport .
As a result , both NMN uptake and the expression of biosynthetic genes nadA and nadB stop ( a ) .
W.T. 500 , = 400 ¬ x 300 ¬ ' \ - NA li NA 20o0 ¬ ~ ~ ~ + NA 0 S 100 0 .
( f ) 4001 NadlF ( R T + ) c Ixz .
¬ Z c 40 80 0 20 40 60 8 40 80 m 120 0 0 120 Time ( min .
Effects of NA and chloramphenicol on NMN uptake .
( a ) A wild-type ( W.T. ) strain ( FT15335 ) with ( - ) and without ( E ) addition of 2 x 10-4 M NA at 20 min , assayed at 43 °C ; ( b ) a nadE strain ( FT13493 ) with ( U ) and without ( E ) addition of 2 x 10-4 M NA at 20 min , assayed at 43 °C ; ( c ) a nadA promoter constitutive mutant strain ( FT15604 ) with ( - ) and without ( O ) addition of 2 x 10-4 M NA at 20 min ; ( d ) , a nad ( R-T + ) pnuC + ( FT15870 ) with ( - ) and without ( E ) addition of 2 x 10-4 M NA at 20 min ; ( e ) protein synthesis by a wild-type strain ( TT15335 ) with ( U ) and without ( O ) addition of chloramphenicol ( CM ) ; ( f ) NMN uptake of a wild-type strain ( FT15335 ) in the presence of chloramphenicol with ( - ) and without ( O ) addition of 2 x 10-4 M NA at 20 min .
800 PnuC * PnuC Nadl + ( R+T + ) NadIV ( R-T + ) v 600 600 x V ¬ 400 400-z z 200 200 ¬ Oi 0 20 20 40 80 1 40 80 1 Time ( min .
Effects of nadI mutations on NMN uptake by pnuC * mutant strains .
( a ) A wild-type ( W.T. ) strain ( pnuC ' nadI + ) ( TT15869 ) with ( U ) and without ( O ) addition of 2 x 1o - ' M NA at 20 min ; ( b ) a pnuC * nadl ( R-T - ) strain ( TT15876 ) with ( U ) and without ( E ) addition of 2 x 1O ' M NA at 20 min ; ( c ) a pnuC * nadI ( R + T - ) strain ( TT15877 ) with ( U ) and without ( E ) addition of 2 x 10 ' M NA at 20 min ; ( d ) a pnuC * nadI + strain ( TT15874 ) with ( U ) and without ( E ) addition of 2 x 1O-4 M NA at 20 min ; ( e ) a pnuC * nadl ( R-T + ) strain ( 1T15875 ) with ( - ) and without ( O ) addition of 2 x 10-4 M NA at 20 min .
DISCUSSION We have presented a model for regulation of pyridine levels ( Fig. 3 ) that includes a single bifunctional protein which acts to vary transcription of biosynthetic genes and to modulate the rate of transport of exogenous pyridines .
The most novel aspect of this model is the idea of varying the activity of an uptake system .
Below we discuss the history of this model , the pieces of evidence that support it , and reasons why this elaborate mechanism might be particularly important to the pyridine pathway .
Previous work has demonstrated that mutants defective in both repression and NMN transport map at a single locus ( nadl ) on the Salmonella chromosome ( 10 , 17 , 20 , 30 , 50 ) .
Foster and co-workers found that the DNA sequence of this locus includes a single open reading frame ( 16 , 17a ) .
On the basis of these sequence data and the phenotypes of mutants , a model was presented in which a single protein acts as repressor of biosynthetic genes and a component of the NMN transport system ( 17a ) .
The accompanying report ( 52 ) provides in-vivo data that the transport and repression functions are provided by a single bifunctional protein .
We present here an independently developed model ( Fig. 3 ) that is similar to that of Foster et al. but includes several specific new features for which we can provide evidence .
The new features and the evidence for each are listed below .
( i ) The nadI ( T ) function is not an essential component of the NMN transport system but rather a regulatory modulator of the activity of the pnuC transport system .
We conclude this because pnuC * mutants transport NMN even if the nadI gene is completely deleted .
Thus , the PnuC protein with only minor modification can independently serve to transport NMN .
( ii ) We provide evidence that the activity of this transport system does in fact vary in response to cell physiology , The regulatory signal is likely to be NAD or NADP , since nadE mutants ( blocked ih NAD synthetase ) are unable to vary NMN transport activity in response to exogenous NA .
Mutations that limit nadE function also cause increased transcription of biosynthetic genes , suggesting that NAD ( or NADP ) is also the effector for the repressor activity of the NadI protein ( 23 ) .
( iii ) The role of the NadI protein in varying NMN transport activity is supported by the fact that pnuC * mutants show unregulated high transport activity and have lost the need for nadI ( T ) function to stimulate activity .
Conversely , nad ( T - ) mutants show unregulated low transport activity .
( iv ) The Nadl protein is probably an allosteric protein .
One form is able to repress biosynthetic genes but not activate transport ; the other can not repress transcription but is able to activate transport .
These two states correspond to the nadI mutant phenotypes : R + T-and R-T .
Two lines of evidence support this conclusion .
The main support is the fact that all nadI ( RS ) simulta - ( superrepressor ) mutants have neously lost transport activity .
This is expected of an allo-steric protein which is locked in the repressing conformation ; it represses under all conditions and fails , under all co ` nditions , to stimulate transport .
An alternative possibility , that repression is lifted when NadI protein binds and is sequestered by transport sites , is rendered unlikely because deletion mutants of pnuC which lack the transport protein show normal transcriptional regulation .
The model for nadl functions is presented in simplest form ( without in Fig. 3 .
In this diagram , the NadI protein NAD ) binds directly to PnuC to activate transport .
It may be more likely that the regulatory protein will prove to be a kinase or ADP ribosyltransferase which activates PnuC by covalent modification .
Covalent protein modification ( phosphoryla-tion-dephosphorylation ) has been shown to be a regulatory mechanism in many bacterial systems such as chemotaxis , control of outer membrane ( Omp ) proteins , the phosphate regulon , and nitrogen assimilation functions of Escherichia coli ( 26 , 36 , 39 , 40 ) .
Regulation of NMN transport would be achieved if the NadI protein varied between a repressor and a modifier conformation .
The PnuC protein might then be active or inactive , depending on its state of chemical modification .
Following NA addition , there is a lag of about 20 min before NMN transport ceases ; this lag may include time for removal or spontaneous breakdown of a ( nadI-catalyzed ) modification that activates the PnuC protein .
We that control of pyridine is important suspect transport to Salmonella spp. .
because it permits cells to assimilate NMN and yet avoid raising internal pools of NMN to excessive levels .
The essential enzyme DNA ligase is known to be inhibited by NMN ( 37 , 38 ) ; some dehydrogenases may also be sensitive to inhibition .
The mechanism described here permits NMN transport only under conditions of NAD limitation .
This would prevent accumulation of a large , potentially inhibitory internal NMN pool following exposure to exogenous NMN .
Regulated transport systems of this type might be expected for any valuable nutrient that is toxic at high concentrations .
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