XanthineMetabolismin Bacillussubtilis:Characterization xpt ... · TABLE1.Bacterialstrainsandplasmids Strainorplasmid Genotypeordescriptiona Sourceorreferenceb Strains B.subtilis 168

JOURNAL OF BACTERIOLOGY,0021-9193/97/$04.0010

Apr. 1997, p. 2540–2550 Vol. 179, No. 8

Copyright q 1997, American Society for Microbiology

Xanthine Metabolism in Bacillus subtilis: Characterizationof the xpt-pbuX Operon and Evidence for Purine- andNitrogen-Controlled Expression of Genes Involved

in Xanthine Salvage and CatabolismLYNGE C. CHRISTIANSEN,1 SIMON SCHOU,2† PER NYGAARD,1 AND HANS H. SAXILD2*

Department of Microbiology, Technical University of Denmark, Lyngby,2 and Department ofBiological Chemistry, University of Copenhagen, Copenhagen,1 Denmark

Received 22 August 1996/Accepted 4 February 1997

The xpt and pbuX genes from Bacillus subtilis were cloned, and their nucleotide sequences were determined.The xpt gene encodes a specific xanthine phosphoribosyltransferase, and the pbuX gene encodes a xanthine-specific purine permease. The genes have overlapping coding regions, and Northern (RNA) blot analysisindicated an operon organization. The translation of the second gene, pbuX, was strongly dependent on thetranslation of the first gene, xpt. Expression of the operon was repressed by purines, and the effector moleculesappear to be hypoxanthine and guanine. When hypoxanthine and guanine were added together, a 160-foldrepression was observed. The regulation of expression was at the level of transcription, and we propose thata transcription termination-antitermination control mechanism similar to the one suggested for the regulationof the purine biosynthesis operon exists. The expression of the xpt-pbuX operon was reduced when hypoxanthineserved as the sole nitrogen source. Under these conditions, the level of the hypoxanthine- and xanthine-degrading enzyme, xanthine dehydrogenase, was induced more than 80-fold. The xanthine dehydrogenase levelwas completely derepressed in a glnA (glutamine synthetase) genetic background. Although the regulation ofthe expression of the xpt-pbuX operon was found to be affected by the nitrogen source, it was normal in a glnAmutant strain. This result suggests the existence of different signalling pathways for repression of the tran-scription of the xpt-pbuX operon and the induction of xanthine dehydrogenase.

Bacillus subtilis is able to take up preformed purine basesand use them for nucleotide synthesis (salvage) and as a sourceof nitrogen (catabolism) (Fig. 1). Purine bases are converteddirectly to purine nucleoside monophosphates by reacting with5-phosphoribosyl-a-1-PPi (PRPP). The phosphoribosylation iscatalyzed by specific purine phosphoribosyltransferases. Ingeneral, adenine phosphoribosyltransferases react only withadenine, whereas 6-oxo-purines (hypoxanthine, xanthine, andguanine) may be phosphoribosylated by phosphoribosyltrans-ferases with activity toward more than one purine base (28).Escherichia coli, for example, contains two 6-oxo-purine phos-phoribosyltransferases, one reacting with hypoxanthine andguanine and another reacting with all three 6-oxo-purines (26).Two 6-oxo-purine phosphoribosyltransferases have been iden-tified in Lactobacillus casei; one reacts with hypoxanthine andguanine, and the other reacts with guanine and xanthine (20).A phosphoribosyltransferase that reacts exclusively with xan-thine has been found in Streptococcus faecalis (22). B. subtiliscontains three purine phosphoribosyltranferase activities, i.e.,adenine phosphoribosyltranferase (APRTase), a hypoxan-thine-guanine phosphoribosyltransferase (H-GPRTase), andxanthine phosphoribosyltransferase (XPRTase) (34). Muta-tions in the B. subtilis genes encoding APRTase (apt), H-GPRTase (hpt), and XPRTase (xpt) have been obtained byselection for resistance towards 2-fluoroadenine, 8-thiogua-nine, and 8-azaxanthine, respectively. The substrate specificity

of H-GPRTase and XPRTase have not been characterized (12,34). The apt and xpt genes have been mapped to 2708 and 1988,respectively, on the linkage map, and the hpt gene has beenlocated at 108 immediately upstream of the ftsH gene (8, 34).Purine bases that are present in the surroundings are taken

up by specific membrane-embedded transport systems (25). InB. subtilis, a specific system for the uptake of the purine baseshypoxanthine and guanine (pbuG) has been partially charac-terized, and pbuG mutants were obtained by selection forspontaneous resistance towards the purine analog 8-azagua-nine. The pbuG gene maps close to the promoter-proximal endof the pur operon at 558 on the linkage map (34). A secondpurine base transport system is specific for adenine (4, 29).Several other purine permeases of procaryotic as well as eu-caryotic origin have been described (25). Aspergillus nidulans,as an example, contains two genes, uapA and azgA, whichencode uric acid-xanthine permease and adenine-hypoxan-thine-guanine permease, respectively, and gene uapC, whichencodes a broad-specificity low-affinity purine permease (10).In E. coli, the purP gene encodes a high-affinity adenine per-mease (7).Rather than being anabolized, purines can be catabolized. B.

subtilis can use either adenine, hypoxanthine, xanthine, uricacid, or allantoin as the sole source of nitrogen (6, 14, 29).Adenine deaminase converts adenine to hypoxanthine, and inthe subsequent aerobic degradation of purines, xanthine dehy-drogenase (XDHase) converts hypoxanthine to xanthine andxanthine to uric acid. Uric acid is degraded via several steps tourea, which is cleaved to ammonia and carbon dioxide (39).The degradation of uric acid is induced only under conditionsof nitrogen limitation (30). It has previously been shown that ina Streptomyces sp., hypoxanthine salvage and hypoxanthine ca-

* Corresponding author. Mailing address: Department of Microbi-ology, Technical University of Denmark, Building 301, DK-2800 Lyn-gby, Denmark. Phone: 45 25 24 95. Fax: 45 88 26 60. E-mail: [email protected].† Present address: Novo Nordic A/S, DK-2880 Bagsværd, Denmark.

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tabolism are subject to nitrogen control. Purine salvage is re-pressed during nitrogen limitation, whereas catabolism is re-pressed under excess nitrogen conditions (41). In the presentstudy, we present data that indicate that xanthine metabolismin B. subtilis is regulated in a way similar to that reported forthe hypoxanthine metabolism in Streptomyces spp. Duringgrowth on purines as a nitrogen source, the expression of thexpt-pbuX operon is repressed, whereas the purine catabolicpathway is induced. Conditions with excess nitrogen have theopposite effect. We also show that salvage of xanthine mostlikely is subjected to nitrogen-modulated purine repressionand that the nitrogen regulation of the level of the XDHaseenzyme is dependent on the glnA gene product (glutaminesynthetase).

MATERIALS AND METHODSBacterial strains, plasmids, and media. The bacterial strains and plasmids

used in this work are listed in Table 1. E. coli strains were grown in L broth andin a phosphate-buffered minimal salts medium (23). B. subtilis strains were grownin L broth and in Spizizen salts minimal medium (34) supplemented with 0.4%glucose, 0.2% glutamate, and thiamine-HCl (1 mg/ml). Growth in liquid mediumwas monitored by measuring the optical density at 450 nm. The growth temper-ature was 378C. When B. subtilis strains were grown in minimal medium con-taining nitrogen sources other than ammonium ions, glutamate was omitted andammonium sulfate was replaced by sodium sulfate (final concentration, 2 g/liter).When used as a nitrogen source, purine bases, allantoin, allantoic acid, and ureawere at a concentration of 0.5 mg/ml. Adenine and hypoxanthine were added toa final concentration of 15 mg/ml when used as a purine source for auxotrophicstrains. Amino acids required by auxotrophic strains were added at a finalconcentration of 50 mg/ml. Plasmids were propagated in E. coli MT102 orMC1000 (dam). The following antibiotics were used at the indicated concen-trations: tetracycline, 8 mg/ml; ampicillin, 100 mg/ml; neomycin, 5 mg/ml; chlor-amphenicol, 5 mg/ml; spectinomycin, 100 mg/ml; lincomycin, 25 mg/ml; erythro-mycin, 1 mg/ml for B. subtilis and 150 mg/ml for E. coli. Allopurinol and8-azaxanthine were added to final concentrations of 0.8 and 2 mM, respectively.DNA manipulations and genetic techniques. Plasmid DNA from E. coli was

isolated by the alkaline-sodium dodecyl sulfate procedure (5), and isolation ofchromosomal DNA from B. subtilis was done as described previously (34).Transformation of B. subtilis (34) and E. coli (16) has been described previously.Treatment of DNA with restriction enzymes, T4 DNA ligase, and Klenow frag-

ment of DNA polymerase was performed as recommended by the suppliers(Gibco BRL, Gaithersburg, Md.).Southern blot analysis. Restriction fragments were separated by agarose gel

electrophoresis, blotted onto a Qiabrane nitrocellulose membrane (QiaGen,Hilden, Germany), and fixed by heating to 808C for 2 h. A digoxigenin DNAlabelling and detection kit (Boehringer GmbH, Mannheim, Germany) was usedfor colorimetric detection of the hybridization products. The recipe supplied bythe manufacturer was followed.DNA sequencing. Nucleotide sequence was obtained by use of the dideoxyri-

bonucleotide chain termination method described by Sanger et al. (31). PlasmidspLK1 and pSS8 were used as double-stranded DNA templates.PCR amplification. A 392-bp DNA fragment used in the construction of the

translational pbuX-lacZ fusion in plasmid pLCC9 was made in a PCR containing4 mM MgCl2, 2.5 U of Taq polymerase (Perkin-Elmer, Branchburg, N.J.), 0.2mM deoxynucleoside triphosphate, 20 ng of pLK1 DNA per ml, and the twoprimers 59-AGAGAAGCTTGAAGCTGGGTGTGC-39, which corresponds tonucleotides 562 to 579 (see Fig. 2), and 59-GAGATCTAGATTTCTCATGAATGAACC-39, which is the complementary sequence of nucleotides 946 to 927(see Fig. 2), each at 20 mg/ml.Primer extension analysis and Northern (RNA) blot procedure. Isolation of

total RNA from B. subtilis and primer extension analysis using reverse transcrip-tase (SuperScript RNase H2 reverse transcriptase; Gibco BRL) were performedas described by Saxild et al. (33). The Northern (RNA) blot procedure used inthis work has been described previously in detail (29).Enzyme assays and uptake of purine bases. Cells for enzyme measurement

were harvested in the mid-logarithmic growth phase and homogenized by soni-cation in 30 mM phosphate (pH 7.5), 1 mM EDTA, and 1 mM dithiothreitol.Cell debris was removed by centrifugation. Purine phosphoribosyltransferaseactivities were assayed by the method of Jochimsen et al. as described previous-ly (8). The XDHase activity was determined in an assay identical to that ofXPRTase, except that PRPP was omitted from the reaction mixture. b-Galac-tosidase was measured by the method of Miller (21). All of the determinationswere repeated at least three times. One unit of specific enzyme activity wasdefined as 1 nmol of substrate converted per min per mg of protein. The rate ofxanthine uptake was determined as described by Saxild et al. (32).

RESULTS

Cloning of the xpt and pbuX genes. A library of B. subtilisgenomic HindIII restriction fragments that were ligated intoplasmid pUN121 (27) was transformed into E. coli SØ609(deoD purD hpt gpt), selecting for tetracycline resistance. Dueto the hpt and gpt mutations, strain SØ609 cannot use hypo-xanthine, guanine, or xanthine as a purine source and has arequirement for guanosine plus adenine (17). Only transfor-mants of strain SØ609 containing plasmids encoding XPRTaseactivity should be able to grow on minimal medium supple-mented with xanthine as the sole purine source. By using thisscreening procedure, a plasmid (pLK1) containing a 4-kbHindIII insert was isolated. SØ609 cells containing pLK1had 85 nmol of XPRTase activity per min per mg of protein,whereas HPRTase and GPRTase activity was less than 0.05nmol/min/mg of protein, which agrees with the observationthat no growth was observed with hypoxanthine or guanine asthe purine source for SØ609 containing pLK1. pLK1 was alsotransformed into E. coli SØ446 (apt deoD purE). Both SØ446and SØ446 containing pLK1 had less than 0.05 nmol ofAPRTase activity per min per mg of protein. These data showthat the xpt gene encodes an enzyme that specifically reactswith xanthine. A functional xpt gene could be subcloned on a2.2-kb SspI fragment (pSS8). Below, we show that most of thepbuX gene was also located on this fragment. The ability tocomplement the purine deficiency of strain SØ609 was re-tained when the orientation of the SspI insert in pSS8 wasreversed (data not shown). This indicated that the xpt genemost likely was expressed from a promoter located on theinsert. When B. subtilis ED194 (xpt purH), which cannot usexanthine as a purine source (34), was transformed with pLK1,transformants that were able to grow on minimal mediumagar plates supplemented with xanthine were formed. SincepUN121 derivatives cannot replicate in B. subtilis, xanthine-positive transformants must result from a double crossoverevent in which the chromosomal xptmutation is substituted for

FIG. 1. Purine salvage, interconversion, and catabolic pathways in B. subtilis.The enzyme-catalyzed steps are represented by their gene symbols. Ade, adenine;Hyp, hypoxanthine; Xan, xanthine; Gua, guanine; apt, adenine phosphoribosyl-transferase; hpt, hypoxanthine-guanine phosphoribosyltransferase; xpt, xanthinephosphoribosyltransferase; ade, adenine deaminase; guaB, IMP dehydrogenase;guaA, GMP synthetase; purA, adenylosuccinate synthetase; purB, adenylosucci-nate lyase; guaC, GMP reductase.

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Genotype or descriptiona Source or referenceb

StrainsB. subtilis168 trpC2 C. AnagnostopoulosSS1 Wild type 168 trpC21

ED138 purM E. FreeseED194 xpt purH trpC2 Saxild and Nygaard (34)ED265 ade-1 trpC2 P. DuckertHH285 trpC2 hpt::erm Laboratory stockLCC1 amyE::xpt-lacZ (Neor) SS1 transformed by pLCC1 digested

with KpnI, Neor

LCC4 pbuX::erm (Err) SS1 transformed by pLCC4 digestedwith SphI, Err

LCC5 xpt::erm (Err) SS1 transformed by pLCC5 digestedwith SphI, Err

LCC16 xpt-11 SS1 transformed by pLCC11, Err

LCC19 pbuX::pLCC9 (Cmr) SS1 transformed by pLCC9, Cmr

LCC20 xpt-11 pbuX::pLCC9 (Cmr) LCC16 transformed by pLCC9, Cmr

LCC25 amyE::xpt-lacZ glnA::spc (Neor Spr) LCC1 transformed by pGLNA14, Spr

LCC28 purR::neo (Neor) SS1 transformed by pMW11LCC29 trpC2 ade-1 amyE::xpt-lacZ (Neor) ED265 transformed by LCC1LCC30 amyE::xpt-lacZ hpt::erm (Neor Err) LCC1 transformed by HH285LCC31 trpC2 ade-1 amyE::xpt-lacZ hpt::erm (Neor Err) LCC29 transformed by HH285

E. coliMT102 F2/araD139 D(argF-leu)7696 D(lac)X74 galU galK hsdR2(r2 m1) mcrB1 rpsL (Strr) Laboratory stockMC1000 araD139 D(ara-leu)7679 dam galU galK D(lac)174 rpsL thi-1 Laboratory stockSØ609 hpt deoD D(pro-gpt-lac) ara thi purD Hove-Jensen and NygaardSØ446 metB rpsL relA spoT supF lamB purE deoD apt Hove-Jensen and Nygaard

PlasmidspBR322 Cloning vector; Apr Tcr Laboratory stockpUN121 Cloning vector; Apr Nilsson et al. (27)pUC7erm Apr Er; carries a 1.1-kb BamHI fragment which contains the erythromycin resistance

gene ermW. De Vos

pUC18 Cloning vector; Apr Laboratory stockpDG268neo Apr Nmr; a vector for constructing transcriptional lacZ fusions designed to integrate in the

chromosomal amyE gene which contains a promoterless lacZ gene and neo resistancecassette in opposite directions flanked by the truncated amyE gene

C. Price

pGLNA14 Apr Spr glnA::spc; carries the glnA gene in which the spectinomycin resistance gene spc hasbeen inserted

S. Fisher

pSGMU37 Apr Cmr lacZ; a vector for constructing translational lacZ fusions which contains a pro-moterless lacZ gene and is designed to integrate in the B. subtilis chromosome by a sin-gle crossover event

J. Errington (13)

pMW11 Apr Nmr purR::neo; carries the gene for the purine biosynthetic operon repressor which isinactivated by the neomycin resistance gene

H. Zalkin

pLK1 4.0-kb HindIII fragment containing xpt-pbuX ligated to pUN121 digested with HindIII This workpSS6 1.5-kb EcoRI fragment from pLK1 containing DNA upstream of xpt as well as the xpt pro-

moter region ligated to pUC18 digested with EcoRIThis work

pSS8 2.2-kb SspI fragment from pLK1 containing xpt-pbuX ligated to pBR322 digested with ScaI This workpLCC1 0.9-kb EcoRI-SspI subfragment from pSS8 containing the xpt promoter region ligated to

pDG268neo digested with HindIII; all sites were blunt ended in a Klenow polymerasereaction prior to ligation; transcriptional xpt-lacZ fusion

This work

pLCC4 1.1-kb BamHI fragment from pUC7 containing erm ligated to pSS8 digested with BglII This workpLCC5 1.1-kb BamHI fragment from pUC7 containing erm ligated to pSS8 digested with HpaI; all

sites were blunt ended in a Klenow polymerase reaction prior to ligationThis work

pLCC7 pSS6 derivative digested with BclI and then blunt ended in a Klenow polymerase reactionprior to religation; frameshift in xpt

This work

pLCC9 0.4-kb PCR fragment containing the first three codons of pbuX in the 39 end; digestedwith HindIII and XbaI and ligated to pSGMU37 digested with HindIII and XbaI; trans-lational pbuX-lacZ fusion

This work

pLCC11 1.1-kb BamHI fragment from pUC7 containing erm ligated to pLCC7 digested withBamHI; frameshift in xpt

This work

a Ap, ampicillin; Cm, chloramphenicol; Er, erythromycin; Ln, lincomycin; Neo, neomycin; Sp, spectinomycin; Str, streptomycin; Tc, tetracycline.b Transformations indicated were of the first strain either with the noted plasmid or with DNA from the second strain.

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the wild-type allele on the pLK1 plasmid. By using this kind ofcomplementation analysis, we were able to conclude that atleast a part of the xpt gene was localized on a 0.9-kb SspI-EcoRI subfragment of pSS8 (data not shown).

Nucleotide and deduced amino acid sequence of the xpt-pbuX operon. The nucleotide sequence of the 2.2-kb SspI frag-ment of pSS8 and 200 bp of flanking sequence from pLK1 wasdetermined and is shown in Fig. 2. Towards the end of our

FIG. 2. Nucleotide and derived amino acid sequences of the xpt-pbuX operon. Underlined sequences indicates potential ribosome binding sites (RBS). Linesbeneath the sequence indicate nucleotides that can participate in the formation of different secondary RNA structures. Sequence 2 can base pair with sequence 3 toform a potential factor-independent transcription terminator stem-loop structure. Sequence 2 can also base pair with sequence 1 to form a second stem-loop structure.The 210 and 235 promoter elements are boxed. The beginning of the xpt-pbuX mRNA is indicated by a bent arrow. A potential factor-independent transcriptionterminator structure downstream of the pbuX gene is also indicated (...,,,). The putative PRPP binding site in the xpt sequence is indicated by asterisks underthe amino acid sequence. Only relevant restriction sites are indicated.


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sequence analysis, a preliminary sequence of the same regionof the chromosome was communicated to us by A. Sorokineand D. Ehrlich (INRA, Jouy-en-Josas, France); except for afew discrepancies, the sequences were identical. The se-quenced region of 2,413 bp shown in Fig. 2 contains two openreading frames with overlapping coding regions. The first read-ing frame encodes a 194-amino-acid protein with a calculatedmolecular mass of 21 kDa and is designated xpt. xpt is followedby a 438-amino-acid reading frame (now designated pbuX forpurine base uptake xanthine) encoding a protein with a calcu-lated molecular mass of 46.2 kDa. Putative translational startsites were found in front of the xpt gene and in the 39 end of xpt,7 nucleotides upstream of the start codon of the pbuX readingframe (nucleotides 926 to 930). Two candidates for factor-independent transcription terminators are located immediatelyupstream of the xpt gene (nucleotides 292 to 332) and down-stream of the pbuX gene (nucleotides 2255 to 2270), respec-tively. The sequences are capable of forming stem-loop struc-tures with a calculated DG value of 220.7 and 217.8 kcal/mol,respectively (36). By using the BLAST algorithm (1), the de-duced amino acid sequence of the xpt and pbuX gene productswas compared to sequences deposited in the nucleotide se-quence data base (GenBank release 30.0). Interestingly, wefound that the Xpt sequence from amino acids 98 to 185, whichcorresponds to the C-terminal half including the putativePRPP binding site (Fig. 2), was 52% identical to the deducedamino acid sequence of a 291-nucleotide-long human cDNAsequence (accession no. Z15627, unpublished data). Further-more, the entire Xpt amino acid sequence showed a 22%overall similarity with the C-terminal part of the PurR repres-sor protein from B. subtilis (42). When the deduced amino acidsequence of the xpt gene product was compared to that ofother purine phosphoribosyltransferases, including APRTaseand GPRTase from E. coli, H-GPRTase from B. subtilis,APRTase from Saccharomyces cerevisiae, and the humanHPRTase gene, no significant similarity between the statedamino acid sequences was found. Only the regions containingthe putative PRPP binding site showed some degree of simi-larity (data not shown). The PbuX amino acid sequenceshowed 24 to 26% identity to the sequence of a xanthine-uricacid permease of Aspergillus nidulans (accession no. X71807[10]) and to the sequence of the uracil permease from B.subtilis (PyrP; accession number M59757) and E. coli (UraA;accession number S34223).Characterization of mutants defective in the xpt and pbuX

genes. For mutant constructions, plasmid pSS8 containing the2,179-bp SspI fragment containing the xpt gene and part of thepbuX gene was used. This plasmid has unique recognition sitesfor the restriction enzymes HpaI and BglII. The HpaI site islocated in the xpt gene, and the BglII site is located in the pbuXgene (Fig. 2). pSS8 was digested with HpaI and BglII andligated with a DNA fragment containing the erm gene as de-scribed in Table 1. The resulting plasmid, pLCC4, has the ermantibiotic resistance cassette inserted into the pbuX gene, andthe direction of the erm transcription is the opposite of that ofthe pbuX gene. In plasmid pLCC5, the erm cassette is insertedinto the xpt gene with the direction of erm transcription parallelto that of the xpt gene. pLCC4 and pLCC5 were linearized withSphI and transformed into B. subtilis SS1, selecting for Err. Err

transformants were established as a result of a double cross-over event in which the plasmid DNA recombined with thechromosome, leaving the erm gene integrated into the xpt(strain LCC5) or pbuX (LCC4) gene. The correct integrationof the erm resistance genes was confirmed by Southern blotanalysis (data not shown). XPRTase activity and xanthine up-take were then determined in wild-type strain SS1 and the two

mutant strains. Strain LCC5 has no detectable XPRTase ac-tivity, and the rate of uptake of xanthine was reduced to aboutone-third of the wild-type level. Strain LCC4 had a normallevel of XPRTase activity but was impaired in xanthine uptake(Table 2). In the wild-type strain (SS1), the Km for xanthinewas determined to be 1 mM (concentration range used, 0.2 to5 mM). However, it was not possible to determine the Km inmutant strain LCC4, because the rate of uptake was almostproportional to the xanthine concentration used. At 5 mM, therate was 30-fold lower than that of the wild type. For reasonsdescribed later, a second xpt mutation was constructed. Theplasmid pSS6 was digested with BclI, which cuts the plasmidonce at nucleotide 404 in the xpt nucleotide sequence (Fig. 2).The cohesive ends were first treated with Klenow DNA poly-merase and then with T4 DNA ligase, resulting in plasmidpLCC7, which has gained a ClaI site at the expense of thenative BclI site. The resulting insertion of 4 extra nucleotides inthe xpt sequence leads to a shift in the xpt reading frame and tothe termination of translation 46 nucleotides downstream ofthe native BclI site. The mutant allele was named xpt-11. AnErr DNA cassette was cloned in the BamHI site of pLCC7,resulting in pLCC11, which was subsequently transformed intostrain SS1 selecting for Err. Since pLCC11 cannot replicate inB. subtilis, Err transformants must result from a recombinationevent in which the plasmid has integrated into the chromo-some by homologous recombination, in this case into the xptlocus. Three of four tested Err transformants were resistant to8-azaxanthine, indicating that they had become XPRTase neg-ative (34). An 8-azaxanthine-resistant transformant was grownfor several generations in the absence of erythromycin, and theculture was screened for Ers clones. Three Ers clones werepicked and tested for 8-azaxanthine resistance and XPRTaseactivity. Southern blot analysis of one of the 8-azaxanthine-resistant clones (LCC16) verified that the wild-type xpt allelehad been exchanged with the xpt-11 frameshift mutation fromplasmid pLCC11 (data not shown). LCC16 lacked XPRTaseactivity and was unable to take up xanthine (Table 2).Mapping of the xpt-pbuX promoter and evidence for an

operon structure. The 0.9-kb SspI-EcoRI (nucleotides 1 to891) fragment covering the upstream region and part of the xptgene was cloned into the HindIII site in front of the lacZ genein plasmid pDG268neo (resulting in pLCC1). By this means, atranscriptional fusion between xpt and lacZ was created. Theintegration of transcriptional fusions in pDG268neo into theamyE gene has been described previously (32). Strain LCC1(amyE::xpt-lacZ) appeared blue on L broth agar plates con-taining 40 mg of 5-bromo-4-chloro-3-indolyl-b-D-galactosideper ml, indicating the presence of a promoter on the 0.9-kbSspI-EcoRI fragment. To localize the transcription start site,

TABLE 2. XPRTase activity and uptake rate of xanthine in aB. subtilis wild type and in xpt and pbuX mutant strainsa

Strain Relevantgenotype

XPRTase activity(nmol/min/mg)

Uptake rate of[14C]xanthine (nmol/min/mg [dry wt])

SS1 Wild type 21 6 1 2.0 6 0.3LCC5 xpt::erm ,0.1 0.6 6 0.2LCC4 pbuX::erm 19 6 2 ,0.1LCC16 xpt-11 ,0.1 ,0.2

a Cells were grown in glucose minimal medium containing NH41 and gluta-mate as the nitrogen source. At an optical density at 450 nm of 0.7, cells wereharvested and XPRTase activity and the uptake rate of xanthine were deter-mined as described in Materials and Methods. Values are means 6 standarddeviations.


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primer extension analysis was performed with two oligonucle-otide primers complementary to a DNA sequence upstream(primer 2; nucleotides 249 to 264) or downstream (primer 4;nucleotides 445 to 460) of the putative transcription terminatorin front of xpt. RNA was purified from cells of strain SS1 grownin minimal medium with ammonium ions and glutamate asnitrogen source and with or without hypoxanthine (Fig. 3). Inthe primer extension reaction containing primer 4, two exten-sion products were clearly detectable in the reaction containingRNA isolated from the culture grown in the absence of hypo-xanthine. When RNA from the hypoxanthine-grown culturewas used, markedly reduced amounts of both products wereobserved. The majority of cDNA synthesis stopped at the Gresidue complementary to the C325 (corresponding to the lastnucleotide in the 39-proximal strand of the terminator stem)(Fig. 2), and a small amount was extended to a position com-plementary to one of the residues between T168 and A170. Inthe reaction containing primer 2, the two RNA preparationsproduced equal amounts of extension product and cDNA syn-thesis terminated at a T residue complementary to the residueA172. Since both primers generated cDNA products, which ex-tend to position A172, we suggest that A172 is the transcription-al start site of the xpt gene. As shown in Fig. 2, A172 is pre-ceeded by a sA-like promoter sequence. We further suggest

that under the experimental conditions used, in vitro formationof the stem-loop structure in the xpt mRNA leader takes placeand that the reverse transcriptase enzyme may terminatecDNA synthesis from primer 4, when it reaches this secondarystructure (37).The length of the xpt transcript was determined in a North-

ern blot analysis in which total RNA was isolated from cells ofstrain SS1 grown in minimal medium containing ammoniumions plus glutamate and with or without hypoxanthine (Fig. 4).The probe was a 461-bp radiolabelled BsaBI-HpaI DNA frag-ment (nucleotides 149 to 610). The probe hybridized to anmRNA with the size of approximately 2.1 kb, indicating thatthe transcription initiating at A172 terminates at a point imme-diately downstream of the pbuX gene, most likely after thestretch of U residues that follow the dyad symmetric sequenceat nucleotides 2255 to 2270. However, in the lane containingRNA from cells grown in the presence of hypoxanthine, the2.1-kb signal was weaker, and a small transcript with a lengthof approximately 150 bp was simultaneously detected. Thelength of this transcript corresponds to the number of nucle-otides from the transcription start site (A172) to the end of thestem of the terminator at position C325. From the primer ex-tension experiment with primer 2 (which hybridizes upstreamof the putative terminator), we may conclude that transcriptioninitiation from the xpt promoter is the same regardless ofwhether hypoxanthine is present in the growth medium. On theother hand, with primer 4, a significant amount of extensionproducts was detected only when RNA from cells grown in theabsence of hypoxanthine was used, indicating that the forma-tion of the putative terminator structure may be purine depen-dent. We conclude that the xpt and pbuX genes constitute anoperon and that transcription termination at the secondarystructure in front of the xpt gene is subjected to purine control.Translational coupling. The coding regions of the xpt and

pbuX genes overlap by 1 nucleotide. This observation could

FIG. 3. Mapping of the xpt-pbuX transcription start point by primer exten-sion analysis. Total RNA was isolated from strain SS1 grown in ammonium-plus-glutamate minimal medium with or without hypoxanthine added. First-strandcDNA synthesis was performed as described in Materials and Methods. Primer2 (nucleotides 249 to 264) and primer 4 (nucleotides 445 to 460) hybridize tosequences upstream and downstream, respectively, of the putative terminatorstructure in front of the xpt gene. The same primers were used to generate thesequencing ladders. Lanes: 1 and 2, extension products produced from RNAisolated from cells grown in the presence or absence of hypoxanthine, respec-tively. The most likely transcription start point (A172) is indicated by a bentarrow. The straight arrow indicates the position of the start point of the longertranscript detected with primer 4. The emphasized sequences are given as thecomplementary sequences of those shown in the sequencing ladders.

FIG. 4. Northern blot analysis of the transcripts initiated at the xpt-pbuXpromoter. Total RNA was isolated from cells of strain SS1 grown in ammonium-plus-glutamate minimal medium containing no purine (lane 1) or 0.5 mg ofhypoxanthine per ml (lane 2). The RNA was separated and transferred to anitrocellulose membrane as described in Materials and Methods. The membranewas probed with a radiolabelled BsaBI-HpaI (nucleotides 149 to 610) DNAfragment. The RNA size marker was the 0.24- to 9.5-kb RNA ladder from GibcoBRL. Arrows indicate the positions of the long transcript (2.1 kb) and the shorttranscript (0.15 kb).


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indicate that efficient translation of the pbuX reading framerequires translation of the upstream xpt reading frame. To anal-yze this further, we constructed a translational pbuX-lacZ fu-sion. A 392-bp PCR product containing the 39 end of the xptreading frame and the first three codons of the pbuX readingframe (corresponding to nucleotides 562 to 946 plus restrictionsites) was constructed as described in Materials and Methods.This product was digested withHindIII and XbaI and ligated toHindIII- and XbaI-digested pSGMU37 DNA (13). This gen-erates the plasmid pLCC9, in which the 3rd codon of the pbuXreading frame was fused with the 14th codon of the lacZreading frame of pSGMU37. pLCC9 was recombined into thechromosome of strains SS1 and LCC16 (xpt-11) by selectionfor Cmr. The resulting strains were named LCC19 [pbuX-lacZ(pLCC9)] and LCC20 [pbuX-lacZ (pLCC9) xpt-11]. StrainsLCC19 and LCC20 were grown in minimal medium contain-ing ammonium ions and glutamate, and the b-galactosidase(pbuX-lacZ fusion) activity was determined. The b-galactosi-dase levels were 1,903 6 45 nmol/min/mg of protein in strainLCC19 and 268 6 22 nmol/min/mg of protein in LCC20(means 6 standard deviations). To analyze whether the de-creased level in LCC20 was due to decreased transcription ofthe downstream pbuX gene, a Northern blot analysis with RNAisolated from strains SS1 and LCC16 was performed. TwoDNA probes were used, a 540-bp AccI-EcoRI fragment cov-ering the xpt gene and a 755-bp BglII-PstI covering most of thepbuX gene. In addition, RNA was isolated from strains LCC19and LCC20 and probed with a 665-bp HpaI lacZ-internal DNAfragment from pSGMU37. The data presented in Fig. 5 showthat the xpt-pbuX transcript of SS1 and LCC16 detected by thexpt- and pbuX-specific probes and the xpt-pbuX-lacZ hybridtranscript of strains LCC19 and LCC20 detected by the lacZ-specific probe all appear identical with respect to amount andlength (2.1 kb for the xpt and pbuX probes and 3.8 kb for thelacZ probe).We conclude that the translation of the pbuX reading frame

is dependent on translation of the upstream xpt reading frameand that the transcription of the pbuX gene is unaffected by thepresence of untranslated upstream mRNA.Purine-controlled expression of the xpt-pbuX operon. As

shown in Fig. 4, the expression of the xpt-pbuX operon ap-peared to be regulated by a purine-controlled transcriptiontermination mechanism. A more detailed analysis of the purineeffect on the expression of the xpt-pbuX operon was performedby determining the XPRTase and b-galactosidase (xpt-lacZfusion) levels in cultures of strain LCC1 grown in the presenceof different nitrogen sources and purine compounds (Table 3).A twofold reduction of the XPRTase and b-galactosidase lev-els was observed upon the addition of adenine to the ammo-nium ion-glutamate cultures. The b-galactosidase level wasfurther decreased by a factor of two when ammonium ions andglutamate were not present in the culture containing adenine.The strongest repression of the xpt-pbuX operon expressionwas observed in cultures containing hypoxanthine, inosine, orguanosine. Inosine and guanosine are phosphorylized insidethe cell by inosine or guanosine phosphorylase, resulting in theformation of hypoxanthine and guanine, respectively (34). Therepressing effect was concentration dependent and was halvedat a 0.1 mM concentration (data not shown). The addition ofxanthine had approximately the same effect as the addition ofadenine. The b-galactosidase level (xpt-lacZ fusion) in the hy-poxanthine- and xanthine-grown cultures was less repressed incells growing in the presence of ammonium ions and glutamatethan in those grown in their absence. No repression was ob-served when cells grew with poor nitrogen sources like gluta-mate or uric acid. In cell extracts from cells growing with

purines as the nitrogen source, it was not possible to measureXPRTase activity because of the rapid oxidation of xanthine.We conclude that the xpt-pbuX operon expression is regulatedby purines and that the control is at the level of transcription.Furthermore, it appears that purine repression is partially re-lieved by conditions of excess nitrogen (ammonium ions orglutamine).The intracellular corepressor. To identify the effector mol-

ecule, the level of expression of the xpt-lacZ fusion in mutantstrains defective in genes involved in the salvage and intercon-version of purines was analyzed (Table 4). No reduction in theb-galactosidase level was observed in cultures of strain LCC29(ade amyE::xpt-lacZ) defective in adenine deaminase growingin the presence of adenine. This indicates that adenine must bedeaminated to hypoxanthine to repress the xpt-lacZ expression.Repression by hypoxanthine was normal in this strain. A strongrepression of the xpt-lacZ fusion expression in strain LCC30(hpt::erm amyE::xpt-lacZ) was observed in the absence of anyexogenously added purine compounds. Strain LCC30, which isdefective in hypoxanthine (guanine) phosphoribosyltransfer-

FIG. 5. Northern blot analysis of the xpt-pbuX transcript from wild-type cellsand from cells containing the xpt-11 frameshift mutation. Total RNA was iso-lated from the indicated strains and subjected to a Northern blot analysis asdescribed in Materials and Methods. (A) RNA from wild-type SS1 (lanes 1 and3) and the xpt-11 mutant strain LCC16 (lanes 2 and 4) was fixed to membranes,which were subsequently probed with a radiolabelled AccI-EcoRI DNA frag-ment covering most of the xpt gene (lanes 1 and 2) and a BglII-PstI fragmentcontaining the 39 end of the pbuX gene (lanes 3 and 4). (B) RNA was isolatedfrom strain LCC19 (lane 1) and the xpt-11 mutant strain LCC20 (lane 2), whichboth contain a translational pbuX-lacZ fusion. The membrane was probed witha radiolabelled HpaI DNA fragment that covers a part of the lacZ gene.


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ase, excretes significant amounts of hypoxanthine into the growthmedium, indicating an increased intracellular formation of hy-poxanthine in the H-GPRTase-deficient mutant strain (34a).In the ade hpt double-mutant strain LCC31, the control ofxpt-pbuX expression was normal and the strain did not excretehypoxanthine. In these experiments, inosine and guanosine wereused as a source of hypoxanthine and guanine, respectively,because hypoxanthine and guanine are only poorly taken up bycells lacking H-GPRTase activity. The addition of xanthine to

strain LCC1 also represses the xpt-lacZ expression (Table 3).Since these purine bases cannot be directly converted to otherpurine bases, we conclude that all three 6-oxo-purines (hypo-xanthine, xanthine, and guanine) may act as corepressors andthat hypoxanthine and guanine exert a much greater effectthan xanthine. When added together, a significantly lower ex-pression was seen. In pair-wise combinations, hypoxanthine,xanthine and guanosine worked synergistically (Table 3). Thestrongest effect was seen when guanosine and hypoxanthinewere added together, and the addition of all three purines hadthe same effect as that of the guanosine-hypoxanthine combi-nation (data not shown). When the concentration of hypoxan-thine and guanosine was reduced four times, the level of xpt-lacZ expression was 4 6 1 nmol/min/mg of protein. To see ifthe purR gene product, which has PRPP as an effector mole-cule (42), is involved in the regulation of the xpt-pbuX operon,the XPRTase activity was measured in strain LCC28(purR::neo), which has a mutation in the gene encoding the puroperon repressor (42). The XPRTase level was 21 6 1 and1.9 6 0.1 nmol/min/mg of protein without and with hypoxan-thine added to the growth medium, respectively. This indicatedthat the PurR-PRPP complex is not involved in the purine-controlled expression of the xpt-pbuX operon.Xanthine degradation. B. subtilis is able to use adenine,

xanthine, hypoxanthine, guanine, uric acid, allantoin, and ureaas sole nitrogen sources (14, 29, 30). We found that B. subtiliscould use allantoic acid as a sole nitrogen source (data notshown). These observations indicate that B. subtilis contains apathway for purine catabolism, as illustrated in Fig. 1. To seewhether purines could serve both as a purine and a nitrogensource at the same time, we incubated strain ED138, which isauxotrophic for purines on plates with xanthine or hypoxan-thine in the absence of other nitrogen sources. After 2 days ofincubation, colonies appeared, indicating that xanthine utiliza-tion for nucleotide synthesis and xanthine degradation to am-monium ions can occur simultaneously. Allopurinol, which is aspecific inhibitor of the enzyme xanthine dehydrogenase, pre-vented growth when added together with hypoxanthine or xan-thine but not when added together with adenine, uric acid, orammonium ions plus glutamate (data not shown). These ob-servations indicated the involvement of xanthine dehydroge-nase in the degradation of hypoxanthine and xanthine. Toinvestigate whether PbuX was involved in the utilization ofxanthine as a nitrogen source, we incubated wild-type cells(strain SS1) and the pbuX mutant (strain LCC4) on platescontaining xanthine as the sole nitrogen source and glucose asthe carbon source. In the concentration range of 3.3 to 0.1 mMxanthine, growth rates were similar; at lower concentrations,strain LCC4 grew less well than strain SS1.The initial rate at which cell extracts of B. subtilis could

convert 8-14C-labelled xanthine to uric acid was used to deter-mine the level of XDHase activity (see Materials and Meth-ods). The PRPP-independent conversion of xanthine to uricacid was induced more than 80-fold in wild-type cells growingwith hypoxanthine as the nitrogen source compared to that ofcells growing with glutamine as the nitrogen source (Table 5).A similar fold increase in induction in the XDHase activity wasobserved in cells growing with adenine or xanthine as thenitrogen source (data not shown). The level of XDHase activ-ity was also analyzed for the mutant strain LCC25 (glnA::spc)defective in glutamine synthetase. In contrast to that of thewild type, the XDHase activity was induced in cells of strainLCC25, which have been growing in the presence of highconcentrations of glutamine. This demonstrates that the regu-lation of the production of XDHase is subjected to nitrogencatabolite repression mediated through the GlnA-dependent

TABLE 3. XPRTase activity and expression of the xpt-lacZ fusionin B. subtilis LCC1 grown in minimal medium supplemented

with various nitrogen sources and purine additionsa

Nitrogensource

Purineaddition

Enzyme activity(nmol/min/mg)

XPRTase b-Galactosidase

NH41 None 28 6 4 246 6 19

Glutamine None 24 6 3 302 6 39NH4

1 1 glutamate None 226 3 235 6 14Glutamine 1 gluta-mate

None 23 6 1 409 6 13

Glutamate None 30 6 7 386 6 79

NH41 1 glutamate Adenine 156 2 (2)b 128 6 9 (2)

NH41 1 glutamate Hypoxanthine NDc 55 6 3 (4)

NH41 1 glutamate Xanthine ND 109 6 8 (2)

NH41 1 glutamate Uric acid ND 240 6 13 (1)

NH41 1 glutamate Guanosine ND 47 6 12 (5)

NH41 1 glutamate Inosine 46 2 (6) 53 6 4 (4)

NH41 1 glutamate Hypoxanthine 1

xanthineND 27 6 1 (9)

NH41 1 glutamate Guanosine 1 xan-

thineND 17 6 3 (14)

NH41 1 glutamate Hypoxanthine 1

guanosineND 1.3 6 0.2 (157)

Glutamate Hypoxanthine 13 6 2 (2) 30 6 5 (8)

Adenine None ND 65 6 5 (4)Hypoxanthine None ND 15 6 4 (16)Xanthine None ND 45 6 2 (5)Uric acid None ND 232 6 19 (1)

a Cells were grown in glucose minimal medium. Ammonium ions were addedto a final concentration of 30 mM, and glutamate and glutamine were added toa final concentration of 0.2%. Purine bases were added to a final concentrationof 0.5 mg/ml (3.3 mM), and guanosine and inosine were added to a final con-centration of 0.3 mg/ml (1 mM).b Numbers in parentheses indicate fold of repression of activity calculated as

the enzyme activity found in the NH41-plus-glutamate culture divided by theactivity found in the culture in question.c ND, not determined.

TABLE 4. Expression of the xpt-lacZ fusion in B. subtilis mutantstrains defective in the purine salvage pathwaya

Strain(relevant genotype)

Purineadded

b-Galactosidase activity(nmol/min/mg)

LCC29 (ade-1) None 224 6 22Adenine 230 6 9Hypoxanthine 67 6 4

LCC30 (hpt::erm) None 12 6 1LCC31 (ade-1 hpt::erm) None 290 6 18

Inosine 21 6 3Guanosine 23 6 5

a Cells were grown in glucose minimal medium supplemented with 30 mMammonium ions and 0.2% glutamate. Purine bases were added to a final con-centration of 3.3 mM, and inosine was added to a final concentration of 1 mM.


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signalling pathway. The b-galactosidase (xpt-lacZ fusion) levelsin both the wild type and the glnAmutant strain were repressedin the presence of hypoxanthine. This indicates that the purine-controlled expression of the xpt-pbuX operon works indepen-dently of the GlnA signalling pathway.

DISCUSSION

For the first time, a gene (xpt) encoding a specific XPRTasehas been cloned and its nucleotide sequence has been deter-mined. A search of the nucleotide sequence data base revealeda high degree of identity between the C-terminal part of theXpt amino acid sequence and the deduced amino acid se-quence of a human cDNA clone. However, previous investi-gations indicate that no specific XPRTase activity in humansexists (19, 24). The B. subtilis xpt gene was found to form abicistronic operon with another unique gene (pbuX) encodinga membrane-embedded high-affinity xanthine transport pro-tein. Other examples of nucleobase and nucleoside permeasesthat form operons with genes encoding enzymes responsiblefor the intracellular conversion of the transported molecule arethe upp-uraA (2) and codBA (9) operons of E. coli, whichencode permeases for uracil and cytosine, respectively, and thedra-nupC-pdp operon of B. subtilis (32), which encodes a py-rimidine nucleoside permease.Evidence for the function of the PbuX protein was obtained

from uptake experiments with the wild type and a pbuXmutantstrain. The results clearly showed defective xanthine uptake inthe pbuX mutant strain. A hydrophobicity profile of the de-duced PbuX amino acid sequence (data not shown) revealed12 to 13 hydrophobic segments characteristic of transmem-brane regions of membrane-embedded proteins (40). We con-structed two xpt mutants, strains LCC5 and LCC16. Since theyare both defective in the synthesis in XMP from xanthineand PRPP, we expected them to be deficient in the uptake ofxanthine. While strain LCC16 was seen to behave as predicted,strain LCC5 exhibited only a threefold reduction in xanthineuptake (Table 2). The reason why strain LCC5 shows suchunexpected behavior might lie in the nature of the mutation ofthe xpt gene. The gene is disrupted by an erythromycin resis-tance gene, erm, and it is possible that the previously reportedtranscription in the 59-to-39 direction relative to the erm geneof the resistance DNA cassette extends into pbuX (32). Thismay cause an increased production of the xanthine permeaseprotein. This result and our findings that xanthine transport issaturable indicate that xanthine transport, like that of other

purine bases in B. subtilis (4, 34), are mediated by facilitateddiffusion across the membrane.Overlapping coding sequences are a common feature of B.

subtilis, and it has often been suggested that such overlaps areaccompanied by the phenomenon known as translational cou-pling, where a downstream gene in an operon is at least par-tially dependent on the successful translation of an upstreamgene. The xpt and pbuX reading frames overlap by 1 nucleo-tide. In this report, we demonstrate that premature termina-tion of translation of the xpt mRNA in the xpt frameshiftmutant strain LCC20 resulted in a sevenfold reduction oftranslation of pbuX-lacZ mRNA compared with that of strainLCC19, which contains the wild-type xpt gene. In addition, weshow that the transcription of the downstream pbuX gene is notaffected in a mutant strain containing the xpt-11 frameshiftmutant allele (Fig. 5). That indicates that no premature tran-scription termination due to the presence of longer segmentsof “naked” mRNA attached to the RNA polymerase takesplace (18).The transcription of the xpt-pbuX operon is subjected to

purine repression. Analysis of the sequence of the xpt leadermRNA indicated that two mutually exclusive stem-loop struc-tures might be formed. The leader sequence is capable offorming a stable factor-independent transcription terminatorstructure, and the 59-proximal end of the terminator stem mayalso form an alternative secondary structure with nucleotideslocated upstream. This second structure may be designated thetranscription antiterminator (Fig. 6). The structure of the xpt-pbuX mRNA leader sequence is very similar to that of themRNA leaders of the trp, pur, and pyr operons (35, 38, 43). Thetranscription termination-antitermination mechanism control-ling the expression of these operons has been described pre-viously (references 35, 43, and 38, respectively). The low-mo-lecular-weight effectors of the xpt-pbuX expression were found

FIG. 6. Secondary-structure predictions of the nontranslated leader se-quence of the xpt-pbuX transcript. Numbers in parentheses indicate the first andlast nucleotides in the shown sequences. The numbering is the same as that usedin Fig. 2. Sequences involved in secondary-structure formation are indicated byheavy lines. Base pairings between G and C residues and between A and Uresidues are indicated by double and single lines, respectively. The lowest freeenergy was calculated as described previously (37). (A) Secondary structurecomprising sequences 1 and 2 (Fig. 2); (B) transcription terminator structureresulting from base pairing of sequences 2 and 3.

TABLE 5. Expression of the xpt-lacZ fusion and the level ofxanthine dehydrogenase (XDHase) activity in B. subtilis wild type

and in a glnA mutant straina

Strain(relevant genotype)

Addition tomedium

Enzyme activity(nmol/min/mg)

b-Galactosidaseb XDHase

LCC1 (wild type) Glutamine 302 6 39 ,0.3Hypoxanthine 15 6 4 24 6 1Glutamine 1 hypo-xanthine

40 6 6 ,0.3

LCC25 (glnA::spc) Glutamine 331 6 44 26 6 6Glutamine 1 hypo-xanthine

20 6 2 32 6 10

a LCC1 and LCC25 were grown in nitrogen-free glucose minimal mediumsupplemented with 0.2% glutamine and/or 3.3 mM hypoxanthine.b b-Galactosidase values for strain LCC1 were taken from Table 3.


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to be hypoxanthine and guanine, with xanthine having someeffect (Table 4). The effectors appear to be the same as thosefor the so-called guanine nucleotide-mediated control of thepur operon expression, which is proposed to be mediated by anRNA-binding regulatory protein (11). It is tempting to spec-ulate that the regulatory proteins of the xpt-pbuX and puroperons are the same. In addition to the guanine nucleotide-mediated transcription termination control, the pur operonexpression is also regulated by the PurR repressor, which is aregulatory protein, that binds to a region upstream of the purpromoter and controls the rate of transcription initiation. Theeffector molecule involved in this regulation is PRPP (42). Wefound no evidence for the involvement in the expression of thexpt-pbuX operon of the PurR-PRPP complex but did find thatthe repression of the xpt-pbuX operon by purine nucleobaseswas quite sensitive to the presence of excess ammonium ions(Table 3). Thus, a 4-fold reduction in expression was seenwhen hypoxanthine was added to cells grown in the presence ofammonium ions, while a 16-fold reduction in expression wasseen when hypoxanthine served as the sole nitrogen source.This implies that the purine control is modulated by the avail-ability of excess ammonium ions.The ability of B. subtilis to grow on different purine com-

pounds has been documented before (30); in this study, wereported the ability of B. subtilis to grow on allantoic acid asthe sole source of nitrogen as well. It was furthermore shownthat XDHase was required for the utilization of hypoxanthineor xanthine as the nitrogen source. Allopurinol did not inhibitgrowth on adenine as the sole source of nitrogen because thedeamination of adenine to hypoxanthine results in the forma-tion of ammonium ions (Fig. 1). The GlnA-dependent signal-ling pathway, which regulates the nitrogen-controlled produc-tion of nitrate or nitrite reductase (15), glutamine synthetase,urease, the nrgAB operon-encoded proteins, and asparaginase(3), also affects the level of XDHase activity, which was high ina glnA genetic background (Table 5). xpt-pbuX expression lev-els in both the wild-type and glnA mutant strains were re-pressed to the same extent in the presence of hypoxanthine,indicating that the purine-controlled expression works inde-pendently of the GlnA signalling pathway.The nutritional state of the B. subtilis cell determines the

fate of xanthine by two control mechanisms, namely, the pu-rine-controlled expression of the xpt-pbuX operon and theGlnA-dependent induction of the XDHase. Although the twomechanisms seem to work independently, they have an elegantcoexistence. Under conditions in which glutamine plus gluta-mate are present, the xpt-pbuX operon is fully expressed,whereas the locus for the XDHase is repressed. Conversely,when ammonium ions are absent and purines are added as thenitrogen source, XDHase is expressed and the xpt-pbuX ex-pression is repressed. When considering the pbuX-encodedxanthine permease, it seems illogical to repress its synthesiswhen purines are present. However, because the permeaseappears to be a high-affinity permease, it is not needed at highconcentrations of xanthine.

ACKNOWLEDGMENTS

We thank Kirsten Hansen for excellent technical assistance. Wethank Howard Zalkin for providing us with plasmid pMW11 and SusanFisher for plasmid pGLNA14.This research was supported by EU contract BIO2-CT95-0278 and

received financial support from the Saxild Family Foundation.

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XanthineMetabolismin Bacillussubtilis:Characterization xpt ... · TABLE1.Bacterialstrainsandplasmids Strainorplasmid Genotypeordescriptiona Sourceorreferenceb Strains B.subtilis 168