purine utilization by klebsiella oxytoca m5al: genes for ring...

JOURNAL OF BACTERIOLOGY, Feb. 2009, p. 1006–1017 Vol. 191, No. 30021-9193/09/$08.00�0 doi:10.1128/JB.01281-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Purine Utilization by Klebsiella oxytoca M5al: Genes for Ring-Oxidizingand -Opening Enzymes�

Scott D. Pope,† Li-Ling Chen, and Valley Stewart*Department of Microbiology, University of California, Davis, California 95616-8665

Received 11 September 2008/Accepted 25 November 2008

The enterobacterium Klebsiella oxytoca uses a variety of inorganic and organic nitrogen sources, includingpurines, nitrogen-rich compounds that are widespread in the biosphere. We have identified a 23-gene clusterthat encodes the enzymes for utilizing purines as the sole nitrogen source. Growth and complementation testswith insertion mutants, combined with sequence comparisons, reveal functions for the products of these genes.Here, we report our characterization of 12 genes, one encoding guanine deaminase and the others encodingenzymes for converting (hypo)xanthine to allantoate. Conventionally, xanthine dehydrogenase, a broadlydistributed molybdoflavoenzyme, catalyzes sequential hydroxylation reactions to convert hypoxanthine viaxanthine to urate. Our results show that these reactions in K. oxytoca are catalyzed by a two-componentoxygenase (HpxE-HpxD enzyme) homologous to Rieske nonheme iron aromatic-ring-hydroxylating systems,such as phthalate dioxygenase. Our results also reveal previously undescribed enzymes involved in urateoxidation to allantoin, catalyzed by a flavoprotein monooxygenase (HpxO enzyme), and in allantoin conversionto allantoate, which involves allantoin racemase (HpxA enzyme). The pathway also includes the recentlydescribed PuuE allantoinase (HpxB enzyme). The HpxE-HpxD and HpxO enzymes were discovered indepen-dently by de la Riva et al. (L. de la Riva, J. Badia, J. Aguilar, R. A. Bender, and L. Baldoma, J. Bacteriol.190:7892–7903, 2008). Thus, several enzymes in this K. oxytoca purine utilization pathway differ from those inother microorganisms. Isofunctional homologs of these enzymes apparently are encoded by other species,including Acinetobacter, Burkholderia, Pseudomonas, Saccharomyces, and Xanthomonas.

Purines and purine derivatives comprise a large portion ofbiomass and are involved in almost every step of life. Not onlya major constituent of nucleic acids, they also are central toenergy transfer and storage (ATP) as well as protein synthesisand signaling (GTP). Plants, animals, and many microorgan-isms use purines and purine derivatives to store and translo-cate nitrogen for assimilation or excretion (96).

Salvage pathways operate to recycle purines, including hy-poxanthine and xanthine, back into nucleoside pools (107).Additionally, some organisms can utilize purines as the solesource of nitrogen and carbon. Adenine and guanine aredeaminated to form hypoxanthine and xanthine, respectively,which then are oxidized to form uric acid (urate at physiolog-ical pH) (Fig. 1). These oxidation steps are catalyzed by xan-thine dehydrogenase, a well-studied molybdoflavoenzyme thatis conserved from bacteria to humans (51). Two sequentialring-opening steps convert urate via allantoin to allantoate(Fig. 1). Subsequent steps, which comprise different pathwaysin different microorganisms (96), convert allantoate to ammo-nium, which is assimilated.

Some organisms express only the latter portion of the purineutilization pathway and cannot use purines or urate as solesources of nitrogen. For example, Escherichia coli K-12 can use

allantoin and its catabolites as the sole nitrogen source, albeitonly under anaerobic conditions (21). Saccharomyces cerevisiaeuses allantoin as a nitrogen storage compound (17). However,the complete pathway is present in other bacterial and fungalspecies, including Bacillus subtilis (84) and Aspergillus nidulans(83).

Molybdoenzymes (excepting dinitrogenase) contain the mo-lybdenum cofactor Mo-molybdopterin (42). Thus, mutations ingenes for molybdenum cofactor biosynthetic enzymes (molgenes in bacteria and cnx in A. nidulans) confer pleiotropicphenotypes: these mutants can utilize neither nitrate nor pu-rines, due to lack of the molybdoenzymes nitrate reductase andxanthine dehydrogenase (74). We previously reported thatKlebsiella oxytoca mol mutants cannot assimilate nitrate butcan utilize xanthine as the sole nitrogen source (32). Thissuggested, as one possibility, that K. oxytoca uses a molybde-num-independent enzyme in place of conventional xanthinedehydrogenase. Results reported here demonstrate that this iscorrect, as insertion mutants blocked specifically in xanthineand hypoxanthine utilization define the structural genes for anapparent two-component Reiske nonheme iron oxygenase.

Here, we report analysis of 12 genes whose products catalyzeconversion of purines to allantoate. Our investigation of theremaining genes, whose products catalyze allantoate utiliza-tion, is ongoing. Results show that several steps in the overallpathway are catalyzed by previously undescribed enzymes.

While this paper was in review, the paper by de la Riva et al.(24), describing the hpxDE, hpxR, hpxO, and hpxPQT genesfrom Klebsiella pneumoniae W70, was posted in the “JB Ac-cepts” section of the Journal of Bacteriology online edition.Results and conclusions concerning these seven genes are con-gruent between the two studies.

* Corresponding author. Mailing address: Department of Microbi-ology, University of California, One Shields Ave., Davis, CA 95616-8665. Phone: (530) 754-7994. Fax: (530) 752-9014. E-mail: [email protected].

† Present address: Molecular Biology Interdepartmental Ph.D. Pro-gram and Department of Microbiology, Immunology & MolecularGenetics, University of California, Los Angeles, CA 90095-1489.

� Published ahead of print on 5 December 2008.

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(Some of the work presented here was submitted byDanielle Carl in 1994 as part of an undergraduate thesis to theCornell University Division of Biological Sciences Honors Pro-gram.)

MATERIALS AND METHODS

Media. Defined, complex, and indicator media for routine genetic manipula-tions were used as described previously (57). Nitrogen-free medium contained0.2% (wt/vol) glucose, 1% (wt/vol) sodium citrate, 0.74% (wt/vol) sodium phos-phate (pH 8), and 1 mM MgSO4 (53). This medium was supplemented withadditional nitrogen sources (10 mM NaNO3 or NH4Cl and 2.5 mM hypoxan-thine, xanthine, urate, allantoin, or potassium allantoate) as indicated. Uratesolutions were prepared as described previously (79). 5-Bromo-4-chloro-3-indo-lyl-�-D-galactopyranoside (X-Gal; 40 �g/ml) was included at 40 �g ml�1 to scorethe Lac phenotypes of MudJ insertion mutants. All nitrogen sources were fromSigma-Aldrich (St. Louis, MO).

Selection for K. oxytoca transformants carrying bla-containing plasmids wasaccomplished with a combination of carbenicillin and ampicillin (Ap) at 800 and60 �g ml�1, respectively (54). Streptomycin (Sm) was used at 500 �g ml�1 forselecting K. oxytoca Smr segregants (102). Other antibiotics used were chloram-

phenicol (Cm) at 50 �g ml�1, kanamycin (Km) at 100 �g ml�1, spectinomycin(Sp) at 50 �g ml�1, and tetracycline (Tc) at 20 �g ml�1.

Defined medium used to grow cultures for �-galactosidase assays and forgrowth yield tests was buffered with 3-[N-morpholino]propanesulfonic acid(MOPS) as previously described (69). Glucose (40 mM) was used as the solecarbon source, and nitrogen sources were added as indicated.

Culture conditions. Liquid cultures and plates were incubated at 30°C (41).Cultures for �-galactosidase assays were aerated at 240 rpm in 10 ml of mediumin 125-ml sidearm flasks. Culture densities were monitored with a Klett-Sum-merson photoelectric colorimeter (Klett Manufacturing Co., New York, NY)equipped with a no. 66 (red) filter. Cultures in the mid-exponential phase (about40 Klett units) were harvested, chilled on ice, and washed with saline. Cell pelletswere stored overnight at �20°C prior to the assay.

Cultures for nitrogen assimilation tests were grown in 5 ml of MOPS mediumin 16- by 175-mm culture tubes, aerated on a roller drum. Nitrogen sources wereadded over a range of growth-limiting concentrations: for nitrate, 0.4 to 1.8 mM;for histidine, 0.2 to 0.9 mM; for xanthine, 0.1 to 0.45 mM; and for guanine, 0.08to 0.36 mM. Culture densities (optical densities at 420 nm) were measured after24 and 48 h.

Phenotypes were determined by growth on plates made with nitrogen-freemedium and microbiological agar (Marine BioProducts, Delta, British Colum-bia) supplemented with the test nitrogen source at a final concentration of 10

FIG. 1. Purine ring oxidation and opening steps. The enzyme proposed to catalyze each step is shown. The K. oxytoca gene for adeninedeaminase was not identified in this study. Dashed lines show reactions that can occur spontaneously.

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mM N atoms. Strains were first streaked for single colonies on plates with addednitrate, which then were replica printed to plates with test compounds (49).Plates for anaerobic nitrogen assimilation tests were incubated in Brewer-Allgeier jars (11).

�-Galactosidase assays. Activity in CHCl3-sodium dodecyl sulfate-permeabi-lized cells was measured at room temperature, approximately 21°C, by monitor-ing the hydrolysis of o-nitrophenyl-�-D-galactopyranoside. Specific activities areexpressed in arbitrary (Miller) units (63). Cultures were assayed in duplicate, andreported values are averaged from two independent experiments.

Strains. Table 1 lists the strains used in this study. Previously, strain M5al wasclassified under Aerobacter aerogenes and subsequently under K. pneumoniae.However, phenotypic properties (such as positive reaction in the indole test)place this strain in the species K. oxytoca (see reference 102). Genetic crosseswere performed by P1 kc-mediated generalized transduction (90).

MudJ, originally termed Mu dI 1734, is a transposase-null bacteriophagetransposon that encodes resistance to Km and is used for isolating lacZ operonfusions (14). Mu cts hP1, a P1 host range variant of bacteriophage Mu (19), wasused as a helper phage for K. oxytoca as described previously (13). Tn10d(Tc)and Tn5d(Sp) are transposase-null transposons encoding resistance to Tc and Sp,respectively (25, 47).

Nomenclature. The genetic nomenclature extends that used by de la Riva et al.(24). The mnemonic hpx (hypoxanthine) is applied to all genes whose productsare involved in utilization of hypoxanthine or its catabolites.

We use Hpx as a general phenotypic designation without reference to aparticular step in the pathway. Specific phenotypes are denoted according to theblocked step (Fig. 1); for example, Hxn� mutants utilize neither hypoxanthinenor xanthine but grow normally with urate and downstream purine catabolites.Similarly, downstream blocks are denoted Urt� (urate), Aln� (allantoin), andAlt� (allantoate).

Transposon insertions. Table 2 lists insertion alleles used in this study. Inser-tion mutagenesis with transposons MudJ (13), Tn5d(Sp) (35), and Tn10d(Tc)(57) was performed as described previously. Insertions were selected on antibi-otic plates, with nitrate as the sole nitrogen source, to preclude isolation ofmutants with general defects in nitrogen assimilation. Colonies were then replicaprinted to antibiotic plates, with purine utilization intermediates (hypoxanthine,xanthine, urate, allantoin, or allantoate) as the sole nitrogen source.

Plasmids. Table 1 lists the plasmids used in this study. Standard methods (57)were used for restriction endonuclease digestion, ligation, transformation, and

PCR amplification (80). Plasmid pVJS3982 was constructed by excising the Kmr

determinant from plasmid pEG5005 with restriction endonuclease EcoRI andinserting the �-Sp integron (30) in its place.

Molecular cloning. In vivo cloning utilized the mini-Mu cloning vectorspEG5005 and pVJS3928 as described previously (53). Lysates prepared from thehpx� donor strains VJSK038 and VJSK3047 were used to transduce hpx insertionmutants lysogenic for Mu cts hP1. Strain VJSK650 (hpxO; Tcr) was the recipientfor cloning with vector pEG5005 (Kmr), and strain VJSK3046 (hpxE; Kmr) wasthe recipient for cloning with vector pVJS3982 (Spr). Antibiotic-resistant trans-formants were selected on complex medium, and colonies were replica printed toduplicate nitrogen-free plates, one containing nitrate and the other xanthine or

TABLE 1. Strains and plasmids

Strain or plasmid Genotype or description Source or reference

K. oxytoca M5al strainsVJSK009 hsdR1 13VJSK014 hsdR1 lac Tn7 13VJSK038 As VJSK014 but mal::Mu cts hP1/pEG5005 13VJSK650 As VJSK014 but hpxO102::Tn10d(Tc) mal::Mu cts hP1 This studyVJSK1371 As VJSK009 but mdr::Tn5 Laboratory collectionVJSK2088 As VJSK009 but lacZ101::Tn10d(Tc) rpsL 102VJSK2966 As VJS2088 but �rhaBS::��(hpxD-lacZ) This studyVJSK2967 As VJS2088 but �rhaBS::��(hpxR-lacZ) This studyVJSK2968 As VJS2088 but �rhaBS::��(hpxD-lacZ) hpxR113::�-Cm This studyVJSK2969 As VJS2088 but �rhaBS::��(hpxR-lacZ) hpxR113::�-Cm This studyVJSK3046 As VJSK009 but hpxE108::MudJ zxx::Mu cts hP1 This studyVJSK3047 As VJSK009 but zxx::Mu cts hP1/pVJS3928 This study

Plasmidsa

pAH144 Smr; ori R6K; pUC18 multiple cloning site 39pEG5005 Apr Kmr; ori pMB1; Mud5005 38pHG165 Apr; ori pMB1; pUC8 multiple cloning site 89pSU18 Cmr; ori p15A; pUC18 multiple cloning site 4pKAS46 Apr Kmr; ori R6K; rpsL�; pSL1180 multiple cloning site 88pVJS1435 As pHG165; (HindIII) 1284 to HindIII 17197 (hpxD-hpxU) This studypVJS1436 As pHG165; (HindIII) 2344 to (HindIII) 12193 (hpxR-hpxB) This studypVJS1454 As pSU18; SalI 11721 to (SalI) 23740 (hpxA-hpxK) This studypVJS2354 As pKAS46 but �rhaBS::lacZ lacY� 103pVJS3928 Apr Kms Spr; as pEG5005 but �-Sp This studypVJS3941 As pHG165; (HindIII) 1 to (HindIII) 8681 (hpxE-guaD) This studypVJS3958 As pHG165; HpaI 164 to HpaI 4180 (hpxE-hpxO) This study

a Sequence coordinates for restriction sites are shown. Sites in parentheses are from flanking Mu DNA present in the pEG5005 vector.

TABLE 2. hpx insertions

Allelea Insertioncoordinatesb

Product ofcodonc Straind

hpxE108::MudJ (Lac�) 707–708 Gly-187 VJSK651hpxE109::MudJ (Lac�) 739–740 His-176 VJSK652hpxD103::MudJ (Lac�) 2337–2338 Nonee VJSK643hpxR113::�-Cm 2617–2618 Thr-60 VJSK2962f

hpxO102::Tn10d(Tc) 4079–4080 Ser-211 VJSK642hpxO112::Tn5d(Sp) 4466–4467 Leu-82 VJSK3034guaD114::pAH144 7602–7603 Leu-335 VJSK2985g

hpxA101::Tn10d(Tc) 11782–11783 Gln-242 VJSK641hpxB104::MudJ (Lac�) 12052–12053 Tyr-79 VJSK644hpxB107::MudJ (Lac�) 12189–12190 Asp-124 VJSK648hpxB105::MudJ (Lac�) 12295–12296 Lys-160 VJSK645hpxB110::MudJ (Lac�) 12515–12516 Thr-233 VJSK681hpxF111::Tn5d(Sp) 17840–17841 Asp-28 VJSK3033

a The Lac phenotype for MudJ alleles is indicated in parentheses.b The insertion or disruption lies between the indicated sequence coordinates.c Codon disrupted.d All are in the VJSK009 strain background except as noted.e The insertion lies 20 nt upstream of the hpxD initiation codon.f VJSK2088 strain background.g VJSK1371 strain background.

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hypoxanthine. Hpx� colonies were isolated, and plasmid DNA was extracted andretransformed to confirm the phenotype.

This in vivo cloning method generates essentially random inserts whoseboundaries are delimited by the ends of bacteriophage Mu DNA (38). Largeinserts from the pEG5005-based clones were subcloned into the pHG165 orpSU18 medium-copy-number vector to generate the plasmids pVJS1435,pVJS1436, pVJS1454, and pVJS3941 (Table 1). Subcloning utilized HindIII orSalI restriction endonuclease sites near the Mu DNA ends. These sites areindicated in parentheses in Table 1. All other sites are native to the K. oxytocahpx DNA region. Sites are denoted according to the nucleotide position withinthe sequenced region (see below).

DNA sequencing. Automated DNA sequencing reactions (82) were performedby the DNA Sequencing Facility, University of California—Davis. Double-stranded templates were sequenced on a model 373A stretch DNA sequencer byusing dye terminator chemistry and AmpliTaq-FS DNA polymerase (PerkinElmer/Applied Biosystems Division, Foster City, CA). Templates were preparedby using QIAprep spin plasmid kits (Qiagen, Inc., Chatsworth, CA).

Several subclones were generated by deleting or by recloning internal restric-tion fragments. Sequences were primed from flanking polylinker sequence insubclones or from oligonucleotide primers synthesized to match the ends ofsequence reads (primer walking). The DNA sequence was determined frommultiple reads on both strands. Sequence information was compiled and ana-lyzed through programs from DNASTAR, Inc. (Madison, WI).

DNA sequence analysis. Gene identifications were based primarily on theresults from BLAST searches (1) conducted through servers operated by theNational Center for Biotechnology Information at the National Library of Med-icine. Proteins with similar sequences are denoted by their locus tags or GenInfoIdentifier (GI) accession numbers, as appropriate. Domains (58) are indicated bytheir database accession numbers: conserved domain (cd), clusters of ortholo-gous groups (COG), or protein family (pfam).

Analysis was facilitated as unpublished whole-genome sequence data for K.pneumoniae strain MGH78578 were released by the Washington UniversityGenome Center. These data were particularly useful for assigning likely promot-ers and sites for translation initiation and termination, which most likely areconserved between the two species. The K. pneumoniae MGH78578 chromo-some sequence is available in GenBank under accession number NC_009648.

Allelic replacement. Two insertion alleles were constructed directly. ThehpxR113::�-Cm allele was made by cloning a BamHI-released �-Cm interposon(30) into the unique BclI site within the hpxR gene. This insertion was cloned intothe conditionally replicating vector pKAS46 and transplanted to the chromo-some by integration-excision allelic exchange as described previously (102). TheguaD114::pAH144 allele was made by cloning an internal fragment spanningguaD codons 125 to 334 into the conditionally replicating vector pAH144. Thesingle-crossover integration was selected by resistance to Sp. The veracity of bothreplacements was confirmed by PCR analysis.

�(hpxD-lacZ) and �(hpxR-lacZ) operon fusions. The divergent hpxD-hpxRtranscription control region was cloned as two approximately 320-nucleotide (nt)PCR-generated fragments with introduced XbaI and BamHI restriction endo-nuclease sites at either end. The endpoints lie within the codons encoding Ala-30and Gln-37 of the hpxD and hpxR coding regions, respectively. Fragments weresubcloned into BamHI- and XbaI-digested vector pVJS2354 to create �(hpxD-lacZ) and �(hpxR-lacZ) operon fusions. The resulting constructs were trans-planted into the rha locus by integration-excision allelic exchange as describedpreviously (103).

Nucleotide sequence accession number. The DNA sequence reported in thispaper is available from the GenBank nucleotide sequence database under ac-cession number EU884423. The sequence spans 23,740 nt, including 287 ntcomprising the 3 portion of a gene homologous to the Serratia marcescens fbpCgene (2) (adjacent to the hpxE gene) and 581 nt comprising the 3 portion of agene homologous to Erwinia carotovora gene ECA2135 (adjacent to the hpxKgene).

RESULTS AND DISCUSSION

Purine utilization. K. oxytoca M5al can use purines, pyrimi-dines, and most amino acids as sole sources of nitrogen (66).The purine rings contain four N atoms (Fig. 1), so we wishedto determine if all four can be assimilated. We measured thegrowth yield of K. oxytoca cultured with limiting amounts ofnitrogen, as described in Materials and Methods. Nitrate (oneassimilable N atom) and histidine (two assimilable N atoms)

(56) served as calibration controls. Results indicate that allfour N atoms were assimilated from xanthine and that all fiveN atoms were assimilated from guanine (Table 3 and data notshown). We conclude that K. oxytoca quantitatively assimilatesall N atoms from purines. An identical conclusion was drawnby de la Riva et al., who used adenine instead of guanine (24).

Insertion mutants. We conducted several screens for trans-poson insertion mutants with specific blocks in purine utiliza-tion, as described in Materials and Methods. Mutants weretested for growth with hypoxanthine, xanthine, urate, allantoin,and allantoate (Fig. 1). Urea was not tested, since urease isencoded at a distinct, well-characterized locus (64). Fourclasses of mutants were identified. Mutants with the Hxn�

phenotype utilized neither hypoxanthine nor xanthine but didutilize urate, allantoin, and allantoate. Thus, these mutants arespecifically blocked in the conversion of hypoxanthine andxanthine to urate (Fig. 1). Similarly, mutants with the Urt�,Aln�, and Alt� phenotypes were specifically blocked in theutilization of urate, allantoin, and allantoate, respectively.

Our analysis of genes for utilizing allantoate (hpxFGHIJK),downstream purine catabolites (hpxWXYZ), and an associatedregulator (hpxU) will be reported separately. Provisional func-tions for these genes are included in the deposited GenBankfile.

We employed bacteriophage P1-mediated generalized trans-duction to backcross each insertion to the wild type in order todemonstrate linkage between the transposon and the Hpx�

phenotype. Genetic crosses (105) with pairs of insertions en-coding different antibiotic resistance phenotypes establishedthe order hpxD-hpxO-hpxB-hpxF (Fig. 2A). Additional crossesnot depicted in Fig. 2A yielded congruent results. Subsequentmolecular genetic analysis confirmed this order (see below andFig. 2B).

As we completed the DNA sequence of the hpx locus (seebelow), we employed whole-colony PCR amplification andDNA sequencing to determine the exact insertion site for eachof the transposons. The results revealed 11 distinct insertions(Table 2 and Fig. 2B). Thus, the insertion mutagenesis fell farshort of saturating the hpx locus. We used allelic replacementmethods to construct two additional null alleles as describedbelow. Overall, analysis of mutant phenotypes in conjunctionwith predictions from DNA sequence analysis allows func-tional assignments for each of the genes as described below.

Gene product identification. We cloned the hpx� genes bycomplementation as described in Materials and Methods. Fur-ther subcloning and complementation analyses localized spe-

TABLE 3. Growth yields from nitrogen sources

Compounda No. ofN atoms OD420/mM N atomsb

Nitrate 1 0.199 � 0.030Histidine 2 0.217 � 0.019Xanthine 4 0.267 � 0.053Guanine 5 0.255 � 0.033

a Cultures of strain VJSK009 were aerated overnight in MOPS defined me-dium with glucose and a range of growth yield-limiting concentrations of theindicated nitrogen source.

b Average final yield for at least five different cultures containing variousnitrogen source concentrations. OD420, optical density at 420 nm.



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cific hpx functions to defined regions of the restriction map asdescribed below. We determined the DNA sequence for the22,872-nt hpx region, and concomitant computer-assisted anal-yses identified 23 genes (Fig. 2B).

Some functional assignments are based on homology toknown proteins. In other instances, however, the proteins hadnot previously been characterized. In these cases, we initiallyattempted to identify orthologs present in genomes of otherspecies (48). However, orthologous proteins do not necessarilyperform identical functions (75); furthermore, it can be diffi-cult to ascertain orthology (48). Therefore, we have identifiedwhat we presume to be “isofunctional homologs” in otherspecies, based not only on overall sequence similarity and

conservation of critical residues but also on genetic clusteringof structural genes with those of other enzymes in the same orrelated pathways (i.e., synteny) (43). These assignments aredescribed in detail below and are summarized in Table 4.

Transcriptional organization. Transcription of most K. oxy-toca genes encoding nitrogen assimilation functions is regu-lated by general nitrogen control (Ntr) acting through the NtrCor Nac activator proteins (7, 55, 62). The NtrC protein acti-vates promoters dependent upon the RpoN (�54) form ofRNA polymerase, which recognizes promoters whose consen-sus sequence is TGGYRYRRNNNYYGCW (where R repre-sents purine, Y represents pyrimidine, and W represents A orT) (55). We identified six potential promoters by visual inspec-

FIG. 2. Klebsiella genes for purine utilization. (A) Genetic map as determined by generalized transduction. Map distances are calculated as 100minus percent cotransduction. The arrow points to the selected marker. (B) Physical map as determined by DNA sequence analysis. Genes aredenoted by arrows according to their encoded products: gray, enzymes; open, transporters; filled, regulators. Filled circles show sites of transposoninsertion, and filled squares show directed allelic replacements. Line arrows indicate predicted RpoN-dependent transcripts. The five geneticmodules are denoted by the substrate upon which their products act. (C) Gene organization in K. pneumoniae MGH78578. See also Table 4. Locustags (KPN_) delimiting the boundary of each unit are shown. Arrowheads indicate relative orientation in the genome.

TABLE 4. hpx genes, proposed functions, and isofunctional homologs

Gene No. ofcodons Proposed function

Isofunctional homologa

KPN_ XCC BTH_I ACIAD W168

hpxE 322 (Hypo)Xanthine hydroxylase reductase 01661 0296hpxD 345 (Hypo)Xanthine hydroxylase NAb 0297hpxR 312 hpxDE transcription activator (LysR type) 01662 0298hpxO 384 Urate hydroxylase 01663 0279c 3540hpxP 460 Purine permease 01664 pucJ, pucKhpxQ 166 OHCUd decarboxylase 01665 0280 2068 3537 pucLe

hpxT 108 5-Hydroxyisourate hydrolase 01666 0278 2072 3538 pucMguaD 440 Guanine deaminase 01791hpxC 495 Allantoin permease 01790 2065 2247 pucIhpxS 238 Transcription regulator (GntR type) 01789 2064hpxA 247 Allantoin racemase 01788 2066 2248hpxB 310 Allantoinase 01787 0282 2067 3536

a Locus tags: KPN_, K. pneumoniae MGH78578; XCC, Xanthomonas campestris ATCC 33913; BTH_I, Burkholderia thailandensis E264 (chromosome I); ACIAD,Acinetobacter sp. strain ADP1; W168, Bacillus subtilis W168 (genes).

b NA, not annotated. The orthologous gene is present at this location but not currently annotated.c Nonorthologous “flavin-containing monooxygenase,” presumably catalyzing the same reaction.d OHCU, 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline.e The region encoding the amino-terminal domain only.



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tion, each of which is conserved in the K. pneumoniaeMGH78578 sequence. These promoters are indicated in Fig.2B as directing synthesis of the following hypothetical tran-scripts, shown by arrows: hpxO, hpxPQT, guaD, hpxC,hpxWXYZ, and hpxFGHIJK. Nucleotide sequence coordinatesfor these potential promoters are included in the depositedGenBank file. Thus, transcription of most hpx genes is likelyactivated directly by phospho-NtrC protein. We emphasizethat we have not examined operon structure through directexperiments.

Modularity of hpx gene organization. Genes apparently in-volved in each step of the overall pathway are clustered to-gether, so the overall hpx locus appears to comprise asupraoperonic cluster (100) containing five distinct modules ofhpx genes plus the guaD and hpxU genes (Fig. 2B). The genecontent and order within modules is essentially identical in theK. pneumoniae MGH78578 genome (Table 4). However, in K.pneumoniae, the five modules are separated into three distinctgroups: hpxEDR–hpxOPQT– 100 kb – hpxKJIHGF–hpxU–hpxWXYZ – 16 kb – hpxBASC–guaD (Fig. 2C). Note that therelative orientations between modules also differ in the twospecies.

For K. oxytoca M5al, both the genetic and the physical mapsindicate that these modules are immediately adjacent. Forexample, the cotransduction frequency between the hpxF111::Tn5d(Sp) and hpxD103::MudJ alleles corresponds to a physicaldistance of roughly 30 kb, as estimated from the formula ofSanderson and Roth (81), approximately twice the actual dis-tance of about 16 kb. Similarly, the calculated distance be-tween the hpxO112::Tn10d(Tc) and hpxB105::MudJ alleles,roughly 15 kb, is approximately twice the actual distance ofabout 8 kb. Thus, at least for the region encompassing the hpxgenes, the genomes of these two Klebsiella species appear to beorganized differently.

Guanine to xanthine: GuaD guanine deaminase. Guanine isdeaminated to form xanthine (Fig. 1). The guaD gene productis most similar to E. coli K-12 guanine deaminase (60% se-quence identity over 97% of its length), the product of theguaD (ygfP; cd01303) gene (61). The GuaD sequence includesa conserved nine-residue motif (P-G-X-V/I-D-X-H-T/V/I-H)also shared by members of the cyclic amidohydrolase family.This motif has been implicated in Mn2� or Zn2� binding inmembers of the family (46, 101). E. coli guanine deaminasecontains Zn2� (61).

We constructed a guaD disruption strain to determine therole of this gene in purine utilization. The resulting mutantfailed to grow on plates with guanine as the sole nitrogensource but exhibited wild-type growth with adenine, hypoxan-thine, xanthine, and urate. The guaD coding sequence is pre-ceded by an apparent RpoN-dependent promoter and thuslikely constitutes a single-gene operon (Fig. 2B).

K. oxytoca M5al also uses adenine as the sole nitrogen source(66). However, the hpx locus does not include the gene foradenine deaminase.

(Hypo)Xanthine to urate: the hpxR-hpxDE module. Hypo-xanthine is hydroxylated to form xanthine, which also is hy-droxylated to form urate (Fig. 1). Xanthine dehydrogenase(purine hydroxylase), a well-studied molybdoflavoenzyme, isthe previously described enzyme catalyzing the sequential ox-idation of hypoxanthine and xanthine to urate (51). Structural

genes for this enzyme have been characterized for Rhodobactercapsulatus (52) and Bacillus subtilis (84). However, no suchgenes are present in the K. oxytoca hpx locus, and none wereidentified in the K. pneumoniae MGH78578 genome sequence.

Three MudJ insertion mutants exhibited the Hxn� pheno-type, failing to grow with hypoxanthine or xanthine as the solenitrogen source but displaying wild-type growth with urate anddownstream purine catabolites. Two of these insertions disruptthe hpxE gene, whereas the third lies 20 nt upstream of thehpxD initiation codon (Table 2) and therefore blocks hpxDEoperon transcription. We also constructed an hpxR insertion todetermine the role of this gene in regulation (see below). ThishpxR::�-Cm mutant also exhibited the Hxn� phenotype. Allfour insertion mutants were complemented to the Hxn� phe-notype by plasmid pVJS3958, a subclone that contains only thehpxRAB region (Table 1). Therefore, the hpxD, hpxE, and hpxRgenes encode functions required for conversion of (hypo)xan-thine to urate, a conclusion reached independently by de laRiva et al. (24). The hpxD and hpxE genes are separated byonly 13 nt and thus likely comprise an operon.

(i) HpxD and HpxE: (hypo)xanthine hydroxylase. Sequencecomparisons indicate that the hpxE and hpxD gene productscomprise the reductase and oxygenase, respectively, of a two-component Rieske nonheme iron aromatic-ring-hydroxylatingoxygenase system. The HpxD sequence is that of a Rieskenonheme iron oxygenase, categorized in the phthalate familyof Gibson and Parales (34), group I of Nam et al. (68), andclass IA of Batie et al. (5). These oxygenases are homomul-timeric aromatic-ring-activating mono- and dioxygenases withbroad substrate specificity. They share relatively low sequenceidentity and range in length from 329 to 446 aminoacyl resi-dues (the HpxD enzyme is 345 residues). The HpxD sequenceincludes a Rieske-type iron-sulfur cluster (consensus sequence,C-X-H-X15-26-C-X2-H; pfam00355; COG2146) near the aminoterminus and a likely nonheme Fe(II)-binding 2-His-1-carbox-ylate facial triad (consensus sequence, H-X4-5-H-Xn-D) nearthe center (reviewed in references 31 and 73). The HpxDsequence shares 29% identity over a 160-residue span with thatof the well-studied phthalate dioxygenase (OphA2 enzyme;GI 4128221; 443 residues) from Burkholderia cepacia DBO1(6, 16).

The HpxE sequence (322 residues) is that of a correspond-ing reductase, categorized in class IA of Batie et al. (5), andincludes the three defining cofactor domains: an amino-termi-nal FMN binding domain (consensus sequences, R-X-Y-S-Land G/S-R-G-G-S), a central NAD binding domain (consensussequence, G-G-I-G-I-T-P; pfam00175) (in HpxE and KshB[95], the first Gly residue is replaced with Ala), and a carboxyl-terminal plant-type iron-sulfur cluster (consensus sequence,C-X2-G-X-CG-X-C-X6-G-X3-HRD-X2-L-X5-A-X7-C-X-S;pfam00111; COG0633) (in HpxE, L-X5 is Q-X6) (16, 18, 67,95; reviewed in reference 60). The HpxE sequence shares 34%identity with the sequence of the phthalate dioxygenase reduc-tase (OphA1 enzyme; GI 13432205; 322 residues) from B.cepacia DBO1 (6, 16) and about 40% identity with those of thevanillate O-demethylase reductases from a variety of species(e.g., the VanB enzyme from Acinetobacter sp. strain ADP1;GI 2271499; 318 residues) (86).

Purines, which resemble two fused aromatic rings, exhibitsome aromatic properties (87). We suggest that the HpxE and



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HpxD enzymes comprise (hypo)xanthine hydroxylase reduc-tase and bifunctional (hypo)xanthine hydroxylase, respectively.This enzyme complex likely functions by the same mechanismsas those described for homologous aromatic-ring-hydroxylat-ing oxygenases, hydroxylating first the C-2 position of hypo-xanthine to yield the enol form of xanthine and second theC-8 position to yield the enol form of urate (Fig. 1). Purinesundergo tautomeric interconversions to shift between therelatively unstable enol form and the more stable keto form(8, 23, 87).

(ii) Transcriptional regulation in the hpxR-hpxDE module.The two hpxE::MudJ insertion mutants expressed LacZ activ-ity, indicating that they form �(hpxE-lacZ) operon fusions. Wecultured these strains in defined medium, with glutamine as aneutral nitrogen source, and measured LacZ-specific activity.Both strains expressed approximately 4,000 Miller units aftergrowth with added xanthine but less than 50 units after growthwith added urate, allantoin, or allantoate and about 10 unitsafter growth with added xanthine plus ammonium. This indi-cates that hpxDE operon transcription is subject to dual controlby global nitrogen regulation (Ntr) and by pathway-specificinduction by (hypo)xanthine (see also below) but not down-stream purine catabolites.

The hpxDE operon shares a 125-nt intergenic region withthe divergently transcribed hpxR gene (Fig. 2B). The HpxRsequence (312 residues) is similar to those of LysR familytranscriptional regulators, many of which regulate degradationpathways for aromatic compounds (93). The HpxR sequenceincludes an amino-terminal helix-turn-helix motif (pfam00126)and a carboxyl-terminal LysR-type substrate-binding domain(pfam03466).

We constructed an hpxR::�-Cm insertion strain to deter-mine the role of this gene in purine utilization. We also con-structed monocopy �(hpxD-lacZ) and �(hpxR-lacZ) operonfusions at a separate locus in order to evaluate expression inhpx� strains. We cultured both hpxR� and hpxR::�-Cm deriv-atives of these strains in defined medium, with nitrate as aneutral nitrogen source, and measured LacZ-specific activity.The results are shown in Table 5. Expression from the �(hpxD-lacZ) hpxR� strain was subject to both Ntr and pathway-spe-cific control, consistent with the results from the hpxE::MudJstrains summarized above. In contrast, expression from the�(hpxD-lacZ) hpxR::�-Cm strain was undetectable, demon-strating that the HpxR protein is essential for activating hpxDE

operon transcription. This is consistent with the Hxn� pheno-type conferred by the hpxR::�-Cm insertion noted above. Ex-pression from the �(hpxR-lacZ) strain was essentially indiffer-ent to added ammonium or hypoxanthine but was elevatedabout threefold in the hpxR::�-Cm insertion strain relative tothe level for the hpxR� strain. This suggests that hpxR tran-scription is subject to weak negative autoregulation.

These results indicate that the HpxR protein is activated bybinding (hypo)xanthine to stimulate hpxDE operon transcrip-tion initiation. The source of Ntr regulation is less clear. NoRpoN-dependent promoter is evident in the hpxD-hpxR inter-genic region in either K. oxytoca M5al or K. pneumoniaeMGH78578. Expression of the �(hpxR-lacZ) fusion was notregulated by Ntr, indicating that Ntr regulation does not act bycontrolling HpxR protein synthesis.

Very similar results were reported by de la Riva et al. (24),who further demonstrated specific binding of HpxR protein tothe intergenic regulatory region. They additionally concludedthat Ntr control is mediated by neither the NtrC nor the Nacproteins (7).

(iii) HpxD, HpxE, and HpxR isofunctional homologs inother species. The HpxD and HpxE sequences are most closelyrelated to sequences from Xanthomonas spp. (63% and 47%identity, respectively), currently annotated as vanillate O-demethylase. Likewise, the HpxR sequence is most closelyrelated to sequences from Xanthomonas spp. (46% identity).The genes encoding these proteins are adjacent to severalother genes involved in purine catabolism (Table 4). There-fore, these genes comprise the hpxR-hpxDE module in Xan-thomonas spp.

Urate to S-(�)-allantoin: the hpxO-hpxPQT module. Urate ishydroxylated to form 5-hydroxyisourate (HIU), which is hydro-lyzed to form 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline(OHCU), which is decarboxylated to form S-(�)-allantoin (92)(Fig. 1). HIU also undergoes spontaneous conversion to race-mic allantoin (45). These reactions comprise the first ring-opening step in purine utilization.

Uricase (urate oxidase) is the previously described enzymecatalyzing the initial oxidation of urate to HIU. Structuralgenes for this enzyme have been characterized for Bacillussubtilis (84) and for a range of eukaryotic species (71). How-ever, no such genes are present in the K. oxytoca hpx locus, andnone were identified in the K. pneumoniae MGH78578 ge-nome sequence.

Two insertion mutants exhibited the Urt� phenotype, failingto grow with hypoxanthine, xanthine, or urate as the sole ni-trogen source but displaying wild-type growth with allantoinand allantoate. Both insertions disrupt the hpxO gene (Table2). Therefore, the hpxO gene encodes a function required forutilization of urate, a conclusion reached independently by dela Riva et al. (24).

The hpxP and hpxQ genes overlap (23 nt), as do the hpxQand hpxT genes (4 nt), and thus likely comprise the hpxPQToperon (Fig. 2B). Apparent RpoN-dependent promoters forboth the hpxO gene and the divergently transcribed hpxPQToperon are present in the 346-nt intergenic region.

(i) HpxO: urate hydroxylase. Sequence comparisons classifythe hpxO gene product (42 kDa) in the �45-kDa aromatic-ringflavoprotein monooxygenase family (pfam01360), members ofwhich share relatively low sequence similarity (40). For exam-

TABLE 5. Effects of hypoxanthine and ammonium on expressionfrom �(hpxD-lacZ) and �(hpxR-lacZ) constructs

Strain Fusionb hpxRc

LacZ sp acta

� Hyx/� NH4

�� Hyx/

� NH4�

� Hyx/� NH4

�

VJSK2966 �(hpxD-lacZ) � 7 42 2,420VJSK2968 �(hpxD-lacZ) � �1 �1 �1VJSK2967 �(hpxR-lacZ) � 30 40 29VJSK2969 �(hpxR-lacZ) � 100 200 180

a Strains were cultured to the mid-exponential phase in MOPS medium, withnitrate as the nitrogen source. Ammonium or hypoxanthine (Hyx) was added asindicated.

b Operon fusion construct integrated at the chromosomal rha locus.c �, hpxR�; �, hpxR113::�-Cm.



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ple, the HpxO sequence shares 28% sequence identity over84% of its length with salicylate hydroxylase (NahG enzyme;GI 4104764; 437 residues) from Pseudomonas stutzeri AN10(9).

Enzymes in this family cleave dioxygen without the use of ametal ion and incorporate one oxygen atom as a hydroxylgroup ortho or para to an existing hydroxyl group while reduc-ing the other oxygen atom to water (33; reviewed in reference40). The HpxO sequence (384 residues) comprises three de-fining cofactor domains: an amino-terminal domain that bindsthe ADP moiety of flavin adenine dinucleotide (FAD) (con-sensus sequence, G-X-G-X2-G-X3-A/G-X6-G; pfam01494), acentral domain that binds the pyrophosphate moiety ofNAD(P)H and interacts indirectly with the pyrophosphate moi-ety of FAD (consensus sequence, D-X5-DG-X5-R; pfam01360),and a carboxyl-terminal domain that binds the ribose moiety ofFAD. Sequences in the last domain vary considerably, save fora conserved G-D sequence (10, 28, 29, 59; reviewed in refer-ence 40).

p-Hydroxybenzoate hydroxylase (PobA enzyme) is the ar-chetype for the �45-kDa aromatic-ring flavoprotein monoox-ygenase family (3). The reaction mechanism initiates withbinding of the substrate (p-hydroxybenzoate). This leads to thereduction of the flavin isoalloxazine ring by NADPH, formingreduced FAD. Dioxygen then binds to the complex and isreduced by two single-electron transfers. The resulting C4a-flavin (hydro)peroxide hydroxylates the substrate (33; reviewedin reference 40). The HpxO enzyme likely uses a similar mech-anism to hydroxylate urate to form HIU (Fig. 1).

(ii) HpxT and HpxQ. Sequence comparisons indicate thatthe HpxT and HpxQ proteins are HIU hydrolase and OHCUdecarboxylase, respectively (15, 44, 77). The HpxT sequence(108 residues; COG2351) is 32% identical to that of HIUhydrolase (PucM protein; 121 residues) from Bacillus subtilis168 (44, 84). The HpxQ sequence (166 residues; COG3195) is27% identical to that of the amino-terminal 165 residueOHCU decarboxylase domains of urate oxidase (uricase) fromBacillus sp. strain TB-90 (GI 21431959) (104) and B. subtilis168 (PucL protein) (84). The Bacillus uricase is synthesized asa precursor of about 500 residues, from which the amino-terminal OHCU decarboxylase is proteolytically cleaved (70).

(iii) HpxP purine permease. The HpxP sequence (460 resi-dues) shares 32% identity over 95% of its length with thehigh-affinity, high-capacity urate-xanthine permease UapA (GI6136091; 615 residues) (36) and 30% identity over 90% of itslength with the general purine permease UapC (GI 1351342;580 residues), both from A. nidulans (26). Additionally, theHpxP sequence is 34% identical to those of the urate per-meases PucJ (449 residues) and PucK (430 residues) from B.subtilis 168. The HpxP sequence meets the criteria for inclusionin the nucleobase ascorbate transporter family (COG2233)(37), including the nucleobase ascorbate transporter signaturemotif (Q/E/P-N-X-G-X4-T-R/K/G), the QH motif in the mid-dle of transmembrane segment 1, and a conserved alanyl res-idue important for function but not specificity (Ala-404 inUapA and Ala-272 in HpxP).

The region spanning residues 378 to 446 in UapA and 336 to404 in UapC (analogous to the region spanning residues 246 to314 in HpxP) determines uptake specificity, with UapA resi-dues Glu-412 and Arg-414 and UapC residues Gln-370 and

Glu-372 possibly forming part of a purine binding site (27).The corresponding residues in the HpxP sequence are moresimilar to those of UapC than to those of UapA: Ser-280HpxP

and Gln-370UapC are both uncharged, with polar side chains,whereas Glu-412UapA is acidic, and Asp-282HpxP and Glu-372UapC are both acidic, whereas Arg-414UapA is basic. Thissuggests that HpxP may be a general purine permease, anappealing hypothesis since this is the only hpx cluster-encodedpermease for transport of guanine, hypoxanthine, xanthine,and urate.

(iv) HpxO isofunctional homologs in other species. TheHpxO sequence is most similar (53% identical) to a proteinsequence annotated as “putative flavoprotein monooxygenaseacting on aromatic compound” from Acinetobacter sp. strainADP1 (locus tag ACIAD3540; GI 49532471; 385 residues).This gene is adjacent to those encoding HIU hydrolase andOHCU decarboxylase (Table 4), supporting its assignment asencoding the isofunctional homolog of HpxO protein. Intrigu-ingly, Xanthomonas genomes contain a gene for a presumptiveflavin-containing monooxygenase (e.g., XCC0279), not homol-ogous to HpxO, adjacent to those encoding HIU hydrolase andOHCU decarboxylase (Table 4). Thus, it appears that at leasttwo different monooxygenases have been recruited into theurate hydroxylase step of the purine utilization pathway.

R-(�)-allantoin to S-(�)-allantoin to allantoate: the hpxC-hpxSAB module. Conversion of allantoin to allantoate is thesecond of the two ring-opening steps in purine utilization (Fig.1). Five insertion mutants exhibited the Aln� phenotype, fail-ing to grow with allantoin or upstream purine catabolites as thesole nitrogen source but displaying wild-type growth with al-lantoate. The insertions disrupt the hpxA and hpxB genes (Ta-ble 2). Therefore, the hpxAB operon encodes functions in-volved in allantoin utilization. However, the hpxA::Tn10d(Tc)insertion was complemented by subclones carrying the hpxBbut not the hpxA gene, indicating that the hpxA gene is notessential for growth on allantoin. Presumably, the hpxA::Tn10d(Tc) insertion is polar on hpxB gene expression.

Allantoinase is the previously described enzyme catalyzingthe conversion of allantoin to allantoate (91). Structural genesfor this enzyme have been characterized for Bacillus subtilis(84) and E. coli (21) and for a range of eukaryotic species (12).However, no such genes are present in the K. oxytoca hpx locus,and none were identified in the K. pneumoniae MGH78578genome sequence.

Most characterized allantoinases are specific for the S iso-mer. However, the nonenzymatic conversion of HIU to allan-toin creates a racemic mixture of R- and S-allantoin (45), andallantoin is present as the racemic mixture in decaying organicmatter (94). Allantoin racemase has been characterized forbacteria (94) and for Candida utilis (72). To our knowledge,the structural gene for an allantoin racemease has not beenidentified.

An apparent RpoN-dependent promoter for the hpxC gene(but not for the hpxSAB operon) is present in the 52-nt hpxCand hpxSAB intergenic region. The hpxS and hpxA genes over-lap (1 nt; TAATG), and the hpxA and hpxB genes are sepa-rated by only 16 nt, so these likely comprise the hpxSABoperon (Fig. 2B).

(i) HpxB: allantoinase. Sequence comparisons suggestedthat the HpxB enzyme catalyzes conversion of allantoin to



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allantoate. The HpxB sequence (310 residues; COG0726)shares 26% identity over 74% of its length with an imidase (291residues; GI 14040045) from Ralstonia eutropha (97). The imi-dase catalyzes the hydrolysis of hydantoin and related com-pounds. Since allantoin is a 5-substituted hydantoin (ureidohy-dantoin), this suggests that the HpxB enzyme acts similarly tothe imidase by hydrolyzing a cyclic C-N bond in the hydantoinring to form allantoate (Fig. 1).

This conjecture was confirmed by Ramazzina et al., whorecently identified the HpxB homolog from Pseudomonas fluo-rescens DSM 50090 as an (S)-allantoin-specific allantoinase(76). They denote this enzyme PuuE.

(ii) HpxA: allantoin racemase. Sequence comparisons indi-cate that the HpxA protein is allantoin racemase. The HpxAsequence (247 residues; COG 4126; pfam01177) shares 45%identity over 96% of its length with 5-substituted hydantoinracemase (HyuE enzyme; 229 residues; GI 266318) fromPseudomonas sp. strain NS671 (98). The HyuE enzyme cata-lyzes racemization of a variety of 5-substituted hydantoins (99).Since HpxB allantoinase is specific for the S enantiomer (76),allantoin racemase could be important for efficient allantoinutilization in natural environments. However, allantoin race-mase likely is not required for colony formation on plates withabundant (racemic) allantoin, explaining why the hpxB� genewas able to complement the polar hpxA insertion (see above).

(iii) HpxC: allantoin permease. The HpxC sequence (495residues; COG1953) shares 22% identity over 85% of its lengthwith allantoin permease from Saccharomyces cerevisiae (Dal4pprotein; 635 residues) and 33% identity over 92% of its lengthwith allantoin permease from Bacillus subtilis W168 (PucI pro-tein; 490 residues).

(iv) HpxS. Sequence comparisons indicate that the HpxSprotein is a member of the GntR superfamily of transcriptionalregulators (COG1802). For example, the HpxS sequence (238residues) shares 28% identity over 58% of its length with theMatR repressor (222 residues; GI 6840861), which controlsexpression of the matABC operon responsible for the uptakeand conversion of malonate to acetyl-coenzyme A in Rhizo-bium leguminosarum (50).

The GntR family is made up of bacterial transcriptionalregulators with similar amino-terminal helix-turn-helix DNA-binding domains and divergent carboxyl-terminal effector-binding and oligomerization domains (93). The GntR super-family has been subdivided into four subfamilies on the basis ofsecondary structure in the C-terminal domain (78). On thebasis of these considerations, the HpxS protein can be classi-fied into the VanR subgroup of the FadR subfamily(COG2186). Many proteins in the FadR subfamily regulateoperons involved in amino acid metabolism. Allantoin resem-bles a dipeptide, and the bond cleaved to yield allantoateresembles a peptide bond (Fig. 1).

(v) Transcriptional regulation in the hpxC-hpxSAB module.The hpxB105::MudJ mutant expressed LacZ activity, indicatingthat this insertion forms a �(hpxB-lacZ) operon fusion. Wecultured this strain in defined medium, with glutamine as aneutral nitrogen source. LacZ-specific activity was approxi-mately 1,600 Miller units after growth with added xanthine andabout 10 units after growth with added xanthine plus ammo-nium. This indicates that hpxSAB operon transcription is sub-ject to global nitrogen regulation (Ntr). LacZ-specific activity

was about 1,050 units after growth with added allantoin, about775 units with urate, and about 400 to 500 units with allantoateor with no addition, suggesting possible pathway-specific in-duction by allantoin or an upstream intermediate. Presumably,the HpxS protein controls pathway-specific transcriptional reg-ulation of the hpxSAB operon.

(vi) HpxA and HpxB isofunctional homologs in other spe-cies. The HpxB allantoinase sequence is most similar (about70% identity) to protein sequences typically annotated as“polysaccharide deacetylase” (76), and the HpxA sequence ismost similar (up to 62% identity) to protein sequences typi-cally annotated as “hydantoin racemase.” Representative ex-amples are shown in Table 4. In the case of Burkholderia spp.and related species, the hpxA and hpxB homologs are presentas an hpxSCAB module within a larger cluster of purine utili-zation genes, including those for HIU hydrolase and OHCUdecarboxylase. In contrast, the Acinetobacter sp. hpxB homologis clustered with the hpxO, hpxT, and hpxQ homologs, encodingenzymes of urate catabolism, whereas the hpxA and hpxChomologs are adjacent to each other at a separate location.This indicates that the HpxA allantoin racemase may functionphysiologically to scavenge exogenous, racemic allantoin. Con-sistent with this notion, the Xanthomonas spp. have hpxB ho-mologs but none for hpxA or hpxC.

Van der Drift et al. identified allantoin racemase in P. fluo-rescens and Pseudomonas putida (among others) but not inPseudomonas aeruginosa or P. stutzeri (94). Database searchesreveal that hpxA homologs are present in genomes of P. fluo-rescens, P. putida, and Pseudomonas syringae but not in P.aeruginosa or P. stutzeri (locus tag Pput_1557 in P. putida F1).This reinforces the conclusion that hpxA encodes allantoinracemase. In these genomes, the hpxA and hpxC genes areadjacent to each other but not to other purine utilizationgenes, as described above for the Acinetobacter sp. (The P.syringae clusters include an hpxS homolog as well.) In contrast,the hpxB gene, encoding allantoinase, is present in the ge-nomes of all five Pseudomonas species examined (locus tagPput_1582 in P. putida F1), clustered with several other genesinvolved in purine utilization.

The HpxA sequence shares 25% identity with the Dcg1pproteins of S. cerevisiae (244 residues; locus tag YIR030C) andCandida albicans (232 residues; locus tag CaO19.244). The S.cerevisiae DCG1 gene was identified as a nitrogen-regulatedgene within the DAL cluster, encoding allantoinase, allantoinpermease, and allantoicase (106), but the function of theDcg1p protein has not been determined. Allantoin racemaseactivity has been characterized for Candida utilis (72). Theanalysis presented here indicates that Dcg1p may function asallantoin racemase.

Conclusions. Microbial genome sequencing projects reveallarge numbers of genes with unknown functions. Here, Kleb-siella purine utilization (Fig. 1) provides numerous examples ofunpredicted enzymes catalyzing an established pathway (96).

Several enzymes were evidently recruited from pathways forcatabolism of aromatic compounds. Aromatic compounds andthe pathways for their catabolism are diverse and widespread,and so, in retrospect, it is unsurprising that such enzymes areinvolved in oxidizing purines (Fig. 1).

Oxidation of (hypo)xanthine to form urate is catalyzed by atwo-component Rieske nonheme iron aromatic-ring-hydroxy-



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lating oxygenase system (HpxE-HpxD enzyme), and oxidationof urate to form 5-hydroxyisourate is catalyzed by an �45-kDaaromatic-ring flavoprotein monooxygenase (HpxO enzyme).These enzymes were identified independently by de la Riva etal. (24). Genome sequence comparisons suggest that a distinctflavin-containing monooxygenase catalyzes the latter step inXanthomonas spp. (Table 4).

Yet another enzyme is involved in xanthine utilization by A.nidulans, for which purine utilization has long been studied(83). Early work established that mutants lacking xanthinedehydrogenase (hxA) or molybdenum cofactor (cnx) exhibitleaky growth with xanthine (but not hypoxanthine) as the solenitrogen source (22). This indicated the presence of a molyb-denum cofactor-independent xanthine alternative pathway, de-pendent on the xanA gene (85). Recently, it was establishedthat XanA protein, a member of the taurine dioxygenase groupof enzymes active on aromatic compounds, is an �-ketogluta-rate-dependent dioxygenase that hydroxylates xanthine toform urate (20, 65).

Finally, our analysis reveals that Klebsiella spp. employHpxA allantoin racemase, related to hydantoin racemases (72,94), as well as the HpxB (PuuE) allantoinase, related to poly-saccharide deacetylases and described recently by Ramazzinaand coworkers (76). Our work in progress indicates furtherthat other previously undescribed enzymes are involved in al-lantoate catabolism (Fig. 2).

ACKNOWLEDGMENTS

We thank Ryan Feathers for his substantial contributions to deter-mining the hpx DNA sequence, Becky Parales and Sydney Kustu forhelpful discussion and critique of the manuscript, and Juan Aguilar forproviding details on the hpx genetic nomenclature. Cornell Universityundergraduate students Jeff Hanna, Ann-Marie Roy, and Wendy Pyoparticipated in the early stages of this work. V.S. is grateful to RobertRabson, Gregory Dilworth, James Tavares, and Sharlene Weatherwax(Energy Biosciences Program; Chemical Sciences, Geosciences andBiosciences Division) for funding basic research in Klebsiella nitrogenassimilation.

S.D.P. was the recipient of a University of California President’sUndergraduate Fellowship. This study was supported by U.S. Depart-ment of Energy grant DE-FG03-99ER20326 from the Office of BasicEnergy Sciences.

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