INTRODUCTION

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Escherichia coli converts exogenous purines (bases or nucleosides) to nucleotides via salvage pathways (Fig. 1) (22). Nucleosides are converted to nucleobases; for example, exogenous guanosine and inosine are degraded to guanine and hypoxanthine, respectively. Adenosine can be converted to two different nucleobases: adenine or hypoxanthine (via inosine). The purine nucleobases are then converted to the corresponding purine mononucleotides by adenine phosphoribosyltransferase (specific for adenine), hypoxanthine phosphoribosyltransferase (specific for hypoxanthine), and guanine phosphoribosyltransferase (which can salvage guanine, hypoxanthine, and xanthine).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Purine salvage pathways in E. coli. The solid lines show known pathways, while the dashed lines indicate reactions demonstrated in this paper. Abbreviations: PRPP, 5'-phospho--D-ribosyl-1-pyrophosphate; FGAR, 5'-phosphoribosyl-N-formylglycinamide; GPRT, guanine phosphoribosyltransferase; HPRT, hypoxanthine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; and sAMP, adenylosuccinate.

Some microorganisms, e.g., Klebsiella pneumoniae, can utilize purines as carbon or nitrogen sources (19). Strains of Escherichia are not thought to degrade most purines, although there is considerable strain-to-strain variation (see references cited in reference 20). Several observations led us to reexamine the ability of E. coli to utilize purines as nitrogen sources. First, we identified five potential ⁵⁴-dependent promoters in a 23-gene cluster at min 65 of the E. coli chromosome. Such promoters often control genes whose products are involved in nitrogen assimilation, such as glutamine synthetase and enzymes of arginine catabolism (13, 17). BLAST analysis suggested that these genes might code for proteins that transport or catabolize purines. (The results of the BLAST searches are described in more detail below.) Therefore, we considered the possibility that nitrogen limitation induces enzymes of purine catabolism. Second, the failure to utilize a particular compound as a sole nitrogen source does not imply that this compound is not catabolized. For example, E. coli cannot degrade pyrimidines as the sole nitrogen source. Nonetheless, in the presence of certain amino acids, ¹⁴CO₂ is produced from [¹⁴C]uracil or [¹⁴C]thymidine (2). A similar situation involves arginine catabolism. Aspartate is not absolutely required for arginine utilization, but aspartate greatly stimulates arginine utilization (17). Because the metabolism of a particular compound may depend on the presence of other compounds, we considered the possibility that this might be the case for purines.

In this paper, we present evidence for purine catabolism. Such catabolism requires aspartate and is not complete. We disrupted two different genes coding for proteins that might catalyze the first reaction in purine catabolism. One mutant eliminated purine catabolism. Despite the limited catabolism of purines, both mutants had an altered phenotype: they were both somewhat sensitive to adenine. We propose that xanthine dehydrogenase participates in purine salvage but not in aerobic purine catabolism.

	MATERIALS AND METHODS

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Strains and their construction. The strains and plasmids used in this work are listed in Table 1.

(i) Strain HX1 (xdhA). BLAST analyses indicate that genes b2866, b2867 (ygeT), and b2868 (the b number is a GenBank identifier) code for proteins with homology to different domains of Drosophila melanogaster xanthine dehydrogenase (XDH). A strain with a deletion of b2866 has a phenotype consistent with a defect in XDH (described below). Therefore, we designated this gene xhdA. A 4.9-kb Acc65I-BglII fragment from Kohara phage 465 containing b2866 (GenBank accession number AE000370) was ligated between the Acc65I and BamHI sites of the cloning vector pWM529. The 2.0-kb AgeI-PvuII region of the insert was replaced with the 1.6-kb EcoRI-HindIII fragment from plasmid pBlue-kan-FRT (Flp recombination target), which contains FRT sites flanking a Kan^r cassette. The FRT sites permit subsequent excision of the region between them. The plasmid was linearized and transformed into E. coli strain K4633 (recD), and recombinants were selected on Luria-Bertani (LB) agar plates containing 50 µg of kanamycin per ml. The mutant allele was transduced from K4633 to W3110 by P1 transduction (10). Finally, the Kan^r insert was excised from the FRT sites using plasmid pCP20, which is a temperature-sensitive plasmid that encodes a site-specific recombinase, as described previously (4). The net result should be an in-frame deletion of codons 70 to 728 of a potential 752-residue protein. The resulting strain was named HX1.

(ii) Strain HX2 (b2881). BLAST analysis indicates that the product of gene b2881 has homology to four of the five domains of D. melanogaster XDH. DNA containing this gene was obtained by PCR amplification of DNA from 300 bp upstream to 700 bp downstream, which resulted in an approximately 4-kb fragment. The PCR primers contained BamHI and EcoRI sites, which allowed insertion of the fragment into the BamHI and EcoRI sites of pWM529. A 2-kb region of the insert was removed between EcoRV and BsiWI sites and replaced with a 1.6-kb SmaI-Acc65I fragment from pBlue-kan-FRT, that contained the Kan^r-FRT cassette. The disrupted allele was introduced into W3110, and the Kan^r-FRT cassette was removed in the same way as for HX1. This resulted in a deletion of codons 161 to 824 of a potential 956-residue protein.

(iii) Strain HX3 (xdhA b2881). The b2881::Kan^r-FRT allele (described for the construction of HX2) was transduced into HX1. The Kan^r-FRT cassette was removed in the same way as for HX1. PCR was used to verify all of the deletions.

Cell growth. The salts for the minimal medium have been described previously (15). In addition, minimal medium contained 0.4% glucose, 0.02% thiamine, and 0.1% of each nitrogen source unless otherwise noted. To measure the growth rates, cells were grown overnight in the medium to be tested, diluted into 10 ml of fresh minimal medium so that the initial turbidity was 10 Klett units (no. 42 filter), and incubated at 30°C at 220 rpm. Turbidity was measured at 1- to 3-h intervals. Generation times are presented as the means from triplicate cultures with the standard deviations.

Measurement of ¹⁴CO₂ from [¹⁴C]adenine degradation. [¹⁴C]adenine (0.5 µCi) (Amersham Pharmacia Biotech) was added to a 10-ml culture. The stock solution contained 287 mCi/mmol, which implies that the final concentration of adenine was 1.7 µM. ¹⁴CO₂ was collected in a 25-ml Falcon tube which contained 0.8 ml of 2M NaOH and which was punctured at its midpoint. The top of the tube was sealed, and the whole tube was inserted into a rubber stopper, which sealed a 250-ml flask that contained the culture medium. After a 24- to 48-h incubation, the NaOH solution was mixed in a 4:1 ratio with Scintisafe Plus fluid (Fisher Scientific), and ¹⁴C was measured in a scintillation counter (Beckman LS6500). Cultures without cells were used to determine the background, which varied from 140 to 160 dpm per culture. This value was subtracted from experimental values, which were at least 15-fold over this background value. The experimental values were normalized to a culture A₆₀₀ of 1.0. The final A₆₀₀ of all of the cultures was slightly above 2.0. All determinations were done in duplicate, with the ranges of values indicated in the figures or the text.

Transcript analysis. Total RNA from E. coli was extracted with hot phenol as described previously (1). The quality of the RNA was monitored by running 2 µl of the RNA preparation in a 0.7% agarose gel. The presence of two sharp bands corresponding to the 16S and 23S rRNAs suggested minimal RNA degradation.

	RESULTS

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Identification of possible genes of purine catabolism: computer analysis of ⁵⁴-dependent promoters. Genes induced by nitrogen limitation frequently possess ⁵⁴-dependent promoters. ⁵⁴ binds to promoters with a distinctive 17-bp sequence: TGGCACG(A/G)NNNNTTGC(A/T). We scanned the E. coli genome for potential ⁵⁴-dependent promoters using the SeqScan program (B. T. Nixon, Department of Biochemistry and Molecular Biology, Pennsylvania State University) (http://www.bmb.psu.edu/seqscan). The sequences are identified using a scoring matrix and are assigned a score ranging from 0 to 100. A score of 100 indicates a perfect one-to-one match, while a score of 0 indicates no matches. The program identifies sites with scores over 60. The complete results of this analysis will be the subject of a separate communication. Some highlights of this analysis are presented to indicate why we became interested in purine catabolism. The scores for the 12 known ⁵⁴-dependent promoters in E. coli range from 70.3 to 95.4, with a mean of 82.7. None of these promoters is within a gene. There are 217 possible ⁵⁴-dependent promoters outside of genes (or open reading frames) and only 48 (including known promoters) with a score greater than 70.

View this table: [in this window] [in a new window]   TABLE 2.   Possible 54-dependent promoters at min 65 of the E. coli chromosome

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Genes at min 65 of the E. coli genome. The diagram shows the arrangement of genes that BLAST analysis suggests are involved in purine catabolism. The locations of the possible 54-dependent promoters are designated A, B, C, C', and D.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Possible purine catabolic pathway in E. coli.

Effect of purines on nitrogen-limited E. coli. The identification of possible ⁵⁴-dependent genes that might code for enzymes of a purine catabolic pathway led us to reinvestigate purine catabolism in E. coli during nitrogen-limited growth. E. coli could use adenine or adenosine, but not hypoxanthine, xanthosine, inosine, or allantoin, as the sole nitrogen source (data not shown). This does not necessarily imply that these compounds are not catabolized. It is possible that there is a low level of purine catabolism that is insufficent to support growth. Therefore, we examined whether purines stimulated growth with 0.1% aspartate as the sole nitrogen source, which supports a generation time of 7.23 ± 0.97 h. The addition of 0.07% hypoxanthine, 0.05% guanosine, 0.1% inosine, and 0.1% xanthosine reduced the generation times (in hours) to 4.80 ± 0.12, 4.15 ± 0.25, 4.33 ± 0.01, and 4.59 ± 0.17, respectively. In contrast, 0.1% allantoin did not stimulate growth (data not shown). This observation does not prove that purines are catabolized. It is possible that purine salvage reduces aspartate consumption for de novo purine synthesis, which results in elevated intracellular aspartate and more rapid aspartate catabolism. The fact that all of the purines stimulated growth to the same extent is consistent with this possibility.

Extent of purine catabolism in E. coli. To determine whether the stimulatory effect of purines results from catabolism or simply purine salvage, we tested for the presence of products of purine catabolism. We first examined whether growth with [U-¹⁴C]adenine produced ¹⁴CO₂, which would be generated from the first two reactions of purine catabolism but not from a salvage pathway. We also examined purine catabolism by K. pneumoniae, which can use purines and allantoin as the sole carbon or nitrogen source (21). For cells grown with 0.1% aspartate as the nitrogen source and 1.7 µM [U-¹⁴C]adenine, duplicate 10-ml cultures of E. coli generated 1,310 dpm of ¹⁴CO₂, whereas similar cultures of K. pneumoniae produced 22,300 dpm. (The range of values was 3% or less of the mean.) These results establish that xanthine is catabolized to allantoin in E. coli. The 17-fold-less-efficient purine degradation in E. coli compared to that in K. pneumoniae may account for the former's inability to utilize most purines as nitrogen sources.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Final cell densities with NH4Cl or adenosine as the nitrogen source. Wild-type K. pneumoniae and E. coli strains were grown in minimal medium with either 0.54 mM NH4Cl or 0.54 mM adenosine at 37°C for 48 h. The experiment was done three times. The error bars indicate the standard deviations.

Putative genes for XDH in E. coli. Despite the absence of extensive purine catabolism, we wanted to know if the purine-dependent growth stimulation was caused by purine catabolism or purine salvage. To distinguish between these possibilities, we focused a genetic analysis on genes that potentially specify XDH, because the XDH reaction initiates purine catabolism and is required for CO₂ formation from purines. Homology searches have a good chance to identify such enzymes because of several unique structural and sequence determinants. The xanthine oxidase family is the largest and most diverse group of molybdenum cofactor (Mo-co)-containing enzymes, which usually transfer an oxygen atom to or from a substrate in a two-electron transfer reaction (7). This family includes xanthine oxidase/dehydrogenase, aldehyde oxidase, 4-hydroxybenzoyl coenzyme A reductase, quinoline oxidoreductase, and CO dehydrogenases.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Xanthine oxidase family genes in E. coli. The reference genes for comparison are the xanthine dehydrogenase (rosy locus) gene of D. melanogaster (A) and the aldehyde oxidoreductase of D. gigas (B). The numbers refer to hundreds of amino acid residues. The boxes immediately below the first line of each section indicate the extents of the domains. The Fe2-S2 domains bind the iron-sulfur clusters, the FAD domain binds FAD, and Mo1 and Mo2 are two separate Mo-co-binding domains. The solid boxes with gene designations above them indicate that there were 36 to 38% identity and 50 to 57% similarity to the reference genes. The open boxes indicate 23 to 27% identity and 37 to 46% similarity. The dashed box for yagS signifies that it is not homologus to the D. melanogaster XDH gene. Instead, it is 24% identical and 37% similar to xdhB.

Phenotypes of HX1 (xdhA), HX2 (b2881), and HX3 (xdhA b2881). (i) CO₂ generation. We examined the effects of disruptions of genes for two potential xanthine-oxidizing enzymes: xdhA, the first gene of a possible operon xdhABC operon, and b2881. We did not disrupt the yag genes. We were unable to directly verify the loss of xanthine dehydrogenase or xanthine oxidase activity, since we could not assay either activity from W3110 or strains with xdhABC or b2881 expressed from a high-copy-number plasmid. Instead, we examined ¹⁴CO₂ production from [U-¹⁴C]adenine by strains HX1, HX2, and HX3 grown in minimal medium supplemented with aspartate and histidine. We added histidine in the hope that it would optimize purine catabolism and result in a more sensitive assay to detect purine catabolism. Histidine inhibits the formation of guanine nucleotides from adenine via ATP to 5-aminoimidazole-4-carboxamide ribonucleotide, which involves the first part of the histidine synthetic pathway. 5-Aminoimidazole-4-carboxamide ribonucleotide is subsequently converted to IMP and finally to GMP (8). If this pathway is inhibited, the only mechanism of GMP formation from adenine is via hypoxanthine. Increased hypoxanthine formation may stimulate purine catabolism. This complex reasoning is apparently correct, since histidine increased ¹⁴CO₂ production from adenine by W3110 from 1,300 dpm per culture to 4,000 dpm per culture (the results are normalized to a constant cell density). Therefore, changes in ¹⁴CO₂ production caused by the deletion of xdhA or b2881 can be more easily observed.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   14CO2 production from [14C]adenine. E. coli strains W3110, HX1 (xdhA), HX2 (b2881), and HX3 (xdhA b2881) were grown in minimal medium containing 0.1% aspartate, 0.03% histidine, and 1.7 µM [U-14C]adenine. Methods and Materials describes all other procedures, including data processing. Each value is the mean of two determinations, and the range of values (error bars) is shown.

(ii) Adenine toxicity. During these experiments, we noticed that the mutants grew slower than the wild-type strain when adenine was present. This was not due to a contaminant in the radioactive adenine, since unlabeled adenine gave a similar effect. Adenine at high concentrations (>0.1%) inhibits the growth of wild-type E. coli (reference 8 and references cited therein). Certain mutants defective in purine salvage are much more sensitive to adenine. An hpt gpt double mutant, which is deficient in both hypoxanthine and guanine phosphoribosyltransferases, fails to grow with 0.002% adenine (8). There are two components to this toxicity in the double mutant. First, adenine (actually adenine nucleotides, which are synthesized from adenine by adenine phosphoribosyltransferase) inhibits de novo purine synthesis. Second, there is inefficient (or mutationally blocked) formation of guanine nucleotides from adenine, since the hpt gpt mutant cannot phosphoribosylate either hypoxanthine or xanthine (8). An important piece of evidence that supports the hypothesis that adenine toxicity results from failure to synthesize GMP is the observation that guanosine reverses the inhibition (8).

View larger version (22K): [in this window] [in a new window]   FIG. 7.   Adenine sensitivity of HX3 (xdhA b2881) and reversal by guanosine. W3110 (wild type) and HX3 were grown exponentially in cultures containing 0.1% NH4Cl as the nitrogen source. These cells were inoculated into fresh medium containing 0.1% NH4Cl with no addition, with 0.03% adenine, or with 0.03% adenine plus 0.06% guanosine. Growth was monitored at 30°C. The experiment was done twice, and the same pattern was observed.

(iii) Growth rates. Surprisingly, HX1 (xdhA) grew faster than wild-type E. coli in nitrogen-limited minimal medium with 0.1% aspartate as the sole nitrogen source: the doubling times were 5.70 ± 0.25 and 7.36 ± 0.31 h, respectively. However, the mutant grew normally in nitrogen-rich ammonia-containing minimal medium (1.92 ± 0.2 and 1.75 ± 0.16 h, respectively).

Transcript analysis of xdhA. We initially studied the 23-gene cluster at min 65 because it contained five possible ⁵⁴-dependent promoters. Such promoters are often associated with expression during nitrogen limitation. However, we detected ¹⁴CO₂ from [U-¹⁴C]adenine from E. coli grown not only in nitrogen-limited minimal medium but also in nitrogen-rich (ammonia-containing) minimal medium and amino acid-rich LB broth (data not shown). These results show that XdhA (the primary enzyme responsible for CO₂ generation) is not solely under nitrogen regulation. To verify this regulation and to locate the transcriptional start site, we mapped the 5' end of the xdhA transcript by primer extension analysis. Transcript mapping was done for xdhA, which appears to be the first gene in its operon. (We did not examine transcription from b2881 because it is not clear where the operon starts.) The results of primer extension analysis of xdhA are shown in Fig. 8. As a positive control, primer extension was also done for the glnA gene, which is actively transcribed under nitrogen-limiting conditions. The expected results for the glnA transcript were observed: there was significantly less transcript in nitrogen-rich ammonia-containing medium. The DNAs made from primer extension of xdhA transcripts were 126 and 134 bases, which correspond to start sites 80 and 88 bases, respectively, upstream from the presumed initiation codon (Fig. 9). (Adjacent sequencing reactions were used to determine the exact sizes of the transcripts, and these results are not shown.) Both transcripts were present for cells grown in nitrogen-limited medium, without or with hypoxanthine (Fig. 8, lanes 4 and 5, respectively), and for cells grown in nitrogen-rich medium (lane 6). The larger transcript, but not the smaller transcript, was also observed from an rpoN (encoding ⁵⁴) mutant grown in LB broth (lane 7, Fig. 8). The smaller transcript might be ⁵⁴ dependent. Its first nucleotide is 14 bases from the potential ⁵⁴ recognition site (promoter A [Table 2]), which is not unusual. However, if this is actually the 5' end of the mRNA, and not a degradation product, then transcription begins in a pyrimidine-rich region, which seems unlikely. The upstream transcript begins in a purine-rich region, which is reasonable. Furthermore, the putative, 10 region (TAAATCTT) is AT rich, which is consistent with an authentic binding site for RNA polymerase. In the absence of results from transcription with purified components or mutational alteration of the putative binding site for ⁵⁴, the results must be considered preliminary. Nonetheless, we suspect that the smaller transcript is a degradation product of the larger and that the potential ⁵⁴-dependent promoter is not functional. In any case, there is clearly transcription independent of ⁵⁴, which is consistent with the conditions in which CO₂ is generated from adenine.

View larger version (57K): [in this window] [in a new window]   FIG. 8.   xdhA start site of transcription. Primer extensions were done using a primer annealing to glnA (lanes 1 to 3) or xdhA (lanes 4 to 7). RNA was extracted from either W3110 grown in various minimal media (lanes 1 to 6) or an isogenic rpoN mutant grown in LB medium (lane 7). The minimal media contained the following nitrogen sources: 0.1% aspartate (lanes 1 and 4), 0.1% aspartate and 0.08% hypoxanthine (lanes 2 and 5), or 0.1% NH4Cl (lanes 3 and 6). Lanes M, size markers.

View larger version (15K): [in this window] [in a new window]   FIG. 9.   Nucleotide sequence upstream of the xdhA structural gene. The first three lines show 240 bases upstream from the xdhA structural gene. The fourth line shows the sequence at the amino-terminal coding region of xdhA. The t1 transcript is the most upstream of two transcripts, while t2 is the downstream transcript. The boxed region shows the possible 54-binding region.

	DISCUSSION

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Purine catabolism in E. coli. We have presented evidence that E. coli catabolizes purines, although not sufficiently to support aerobic growth as sources of nitrogen (with the exception of adenine and adenosine). The best evidence for such catabolism is the generation of ¹⁴CO₂ from [¹⁴C]adenine, which implies functional XDH and uricase activities, and the production of allantoin. It has recently been shown that allantoin can be used as the sole nitrogen source in anaerobically grown E. coli (5). Cusa et al. (5) showed that E. coli contains allantoinase, allantoate amidohydrolase, and ureidoglycolate dehydrogenase, which collectively catalyze the conversion of allantoin to oxaluric acid. Such a metabolism implies that E. coli possesses most of the enzymes of purine catabolism (Fig. 3). The inability to use purines aerobically might be due to inadequate transport, weak promoters, or the absence of appropriate regulation.

XDH activity and purine salvage in E. coli. XDH catalyzes two reactions: the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid. The second reaction is the first committed step in purine catabolism. It diverts purines away from the salvage pathways. ¹⁴CO₂ production from [U-¹⁴C]adenine results from the activity of uricase on uric acid and implies XDH activity, which is required for uric acid formation. Deletion of xdhA eliminated ¹⁴CO₂ production, which implies that XdhA is required for this activity. All known XDHs have domains that bind Fe-S clusters and FAD, and separate genes can specify these domains. The two genes just downstream of xdhA code for proteins with homology to the FAD-binding domain and the iron-binding domain of D. melanogaster XDH, respectively. Therefore, we suggest that b2867 (ygeT) and b2868 should be renamed xdhB and xdhC, respectively. We propose that xdhA, xdhB, and xdhC code for components of a heterotrimeric XDH.

XDH and nitrogen limitation. We initiated this study because a computer analysis of ⁵⁴-dependent genes suggested that nitrogen limitation might induce a purine catabolic pathway. However, our results do not provide evidence that purine catabolism produces nitrogen for cell growth. Nonetheless, there is a link between nitrogen limitation and purine metabolism. An xdhA mutant grew faster than wild-type E. coli with aspartate as the sole nitrogen source. A possible explanation for such an effect is that blocking the conversion of hypoxanthine to xanthine results in a higher intracellular level of aspartate, which can now be more efficiently utilized. However, there is no simple explanation that could account for the differences in the aspartate concentration, especially since the extracellular concentration is high (aspartate is the nitrogen source in the medium), which should imply that the intracellular concentration is also high. An alternate explanation is that accumulation of an intermediate in purine salvage stimulates aspartate catabolism. At this time, it is not clear why HX1 grows faster with aspartate as the nitrogen source.