INTRODUCTION

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It has long been known that Escherichia coli and similar organisms cannot utilize exogenously supplied oxidized forms of folic acid or its derivatives. A blockade of dihydrofolate synthesis by inhibitors of dihydropteroate synthase (DHPS) (such as sulfathiazole or other sulfonamide derivatives) or of dihydrofolate reductase (DHFR) (aminopterin or trimethoprim) can be circumvented only by supplementation with the end products of folate metabolism, i.e., purines, thymidine, glycine, methionine, and pantothenic acid (14, 37). Even then, growth is slow, partly because of the failure to formylate methionyl-tRNA^fMet for the initiation of protein synthesis (2, 14).

The failure of E. coli to utilize oxidized folate derivatives directly may be due to a lack of transport of these compounds or the failure to reduce them to dihydro or tetrahydro derivatives once they are transported. As illustrated in Fig. 1, folate compounds are synthesized in reduced (dihydro) form, beginning with dihydropterin precursors (39). Following the conjugation of dihydropterin pyrophosphate with p-aminobenzoate to form dihydropteroate, glutamate is added to produce dihydrofolate. Dihydrofolate is then reduced by DHFR to yield tetrahydrofolate, the active one-carbon carrier for cellular metabolism. Bacterial species that require folate derivatives for growth, such as Lactobacillus casei, have elaborate transport systems specific for folate (18). Inhibitory folate analogs, such as aminopterin or methotrexate, are effective against E. coli at relatively high concentrations (24), suggesting that at least passive transport of these compounds may occur. However, once entry into the cell is gained, E. coli DHFR is ineffective in reducing folate to dihydrofolate (25), a fact which prevents the direct entry of oxidized folate derivatives into pools of reduced folates.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Abbreviated scheme for tetrahydrofolate biosynthesis from GTP and chorismate. Compounds to the left of each reaction arrow are additional substrates of the reaction, and compounds to the right are additional products of the reaction. H2, dihydro; H4, tetrahydro; pABA, p-aminobenzoate; PP, pyrophosphate.

Another mechanism by which oxidized folates might be utilized is via catabolic or salvage pathways that could reutilize portions of a folate derivative following degradation. In this regard, little is known about bacterial folate salvage, except for organisms that utilize preformed folates. Additionally, the utilization of the pterin product of degradation, namely, pterin or pteroate, might be limited by the same transport or reduction problem cited above.

The p-aminobenzoate moiety of dihydrofolate is synthesized de novo from chorismate and is easily transported into and out of E. coli cells (12, 16, 17, 23). p-Aminobenzoate auxotrophs can be supplemented by the addition of exogenous p-aminobenzoate, and wild-type cells excrete sufficient p-aminobenzoate to supplement nearby p-aminobenzoate auxotrophs.

We have examined whether p-aminobenzoate auxotrophs can utilize p-aminobenzoate derived from exogenously supplied folate derivatives; we found mutant strains of E. coli that could utilize p-aminobenzoyl-glutamate, a degradation product of oxidized folate compounds. Single-base-pair mutations that allowed growth on p-aminobenzoyl-glutamate were found in an intergenic region between divergently transcribed genes, one a lysR homolog (here designated abgR, for p-aminobenzoyl-glutamate) and the other the first gene in an apparent operon containing three genes, two homologous to aminoacyl hydrolases (abgA and abgB) and a third gene homologous to permeases (abgT). A 4.2-kb tandem duplication containing abgB and abgT fused to fnr also allowed growth on p-aminobenzoyl-glutamate. Finally, plasmids containing wild-type abgT alone could confer the ability to grow on p-aminobenzoyl-glutamate to wild-type E. coli strains.

	MATERIALS AND METHODS

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Microbiological methods. The bacterial strains used in this study are listed in Table 1. Minimal medium, LB medium, and NCE medium were prepared as described previously (7). Antibiotic and amino acid final concentrations were also as recommended in reference 7. For routine purposes, p-aminobenzoate and p-aminobenzoyl-glutamate were added at 0.7 and 1.8 µM, respectively.

Genetic mapping and transduction. Mutations that allowed growth on folate compounds were mapped by conjugation with the Hfr strains and protocols described by Singer et al. (35). Bacteriophage P1 transductions were performed as described by Miller (21).

Molecular methods. E. coli chromosomal DNA was prepared by established methods (30, 38). Small-scale plasmid samples were prepared either by the rapid alkaline lysis technique of Birnboim and Doly (3) or by the rapid boiling technique (30). Bacteriophage  DNA was prepared as described previously (30).

Enzyme assays. Crude extracts were prepared as previously described (23). Hydrolysis of p-aminobenzoyl-glutamate to p-aminobenzoate was determined by incubating 1-ml volumes containing 50 mM Tris-HCl (pH 8.5), 10 mM MgSO₄, 10 mM -mercaptoethanol, and 50 µM p-aminobenzoyl-glutamate with 0.1 to 1.5 mg of protein for 60 min at 37°C. Reactions were terminated with the addition of 0.1 ml of 1 N HCl, and p-aminobenzoate was extracted and quantitated spectrophotofluorometrically as previously described (23). One unit of activity was defined as the amount of protein required to hydrolyze 1 nmol of p-aminobenzoyl-glutamate in 1 h at 37°C.

Bioautography. Bioautography was carried out with E. coli BN101 and BN1001 as test strains. Pyrex baking dishes (21 by 27 cm) dishes were prepared by pouring a layer (200 ml) of solid minimal medium supplemented with tryptophan and cysteine, followed by a layer (100 ml) of the same medium seeded with 5 × 10⁸ cells of the test strain per ml. The top layer was also supplemented with 0.15 mM isopropyl-thio--D-galactoside and 10 µM 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) to aid in the visualization of growth zones. Samples were dissolved and filter sterilized prior to application to Whatman 3MM chromatography paper (15 by 20 cm). The chromatogram was developed by ascending chromatography in 0.1 M potassium phosphate (pH 7.0) until the solvent had reached 2 cm from the top. The chromatogram was air dried, and material was allowed to transfer to solid medium for 2 h. The chromatogram was lifted, and the dish was covered with foil and incubated at 37°C. Samples and quantities used were as follows: p-aminobenzoate and p-aminobenzoyl-glutamate, 7 nmol; folic acid, 110 nmol; folinic acid, 110 nmol; and pteroic acid, 80 nmol.

Construction of interruptions in abgA, abgB, and abgT. To test the roles of abgA, abgB, and abgT in p-aminobenzoyl-glutamate utilization, each open reading frame was interrupted with a kanamycin resistance cassette and then transferred to the chromosomes of E. coli BN1125 (abg-1) and BN1126 (abg⁺). pMJH123 was digested with PstI and ligated with the PstI DNA fragment containing the kanamycin resistance determinant derived from pMB2190. The product (abgT::kan) was designated pMJH132. pMJH124 was partially digested with PstI and similarly ligated with the PstI DNA fragment containing the kanamycin resistance determinant. Plasmid products containing the kanamycin resistance cassette inserted into abgA and abgB were isolated. The EcoRI fragments were recloned into the EcoRI site of pBSIISK+ to yield pJMG111 (abgB::kan) and pJMG112 (abgA::kan).

	RESULTS

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Selection and characterization of abg mutants. To obtain E. coli strains that could grow on folate or folate-related compounds, a pabA derivative of E. coli (BN101) was mutagenized with diethyl sulfate and selected for growth on medium supplemented with folic acid or folinic acid. Because the growth requirement for these compounds on solid medium was high (>10 µM) and the requirement for p-aminobenzoate supplementation was low (<30 nM), we sought to test the strains for growth on potentially contaminating material present in the commercially supplied supplements.

Growth response of strains to p-aminobenzoyl-glutamate. E. coli BN101 and BN1103 grew poorly on p-aminobenzoyl-glutamate concentrations below 10 µM, and bioautography showed that growth on higher concentrations was due to contaminating p-aminobenzoate in commercial preparations. However, the abg mutant strains could grow on much lower levels of p-aminobenzoyl-glutamate (Table 3). The minimum concentration required for full growth was determined by inspection of colony size on solid minimal medium containing different concentrations of p-aminobenzoyl-glutamate (ranging from 0.4 nM to 200 µM). While growth became limited for the parental strain at below 20 µM, the growth of E. coli BN1001, BN1002, BN1003, or BN1005 was not limited until the p-aminobenzoyl-glutamate concentration fell below 1 µM. The growth of E. coli BN1004, on the other hand, was not limited until the p-aminobenzoyl-glutamate concentration fell below 0.1 µM. Two classes of mutations were evident from this analysis, one that increased the sensitivity to p-aminobenzoyl-glutamate approximately 20-fold and one that increased the sensitivity approximately 200-fold over the concentration required by the parent. Growth limitation in response to p-aminobenzoate for all strains was not evident until the concentration fell below 0.02 µM.

Mutant strains contain unaltered DHPS. Although p-aminobenzoyl-glutamate is not used as the normal substrate for dihydrofolate synthesis in vivo (28), it has been reported that DHPS from E. coli as well as other microorganisms can use p-aminobenzoyl-glutamate as a substrate in vitro in place of p-aminobenzoate and thereby bypass dihydropteroate to produce dihydrofolate directly (29) (Fig. 1). To test if the p-aminobenzoyl-glutamate utilization mutants contained altered DHPS that might facilitate the in vivo utilization of p-aminobenzoyl-glutamate, DHPS from mutants BN1001, BN1004, and BN1005 was assayed to determine if it could use either p-aminobenzoyl-glutamate or p-aminobenzoate with an efficiency different from that of parental E. coli BN101 DHPS. As shown in Fig. 2, there was no apparent difference in the K_m for p-aminobenzoate, nor was there any apparent difference in the ability of p-aminobenzoyl-glutamate to compete with p-aminobenzoate for incorporation into dihydropteroate. Since no difference in DHPS activities was evident, we concluded that if p-aminobenzoyl-glutamate were utilized directly, it was by a mechanism not related to an altered DHPS.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Characterization of DHPS from E. coli BN101 and abg derivatives. (a) Comparison of Kms of DHPS from E. coli BN101 (), BN1001 (), and BN1004 (). (b) Comparison of the ability of p-aminobenzoyl-glutamate (p-ABG) to compete with 14C-labeled p-aminobenzoate in the DHPS reaction in E. coli BN101 (), BN1001 (), BN1004 (), and BN1005 (). p-ABA, p-aminobenzoate; v, velocity.

Mapping and cloning of abgT. The abg mutation of one of the strains (E. coli BN1004) was mapped by Hfr conjugation to between 25 and 35 min and by P1 transduction to approximately 30 min on the E. coli chromosome. The abg allele was 50% cotransducible with the zda-3061::Tn10 marker of E. coli CAG12081, which is located at approximately 29.5 min (35). The cotransduction frequencies for the abg alleles from E. coli BN1001, BN1002, BN1003, and BN1005 were similar. In addition, abg Tc^r recipients of the mutant strains were used to backcross the abg allele to either E. coli BN101 or E. coli BN1103, confirming that no additional mutation distant from this locus was necessary for the growth phenotype, nor was the phenotype dependent on another factor in the E. coli BN101 genetic background.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Map of the 7,266-bp region of the E. coli chromosome from bp 1,396,431 to bp 1,403,697, shown in the inverse orientation. Restriction sites used for subcloning purposes are illustrated, as are the extents of known reading frames. DNA fragments used in this study to localize abgT and to construct interruptions in abg genes are illustrated, and the vectors in which they are cloned are indicated. The vector names (leftmost column) and plasmid names are shown to the left of the map. Triangles denote the positions of kanamycin resistance cassette insertions. The ability of each plasmid to confer p-aminobenzoyl-glutamate utilization is indicated at the right.

Nucleotide sequence of a gene allowing p-aminobenzoyl-glutamate utilization. To sequence the 2.3-kb KpnI-HindIII insert of pMJH128, a sequence strategy involving both ordered deletions (15) and synthetic oligonucleotides was used. The entire sequence was completed on both strands, and all sites and primer sequences were overlapped. The nucleotide sequence of a 2,266-bp DNA fragment containing one complete and two partial open reading frames was determined (Fig. 3). Database searches with BlastP (1) identified the 3'-terminal open reading frame as ogt, encoding a DNA repair enzyme, O⁶-alkyl-guanine-DNA alkyltransferase (26). The upstream open reading frame was present on the complete sequence of the E. coli genome and had been given the designation ydaH, indicative of an open reading frame of unknown function (4).

Identification of mutations leading to p-aminobenzoyl-glutamate utilization. To determine if the mutations resulting in p-aminobenzoyl-glutamate utilization were in the regulatory region of the putative abg operon, a 1,105-bp DNA fragment spanning the abgR-abgA intercistronic region was amplified from the parental E. coli BN101 DNA and mutant abg chromosomal DNA preparations by PCR. The nucleotide sequences of the PCR products were determined directly and showed that the sequences from the parental strain and BN1004 were identical to the published wild-type sequence (Fig. 4), while the products derived from BN1001, BN1002, BN1003, and BN1005 each contained one nucleotide difference. Extending the sequence determination of BN1001 to include abgA and abgB did not reveal any additional sequence differences. In the four strains analyzed, two nucleotide differences were found. E. coli BN1001, BN1002, and BN1003 each contained a C-to-A transversion at nucleotide position 984 from the SalI site, while E. coli BN1005 had a C-to-T transition at position 1008. Since both of these alterations lay outside potential open reading frames, we searched for possible transcriptional regulatory sequences that might be affected by the mutational changes. In Fig. 4 we have noted two regions of dyad symmetry containing the half-site sequence -GATAA-, within which is contained the T-N₁₁-A feature typical of LysR family protein-binding sites (11), and a match to the catabolite activator protein consensus sequence (8). The most obvious possibility from inspection of the nucleotide sequence is that the mutations may lie in the 10 and 35 regions of the putative abgR promoter. Although no direct evidence concerning the effects of the mutations on the expression of abgR has been found, we hypothesize that the mutations alter the expression of abgT, consistent with the observations made above that indicate that only abgT is required for p-aminobenzoyl-glutamate utilization.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Nucleotide and amino acid sequences of the abgR-abgA intercistronic region. The positions of mutations resulting in the p-aminobenzoyl-glutamate utilization phenotype are shown in large bold letters above the sequence. Putative LysR-type protein-binding sequences are shown as dotted lines above the sequence. Putative 10 and 35 sequences of the abgR and abgA promoters are overlined or underlined. The CAP consensus sequence is also indicated above the sequence (lowercase letters indicate nucleotides that are not as strongly conserved as the capitalized nucleotides). Numbering is from the SalI site prior to abgR.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Structures of the wild-type (a) and duplicated (b) regions of E. coli BN1004. The HindIII site within ogt is illustrated, as are the primer hybridization sites used to delimit the extent of duplication. The 12-bp direct repeat sequences within abgA and fnr are illustrated. It can be seen from panel b that only primer pair pri1-pri2 could yield a PCR product from DNA containing a duplication. The source of the 4.2-kb HindIII fragment containing abgT is also evident.

Hydrolysis of p-aminobenzoyl-glutamate. The protein products of the two open reading frames upstream of abgT (here designated abgA and abgB) showed sequence similarity to one another and sequence similarity to a family of aminoacyl aminohydrolases. Outside the E. coli sequences, the greatest similarity was between AbgA and Enterobacter agglomerans indole-3-acetyl-aspartic acid hydrolase (6), but of greater interest was the similarity of each to Pseudomonas sp. strain RS-16 carboxypeptidase G₂ (22), an enzyme capable of cleaving folate to pteroate and glutamate and p-aminobenzoyl-glutamate to p-aminobenzoate and glutamate (33). Based on these similarities, we hypothesized that p-aminobenzoyl-glutamate utilization conferred by transport of the supplement into the cell by AbgT might be accompanied by hydrolysis to p-aminobenzoate and glutamate by AbgA and AbgB, either alone or in combination.

Effects of interruptions in abgA, abgB, and abgT. The phenotypic effects of interruptions in abgA, abgB, and abgT were tested by constructing strains in which each open reading frame was interrupted with a kanamycin resistance cassette. The cassettes were inserted at the PstI sites present in each of the genes. Recombination of the interrupted genes into an abg⁺ strain had no effect on the growth properties of the strains on supplemented minimal medium. However, the introduction of each interrupted gene into an abg-1 host resulted in a reduction in the ability to utilize p-aminobenzoyl-glutamate effectively (Table 5). As anticipated, the strains containing the abgT::kan interruption (BN1143 and BN1153), with the kanamycin resistance determinant transcribed in the same direction as abg, failed to grow on medium supplemented with p-aminobenzoyl-glutamate. Similarly, abg-1 strains containing abgA::kan (BN1142 and BN1152) and abgB::kan (BN1141 and BN1151) also failed to utilize p-aminobenzoyl-glutamate. These data might be explained by polarity of the kanamycin resistance cassette for abgT expression, since the direction of kan transcription is opposite that of abgT transcription in the abgA::kan and abgB::kan interruptions.

	DISCUSSION

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E. coli pabA strains able to utilize p-aminobenzoyl-glutamate were obtained following mutagenesis and selection on folate compounds. The p-aminobenzoyl-glutamate utilization phenotype depended on a single gene, abgT, a homolog of transport genes that apparently was responsible for the transport of p-aminobenzoyl-glutamate into the cell. abg⁺ strains that contained interrupted abgT did not yield p-aminobenzoyl-glutamate utilization derivatives following mutagenesis, and abg-1 strains lost their ability to use p-aminobenzoyl-glutamate when abgT was disrupted. Plasmid-borne, wild-type abgT could confer p-aminobenzoyl-glutamate utilization on abg⁺ hosts, and this ability was lost when abgT was disrupted.

The nucleotide sequence of the abgT allele from an abg-1 strain was identical to that of the wild-type allele, and the cloned wild-type gene was capable of conferring p-aminobenzoyl-glutamate utilization on pabA strains. These observations suggested that wild-type abgT was normally cryptic or not expressed under conditions of growth on minimal medium. Altering the expression of abgT from its wild-type context appears to have been the mechanism for obtaining the growth phenotype. Data from the E. coli genome sequence suggested that abgT lay in an operon with two other genes upstream and possibly also ogt downstream. Since no promoter-like sequences were observed near abgT, it seemed likely that the abgT regulatory signals lay several kilobases upstream, probably near the region of the divergently transcribed LysR-type regulatory gene.

Three different types of mutations were detected in the collection of five abg strains analyzed. Two different single-base-pair mutations in the abgR-abgA intergenic region resulted in an approximately 20-fold-increased ability to utilize p-aminobenzoyl-glutamate. The growth phenotype was accompanied by a small but reproducible increase in the expression of p-aminobenzoyl-glutamate hydrolysis activity detectable in crude extracts of one of the mutant strains (BN1005). The location of the mutations in or near the abgR promoter suggested that alteration of the expression of abgR may have altered the expression of abgT, which in turn resulted in the p-aminobenzoyl-glutamate utilization phenotype. BN1004 contained a 4.2-kb duplication of DNA which placed a second copy of abgB and abgT under the control of the fnr promoter. The anomalous expression of the abg genes resulted in a 200-fold increase in the ability to use p-aminobenzoyl-glutamate and a 5-fold increase in the amount of p-aminobenzoyl-glutamate hydrolase activity in crude extracts. The level of sensitivity to p-aminobenzoyl-glutamate was equivalent to that seen in strains containing a high-copy-number plasmid carrying abgT. Since abgB was duplicated and expressed from the fnr promoter, while abgA was not, we concluded that abgB encoded measurable p-aminobenzoyl-glutamate hydrolase activity but that abgB was not essential for the p-aminobenzoyl-glutamate utilization phenotype.

The utilization of p-aminobenzoyl-glutamate may have occurred by either of two mechanisms. First, DHPS may have used p-aminobenzoyl-glutamate as a substrate directly, forming dihydrofolate as the product and bypassing the normal dihydropteroate intermediate in the dihydrofolate biosynthetic pathway. While the direct in vivo incorporation of p-aminobenzoyl-glutamate into dihydrofolate has not been observed so far for E. coli, DHPS has been shown to be able to use p-aminobenzoyl-glutamate in place of p-aminobenzoate in vitro (29), albeit with a higher K_m for p-aminobenzoyl-glutamate than for p-aminobenzoate. However, it is possible that increased transport of p-aminobenzoyl-glutamate into the cell by increased expression of abgT may have raised the intracellular level sufficiently to make the reaction proceed well enough to support growth.

A second possibility was that the transported p-aminobenzoyl-glutamate was first hydrolyzed to p-aminobenzoate and glutamate by intracellular aminoacyl aminohydrolases and then p-aminobenzoate was incorporated into dihydropteroate in the normal biosynthetic pathway. In support of this idea, we observed that abg strains growing on p-aminobenzoyl-glutamate had the ability to cross-feed the p-aminobenzoate requirement of the parental strain, suggesting hydrolysis of p-aminobenzoyl-glutamate and excretion of p-aminobenzoate. In addition, we demonstrated that crude extracts derived from abg strains had an elevated capacity to hydrolyze p-aminobenzoyl-glutamate to p-aminobenzoate in vitro. However, disruption of abgA and abgB did not reverse the p-aminobenzoyl-glutamate utilization phenotype in strains expressing abgT, indicating that if hydrolysis were required, other activities in the cell were capable of p-aminobenzoyl-glutamate hydrolysis.

The selection for growth on p-aminobenzoyl-glutamate appeared to have resulted in the altered regulation of an operon that resulted in an increased capacity to transport and hydrolyze p-aminobenzoyl-glutamate. It is likely that the abg operon is expressed under specific growth conditions, perhaps in response to an effector of abgR. In this regard, we note that sequences in the abgR-abgA intercistronic region include segments similar to binding sites for LysR-type regulators (11) and for CAP (8). Alternatively, it is possible that the p-aminobenzoyl-glutamate utilization phenotype arose from recruitment of a transport protein from an operon whose normal function may not be involved in p-aminobenzoyl-glutamate or folate metabolism. For example, the wild-type function of the operon may be a peptide permease and hydrolysis system regulated by the presence of peptides in the medium. If such a system had a broad substrate specificity, then it might be able to transport and hydrolyze p-aminobenzoyl-glutamate under conditions of altered regulation. As has been shown with other operons in E. coli and other bacteria, altered-regulation phenomena are a common first step in the evolution of new metabolic pathways in bacterial systems (13).