INTRODUCTION

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Both the role of anthranilate as an intermediary metabolite in tryptophan degradation and the existence of bacteria able to use anthranilate as a sole source of carbon and energy have been known for many years (22). Nevertheless, the first step in the aerobic degradation of anthranilate by bacteria remains poorly characterized. Early attempts to purify the multicomponent enzyme responsible for the initial oxidation step proved to be unsuccessful. These attempts did, however, establish that anthranilate was oxidized when two distinct Pseudomonas protein fractions and Fe²⁺ were present (26). Catechol was identified as the product of anthranilate dihydroxylation, and the corresponding enzyme was shown to be a di- rather than a mono-oxygenase (27, 46). No further information about this anthranilate 1,2-dioxygenase (deaminating, decarboxylating; EC 1.14.12.1) has been reported in nearly 30 years.

Recently there has been renewed interest in microbial dioxygenases (4, 6). This interest has stemmed in part from their importance in degrading a vast array of aromatic compounds in the environment, in part from a fundamental concern about their biochemistry, and in part from their potential use in bioremediation. Compounds similar in structure to anthranilate, such as benzoate (Fig. 1) or 2-chlorobenzoate, are substrates of multicomponent ring-hydroxylating dioxygenase systems (13, 18, 34). Several classes of these dioxygenases have been defined based on the number of components, the types of iron-sulfur clusters involved, and the types of flavin cofactors (4, 6). The chromosomal benABC genes of the soil bacterium Acinetobacter sp. strain ADP1 encode a class IB dioxygenase that converts benzoate to a nonaromatic diol. Further catabolism of this diol yields catechol, which is also the product of anthranilate oxidation. The pathways for anthranilate and benzoate degradation by strain ADP1, therefore, are convergent (Fig. 1). By using a strategy based on previous studies of benzoate and catechol degradation by ADP1 (14, 47), it was possible to select mutants unable to convert anthranilate to catechol.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Degradation of anthranilate and benzoate via the -ketoadipate pathway. Relevant compounds, genes, and enzymes are indicated.

In this report, the characterization of one such ADP1-derived anthranilate dioxygenase mutant is described. The natural transformability of the Acinetobacter strains used in these studies facilitated the identification of the wild-type antABC genes, which restored growth of the mutant with anthranilate as the sole carbon source. These ant genes, shown to encode anthranilate dioxygenase, were homologous in sequence to the benzoate dioxygenase-encoding benABC genes. Comparisons between the regulation and functions of the antABC genes and those of their better-studied ben counterparts were made. Whereas the formation of catechol from anthranilate required only the dioxygenase-catalyzed step, catechol formation from benzoate requires a second enzymatic step that is catalyzed by the benD-encoded dehydrogenase (Fig. 1) (33). Studies of the similar, but distinct, ant- and ben-encoded multicomponent dioxygenases may reveal key features of enzyme substrate specificity and catalytic efficiency.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. Acinetobacter strains are listed in Table 1. Acinetobacter strains were derived from BD413, also designated ADP1 (24). Taxonomic confusion has led to the Acinetobacter calcoaceticus species designation for ADP1 being discontinued until further characterization is complete (11a). Escherichia coli DH5 (Gibco BRL), S17-1 (43), and BL21(DE3) (44) were used as plasmid hosts. Bacteria were cultured in Luria-Bertani broth and minimal medium at 37°C as previously described (39, 42). Carbon sources were added to minimal medium at the following final concentrations: 10 mM succinate, 3 mM benzoate, 3 mM 4-hydroxybenzoate, 3 mM cis,cis-muconate, 2.5 mM anthranilate, or 2 mM catechol. Antibiotics were added as needed at the following final concentrations: tetracycline, 6 µg/ml; kanamycin, 20 µg/ml; streptomycin, 25 µg/ml; spectinomycin, 25 µg/ml; and ampicillin, 150 µg/ml for Acinetobacter strains and 80 µg/ml for E. coli.

DNA manipulation and plasmid construction. Standard methods were used for all chromosomal and plasmid DNA purifications, restriction enzyme digestions, electrophoreses, ligations, and E. coli transformations (39). Plasmids are described in Table 1 and Fig. 2. To isolate a chromosomal ADP1 DNA fragment carrying the ant genes, a recombinant plasmid library was constructed. Chromosomal ADP1 DNA was digested with HindIII. Following electrophoretic separation, fragments of various size ranges were purified from a 0.7% agarose gel with the GeneClean purification kit (Bio101). A fraction of DNA in the 4- to 6-kbp size range was ligated to the cloning vector pZero2.1 (Invitrogen) and used to transform E. coli DH5. This vector enables transformants with recombinant plasmids to be selected on medium with kanamycin. Plasmid pBAC103 (Fig. 2) was one of the recombinant plasmids generated. Subclones of pBAC103 and pBAC163, whose isolation is described below, were constructed by standard methods (39).

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Restriction map of the 6.5-kbp chromosomal antABC region. The locations of genes, and their transcriptional directions, are shown relative to some of the restriction endonuclease recognition sites. Lines indicate the DNA regions contained on recombinant pBAC plasmids. Plasmids shown in boldface and marked by an asterisk were constructed with the pUC19 vector, and all others were constructed with the pZero2.1 vector. The insertion sites of either omega or lacZ cassettes correspond to those in strains and plasmids described in Table 1.

Acinetobacter transformation, strain construction, and chromosomal mapping. Naturally competent Acinetobacter strains were transformed by linear DNA, plasmid DNA, or crude DNA lysates as previously described (16, 23, 32). Some plasmids were introduced into Acinetobacter recipients by conjugation from E. coli S17-1 (32, 43). Plasmids carrying modified regions of ADP1 DNA on replicons that are not stably maintained in Acinetobacter were used to transform recipients, yielding new strains in which homologous recombination had replaced the wild-type chromosomal allele with the modified version. Strains ACN80 and ACN86 were constructed by this method with the antC-disrupted plasmids pBAC112 and pBAC143, respectively. The latter plasmid was used to generate ACN87 with ACN129 as the recipient strain. ACN103 was constructed by similar methods with chromosomal disruptions of both antC and benM (8). Strains constructed with ADP1 as the recipient include ACN88 (by using pBAC141), ACN106 (by using pBAC156), ACN107 (by using pBAC162), ACN130 (by using pBAC176), ACN169 (by using pBAC194), and ACN204 (by using pBAC141). Strain ACN194 was generated by using ACN107 as the recipient and pBAC244 as the donor DNA. This plasmid was also the source of the disrupted ORF3 allele in ACN191. Southern hybridization analyses confirmed that the mutated plasmid alleles replaced the corresponding wild-type regions of the chromosome without integration of the entire plasmid.

Isolation of chromosomal DNA in the region upstream of antA. In ACN88, the S chromosomal insertion lies in ORF2 and introduces an EcoRI recognition site not present in the wild-type strain (Table 1 and Fig. 2). Chromosomal DNA from ACN88 was digested with EcoRI and ligated to EcoRI-digested DNA of the cloning vector pUC19. Following transformation of an E. coli host with the ligated DNA, a single transformant with resistance to streptomycin and spectinomycin, characteristic of the ORF2::S allele, was obtained. This transformant harbored plasmid pBAC163 (Fig. 2), and subsequent Southern hybridization and DNA sequence analyses confirmed that pBAC163 carries DNA from the chromosomal region upstream of antA.

Southern hybridization and DNA sequence analyses. Southern hybridization analyses were performed as previously described (16). DNA probes were labeled with digoxigenin by random priming, and probes were detected with antidigoxigenin-alkaline phosphatase conjugates and chemiluminescent substrates according to the Genius System instructions (Boehringer Mannheim Corp.).

Determination of the sequence of the mutation in the antA5024 allele. Two different approaches were used to identify the mutation(s) causing the temperature-sensitive defect in AntA. In the first approach, the mutant allele was amplified from purified chromosomal DNA by using oligonucleotides 5 and 10 (Table 2) as primers in a PCR with Pfu DNA polymerase according to the instructions of the polymerase supplier (Stratagene). Dimethyl sulfoxide was added to comprise 10% of the total reaction volume. The PCR-amplified DNA product was gel purified by using the QIAquick Gel Extraction kit (Qiagen) and digested with ClaI and NsiI. This DNA was ligated to the cloning vector pUC19 (49), which had been digested with AccI and PstI to form pBAC147. The sequence of Acinetobacter DNA of pBAC147 was determined with primers (from Promega) that recognized the pUC19 vector as described above.

AntABC and -galactosidase assays. To measure anthranilate dioxygenase activity, cell extracts were prepared by sonication as previously described for other enzymes of the -ketoadipate pathway (42). Anthranilate dioxygenase was assayed spectrophotometrically by monitoring the decrease in anthranilate concentration as indicated by A₃₁₀ (26). Protein concentrations were determined by the method of Bradford (5), using bovine serum albumin as the standard.

Metabolite monitoring by HPLC. For metabolite monitoring, E. coli cultures (10 ml) were grown overnight on M9 medium with glucose as a carbon source (39). Acinetobacter cultures were similarly grown on minimal medium with either 10 mM succinate or 4 mM anthranilate as the carbon source. Since ADP1(pBAC189) was unable to use anthranilate as the sole carbon source, it was grown with both 10 mM succinate and 2 mM anthranilate. When appropriate, ampicillin was added for plasmid maintenance. Cultures were diluted with 15 ml of the minimal medium used for overnight growth, and in some cases, 250 µM isopropyl--D-thiogalactopyranoside (IPTG) was added to induce gene expression from the vector's lac promoter. Monitoring was initiated after the addition of approximately 1 mM anthranilate.

Nucleotide sequence accession number. The sequence of the 6,529-bp antABC region was deposited in the GenBank database (accession no. AF071556).

	RESULTS

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Isolation of a mutant, ACN26, unable to degrade anthranilate. The endogenous accumulation of cis,cis-muconate (Fig. 1) is toxic (14). Therefore, the presence of a compound that can be converted to this metabolite prevents growth of strains in which the subsequent metabolism of cis,cis-muconate is blocked, even when an additional carbon source is available (14). Selection for growth under these conditions can yield mutants that are unable to form cis,cis-muconate. For example, the presence of benzoate in the growth media of catB deletion strains has selected mutants that do not convert benzoate to cis,cis-muconate (47). It seemed likely, therefore, that a similar strategy could be used to isolate spontaneous mutants unable to catalyze the initial hydroxylation of anthranilate. Strain ADP205 (Table 1), which cannot degrade cis,cis-muconate because of a catMBC deletion, was grown on solid medium with 3 mM 4-hydroxybenzoate as the carbon source in the presence of 1 mM anthranilate, the potential source of the toxic intermediate. Mutations preventing the conversion of anthranilate to catechol, or those preventing the conversion of catechol to cis,cis-muconate, should allow growth. Whereas mutations of the first type were of interest, mutations of the second type predominated as indicated by catechol production (data not shown).

Different mutations affect cis,cis-muconate and anthranilate metabolism. ACN26 can degrade endogenous cis,cis-muconate since it grows on benzoate. Mutations in the mucK gene, encoding a transport protein, are known to prevent growth on exogenous cis,cis-muconate (47). Therefore, the effect of introducing a wild-type mucK gene into ACN26 was tested. ACN26 was transformed with DNA from plasmid pADPW4, carrying mucK, and a resultant strain was designated ACN84. ACN84 grew with cis,cis-muconate as the sole carbon source, indicating that ACN26 carries a defective mucK gene. ACN84 remained unable to grow on anthranilate as the sole carbon source at 39°C. It is not clear why a mutation in mucK was isolated in the selection for a strain unable to convert anthranilate to cis,cis-muconate. Since similar selections led to the isolation of strains with mutations in both mucK and the structural genes encoding biodegradative enzymes (47), it may be that cis,cis-muconate exported from adjacent cells under these isolation conditions contributes to selective pressure for loss of the MucK uptake activity.

Isolation of the ant genes. A fraction of 4- to 6-kbp HindIII-digested ADP1 DNA was able to transform ACN26 to grow on anthranilate. Following ligation to a vector, this DNA was introduced into E. coli as described in Materials and Methods. The recombinant plasmid-bearing colonies were patched individually to solid anthranilate medium onto which ACN26 cells had been spread. E. coli, which does not use anthranilate as a carbon source, can donate plasmid DNA in the natural transformation of ACN26. Plasmid DNA able to repair the ACN26 mutation should allow growth. The recombinant plasmids were not used in trans to complement ACN26 directly, because the cloning vector is not stably maintained in Acinetobacter, thereby making it difficult to retrieve DNA that restored growth by homologous recombination. Of several hundred E. coli colonies screened, one carried a plasmid, pBAC103, that restored growth of ACN26 on anthranilate. This plasmid, with 4.9 kbp of ADP1 DNA, and subclones derived from it were used for DNA sequence determination (Fig. 2) (see Materials and Methods). Sequence analyses revealed several ORFs (Fig. 2), three of which were designated antABC based on their homology to a number of dioxygenase-encoding genes, described below.

Homology between AntABC and aromatic ring-hydroxylating dioxygenases. By database searches with the deduced amino acid sequences, the 54-kDa AntA and 19-kDa AntB were found to be homologs of the large and small subunits, respectively, of the terminal oxygenases of class IB dioxygenases. The 39-kDa AntC resembled the reductase components associated with these oxygenases. In pairwise alignments of the Ant proteins with their counterparts, the percentage of identical aligned residues ranged from 33 to 46% (Tables 3 to 5). Enzymes most similar to AntABC included the highly specific benzoate dioxygenase and a toluate dioxygenase, with broad specificity, that dihydroxylates both benzoate and substituted benzoates. The former is encoded by the ADP1 chromosomal benABC genes, and the latter is encoded by the Pseudomonas putida xylXYZ genes of the TOL plasmid pWW0 (19, 34). Also homologous to antABC were the cbdABC genes, isolated from a conjugative plasmid of Burkholderia cepacia (18). The CbdABC proteins catalyze the dihydroxylation of 2-halobenzoates, yielding catechol. Additional homologs of antA and antB included the B. cepacia tftA and tftB genes, which encode the large and small subunits of a 2,4,5-trichlorophenoxyacetic acid terminal oxygenase (10). The proposed roles of AntABC are analogous to those of BenABC (Fig. 3).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Proposed functions of the ant gene-encoded proteins. By comparison with the functions of the ben gene-encoded benzoate dioxygenase, indicated in the boxes, the antAB genes encode the terminal dioxygenase and benC encodes the reductase of a class IB dihydroxylating multicomponent dioxygenase.

Identification of the mutation that prevents growth on anthranilate. Plasmid pBAC116 (Fig. 2) transformed ACN26 and ACN84 to grow with anthranilate at 39°C, thereby localizing their mutations to a 298-bp region between the ClaI and NsiI restriction recognition sites of the antA gene. The corresponding region of ACN84 was isolated by gap repair methods (17), and DNA sequence determination revealed a point mutation in the 43rd codon of the antA structural gene that changed a T to an A (position 2307 in the sequence under GenBank accession no. AF071556). This same mutation was identified independently by PCR amplification of the chromosomal region with Pfu DNA polymerase (see Materials and Methods). As illustrated in Fig. 4, this mutation should cause a conserved methionine residue to be replaced by a lysine in the N-terminal region of the mutant protein.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Alignment of dioxygenase protein sequences in the N-terminal regions of the  subunits. A substitution of K for M in AntA causes a temperature-sensitive mutant enzyme that is dysfunctional at high temperatures. Numbers indicate the amino acid position of the adjacent residue. Residues identical to those of AntA are shown in white on a black background.

The antABC genes enable catechol to be formed from anthranilate. In E. coli, expression of the antABC genes, on plasmid pBAC190 (Table 1), allowed 1 mM anthranilate to be converted to catechol in approximately 4 h (Fig. 5A). A diol intermediate was not detected by the HPLC monitoring techniques. An isogenic strain carrying only the cloning vector did not metabolize anthranilate under identical conditions (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Compounds in culture supernatant fractions. (A) Anthranilate was converted to catechol by E. coli BL21(DE3)(pBAC190) with the ADP1 antABC genes in trans. (B) During anthranilate consumption (top) by Acinetobacter strain ADP1 or by ADP1(pBAC189), with the antABC genes in trans, catechol and/or cis,cis-muconate accumulated (bottom). Prior to anthranilate addition at time zero, the growth substrate for ADP1 was anthranilate, whereas ADP1(pBAC189) was grown with both anthranilate and succinate. Concentrations were determined by HPLC methods.

Anthranilate-mediated induction. Anthranilate-grown ADP1 consumed anthranilate immediately upon its addition to the culture, whereas with succinate-grown cells there was a delay of approximately 2 h before consumption was detected (data not shown). It appeared that the anthranilate degradation pathway, like similar pathways, was inducible. Consistent with this, anthranilate dioxygenase activity in cell extracts of anthranilate-grown wild-type cells was approximately 0.03 µmol per min per mg of protein. In succinate-grown cells, the activity was undetectable (less than 0.005 µmol per min per mg of protein).

Three ORFs near antABC are not required for anthranilate degradation. Since genes encoding transcriptional activators are often adjacent to the targets of their regulation, the DNA upstream of antA was isolated on pBAC163 as described in Materials and Methods. The small, 417-bp ORF2 (Fig. 2) immediately upstream of antA was disrupted on the chromosome of the wild-type strain and that of a strain with the antA::lacZ fusion to generate strains ACN204 and ACN120, respectively. Strain ACN204 was able to use anthranilate as the sole carbon source. Moreover, the ORF2 disruption of ACN120 did not alter the ability of anthranilate to induce expression of the lacZ fusion. The presence of anthranilate in the growth medium of ACN120 increased LacZ levels to 15,494 ± 1,781 Miller units, compared to 64 ± 37 Miller units in the absence of added inducers. ORF2, therefore, did not appear to regulate transcription of the antABC genes. In addition, disruption of ORF2 did not have a cis-acting effect on transcriptional regulation.

Localization of the ant genes on the ADP1 chromosome. To map the ant genes by previously described methods (16), a NotI recognition sequence was introduced into the 3' end of antC by using pBAC112 to replace the wild-type allele and generate ACN80 (Table 1). Following the NotI digestion of genomic DNA and electrophoretic separation by transverse alternating-field electrophoresis, the sizes of the six wild-type and seven ACN80 DNA fragments were compared. In ACN80, the wild-type 1,090-kbp fragment was replaced by two fragments, not present in the wild type, whose combined sizes were equal to 1,090 kbp. A labeled probe made from DNA upstream of antC between the ClaI sites in antA and antB (Fig. 2) hybridized to the wild-type 1,090-kbp NotI fragment and to an 810-kbp NotI fragment of ACN80. In addition, this probe hybridized to a 490-kbp NotI fragment of a trpD mutant, ACN30, in which there is a NotI recognition site at map position 3290 (hybridization data not shown). Collectively, these data located the antC gene at map position 3500 and indicated its transcriptional direction to be clockwise, as depicted in Fig. 6.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Genome map of ADP1. The positions of genes are shown relative to the six wild-type NotI recognition sites in the chromosome. The chromosomal S insertion of ACN80 introduced a NotI site at map position 3500, localizing the position of antC as shown. The asterisk marks the position of the hybridization probe indicating the direction of transcription as shown. Clusters 1 and 2 mark the relative positions of large supraoperonic regions containing numerous genes for aromatic compound degradation; only one gene of each cluster is indicated.

Substitution of BenC for AntC. Despite the S insertion in antC, strain ACN80 was able to grow with anthranilate as the sole carbon source. A second antC disruption strain, ACN86 (Fig. 2; Table 1), was constructed and was also able to grow at the expense of anthranilate. When antC was disrupted in certain strains carrying mutations in the ben gene region, however, the ability to grow with anthranilate as the sole carbon source was lost. Specifically, combinations of the antC::S5086 allele with either the benC::K5106 allele (ACN115), the (benB-benC5129) allele (ACN87), or the benM::K5008 allele (ACN103) eliminated growth at the expense of anthranilate. The benM gene encodes a transcriptional activator of benC expression (8). It appears, therefore, that in the absence of antC, benC was required for growth on anthranilate, most likely because the BenC reductase can substitute for AntC in anthranilate oxidation.

	DISCUSSION

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The isolation and analysis of the antABC genes establish that anthranilate dioxygenase is evolutionarily related to class IB dihydroxylating enzyme systems. Dihydroxylating enzymes have been classified according to the number and types of proteins in the multicomponent complex (4, 6). Class I enzymes are comprised of a terminal oxygenase and a flavin-containing NADH-dependent reductase. In class IB enzymes the flavin cofactor is flavin adenine dinucleotide, and the reductase contains a plant-type [2Fe-2S] cluster and a presumed mononuclear iron center. The terminal dioxygenase contains a Rieske-type [2Fe-2S] cluster. In anthranilate dioxygenase, there are two subunits in the terminal oxygenase, the larger of which, the  subunit, is presumed to contain both the Rieske-type iron-sulfur and mononuclear iron centers.

In addition to the anthranilate dioxygenase-dependent route for anthranilate catabolism (21), there are at least two other evolutionarily distinct aerobic pathways for degrading this compound. Some eukaryotic microbes use a flavoprotein monooxygenase, anthranilate hydroxylase, to convert anthranilate to 2,3-dihydroxybenzoate (36). In addition, a novel pathway for the aerobic degradation of anthranilate by a denitrifying Pseudomonas strain, which has some characteristics of both aerobic and anaerobic degradative routes, has been described (1, 2).

Anthranilate dioxygenase and the homologous cbd-encoded enzyme produce catechol from their substrates, whereas the xyl- and ben-encoded dioxygenases yield nonaromatic cis-diols that are converted to catechol in subsequent NAD⁺-dependent dehydrogenase-mediated steps. The direct formation of catechol most likely results from the spontaneous decarboxylation and loss of the ortho substituent when these steps are energetically favorable. No cis-diol is detected during CbdABC-mediated 2-halobenzoate degradation (13, 18), and, similarly, none was detected during anthranilate catabolism (Fig. 5). The inference that the chemical nature of the substrate determines whether a catechol or a cis-diol will form is consistent with observations by Nakatsu et al. (30) that the requirement for the CbaC dehydrogenase, following hydroxylation by the CbaAB chlorobenzoate dioxygenase, depends on the substrate. In this example, a cis-diol is formed when the substrate is 3-chlorobenzoate, but when the substrate is 3,4-dichlorobenzoate, elimination of HCl occurs spontaneously, obviating the need for a dehydrogenase (30).

Comparisons of anthranilate and benzoate dioxygenases. The presence of benC allowed strains lacking antC to degrade anthranilate, suggesting that the BenC reductase was able to transfer electrons from NADH to the AntAB terminal oxygenase component. Amino acids of class IB reductases that are predicted to bind flavin adenine dinucleotide, the [2Fe-2S] cluster, and those predicted to bind NADH are conserved between AntC and BenC (6, 34). Amino acids in these reductases that define substrate specificity, like those involved in protein-protein contacts with the terminal oxygenases, have yet to be determined. Reductases, including BenC, can donate electrons from NADH to artificial electron acceptors, demonstrating that the cognate oxygenase is not required for electron transfer. Moreover, electron transfer from several reductase components to a terminal oxygenase other than the authentic partner has previously been inferred (6, 10).

Expression and chromosomal organization of the antABC genes. The clustered antABC genes may be cotranscribed, as are their ben counterparts (8). The coding regions of antA and antB are separated by only 2 nucleotides, and those of antB and antC are separated by 11 nucleotides. Studies of a chromosomal antA::lacZ transcriptional fusion, described above, indicated that antA expression is inducible by anthranilate itself and that the approximately 300-nucleotide region upstream of the antA translational start signal is probably sufficient for transcriptional control. Comparison of this region with that upstream of benA did not reveal any obvious regulatory signals. Studies of regulatory mutants suggested that neither of the LysR-type activators known to control benzoate and catechol degradation, BenM and CatM, controls ant gene expression (7).