INTRODUCTION

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In the soil bacterium Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus), the enzymes needed for the conversion of benzoate to tricarboxylic acid cycle intermediates via the -ketoadipate pathway are encoded by the ben and cat genes (Fig. 1 and 2) (11). Although cat gene expression has been characterized, little is known about the regulation of the adjacent ben genes. The ben-encoded enzymes convert benzoate to catechol, a compound subsequently degraded by cat-encoded enzymes (26). During growth with benzoate, tight regulation of metabolic flow prevents the accumulation of most intermediary compounds (7). One metabolite whose transient production from benzoate can be detected, however, is cis,cis-muconate, the product of catechol ring cleavage and the inducer of all of the cat-encoded enzymes.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   The -ketoadipate pathway of Acinetobacter sp. strain ADP1. Compounds and genes relevant to these studies are indicated by boldfaced and italicized text, respectively.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Restriction map of the chromosomal ben-cat region. The locations of genes, and their transcriptional directions, are shown relative to some of the known restriction endonuclease sites: EcoRI (E), EcoRV (RV), KpnI (K), SalI (S), HindIII (H), BsaAI (B), PvuII (P), NsiI (N), and SphI (Sp). Triangles mark the insertion sites of S (29), K (6), and the lacZ::Kmr cassette (18) of strains listed in Table 1. Below the map, the DNA sequence of the benM-benA intergenic region was aligned with the corresponding catM-catB intergenic region, with identical nucleotides indicated (:). The known CatM binding site (30) was used to predict a possible BenM binding site that has the consensus sequence (T-11 nt-A) and dyad symmetry (ATAC, GTAT) involved in binding LysR-type activators. An adjacent region with similar sequences is underlined. Circled nucleotides indicate mutations identified in strains ACN147 and ACN149 (GA) and in strain ACN146 (TA).

cis,cis-Muconate regulates its own formation and degradation. Acting on the LysR-type transcriptional regulator CatM, this compound activates transcription of catA and the catBCIJFD genes (30). Moreover, cis,cis-muconate derived from benzoate can prevent the degradation of an alternative carbon source, p-hydroxybenzoate, by an as yet undefined regulatory mechanism (7). The accumulation of cis,cis-muconate during growth with benzoate most likely reflects differential expression of catA and the catBCIJFD genes, which are physically separated on the ADP1 chromosome (Fig. 2). Unlike the other cat genes, catA is expressed in response to benzoate as well as cis,cis-muconate (23). Benzoate in the absence of its metabolism, however, yields only partial induction of CatA. In a similar fashion, partial induction of the ben-encoded enzymes can be mediated by cis,cis-muconate (24). Full induction of these enzymes, however, requires growth with benzoate.

The ability of both cis,cis-muconate and benzoate to induce CatA and the Ben enzymes, albeit to different levels, suggests interconnected regulation of the corresponding genes. The goal of the current investigation was to determine the mechanisms and inducers of ben gene expression and to clarify the basis of benzoate-mediated expression of catA. The first step in this process was to characterize a genetic region adjacent to benA that appeared to control cat gene expression in a catM mutant strain (30). As described in this report, the region contains a gene immediately upstream of and divergently transcribed from benA, designated benM, that encodes a LysR-type transcriptional regulator with striking similarity to CatM. The ability of BenM to regulate the ben and cat genes in response to different inducers was tested. In addition, the effect of benM disruption on metabolic flow was examined by using nuclear magnetic resonance (NMR) techniques to monitor benzoate degradation (7).

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Acinetobacter strains and plasmids are described in Table 1 and Fig. 2. All Acinetobacter strains were derived from BD413 (14), also designated ADP1. Confusion about the taxonomic classification of ADP1 has prompted the use of Acinetobacter sp. until further characterization is complete (9). Escherichia coli DH5 (Gibco BRL) and S17-1 (35) were used as plasmid hosts. Bacteria were cultured in Luria-Bertani (LB) broth and minimal medium (MM) at 37°C as previously described (32, 34). Carbon sources added to MM, at the final concentrations specified, were 10 mM succinate, 3 mM benzoate, 3 mM cis,cis-muconate, 2.5 mM anthranilate, and 2 mM catechol. Antibiotics were added as needed at the following final concentrations: tetracycline, 6 µg/ml; kanamycin, 25 µg/ml; streptomycin, 25 µg/ml; spectinomycin, 25 µg/ml; and ampicillin, 150 µg/ml for Acinetobacter strains and 50 µg/ml for E. coli.

DNA manipulations and plasmid constructions. Standard methods were used for chromosomal and plasmid DNA purifications, restriction enzyme digestions, electrophoresis, ligations, and E. coli transformations (32). Plasmids carrying DNA fragments upstream of benA were generated from pIB1351 and pIB1352 (24). The sole SalI ADP1 DNA fragment of the pIB1351 or pIB1352 was deleted to form pIGG5 or pIGG26, respectively. The two KpnI ADP1 DNA fragments of pIB1351 were deleted to form pIGG23. The sole HindIII ADP1 DNA fragment of pIB1351 was deleted to form pIGG24. The DNA fragment between the EcoRV and KpnI sites of pIGG23 was ligated to pUC19 between the vector's HincII and KpnI sites to form pBAC64.

Strain construction by interposon mutagenesis and allelic replacement. A linear DNA fragment carrying either the benM::S4036 or the benM::K5008 allele was generated by restriction endonuclease digestion of plasmid pIGG27 with PvuII or plasmid pBAC12 with BglI, respectively. Each DNA fragment with an  cassette was electrophoretically separated and purified from an agarose gel with the GeneClean purification kit (Bio101). As described previously (20), the DNA fragments were used to transform and to replace the corresponding chromosomal regions of recipient Acinetobacter strains ADP1 and ISA13, generating ISA36 and ACN9, respectively. Similarly, a linear DNA fragment carrying the benA::lacZ fusion was generated by digestion of pBAC54 with restriction endonuclease Asp718 and used to transform ADP1, thereby generating strain ACN32.

Isolation of plasmids with the benA operator/promoter regions of mutants. The gap repair method of Gregg-Jolly and Ornston (9a) was used to isolate the chromosomal DNA of selected mutants corresponding to the region between K₁ and K₂ in Fig. 2. Plasmid pIGG16 was constructed by ligating ADP1 DNA fragments E₁ to K₁ and K₂ to RV₂ (Fig. 2) into the vector pRK415 (16). Recipient strains were transformed with pIGG16, linearized at the sole KpnI site, and homologous recombination in vivo generated plasmids carrying the benM-benA intergenic regions of the mutants. The intergenic regions between benM and benA were sequenced as described below from plasmids isolated by the gap repair method, using two oligonucleotide primers, 5'-CAAGATTTTGAATTTGTCGGC-3' and 5'-GCTAGTATTAATGACGGGAAT-3', purchased from National Biosciences Inc.

DNA sequence analysis. The DNA sequence of plasmids pIGG5, -23, -24, and -26 and pBAC64 were determined with double-stranded templates and sequencing primers (Promega) which recognize the pUC19 cloning vector. An automated DNA sequencer (ABI373A; Applied Biosystems, Inc.) was used in the University of Georgia Molecular Genetics Instruments Facility. In addition, two oligonucleotide primers, 5'-GCTCTACTGGAATTGGTT-3' and 5'-GCTCAGTAAAACCTTCAT-3' (Genosys Biotechnologies), were used with the pIGG5 DNA template. DNA sequences were analyzed with the Wisconsin Genetics Computer Group programs (5).

CatA and -galactosidase (LacZ) assays. Catechol 1,2-dioxygenase (CatA) activity was measured in cell extracts of cultures grown in LB medium with or without 2 mM benzoate or 2 mM cis,cis-muconate as described previously (30). CatA activity was assayed spectrophotometrically by the increase in A₂₆₀, indicative of cis,cis-muconate formation (25). For LacZ assays, cultures were grown overnight in 5 ml of LB with or without 3 mM benzoate, 3 mM cis,cis-muconate, 2.5 mM anthranilate, or 2 mM catechol. Cells were lysed with chloroform and sodium dodecyl sulfate, and -galactosidase activities were determined as described by Miller (19).

Metabolite monitoring by HPLC. To monitor benzoate metabolism, 1-ml culture samples were centrifuged to pellet cells. Any cells remaining in the supernatant fractions were removed by passage through a low-protein-binding, 0.22-µm-pore-size syringe filter (MSI). A 10-µl sample of the filtrate was analyzed on a C₁₈ reversed-phase high-performance liquid chromatography (HPLC) column (Bio-Rad Laboratories). Elution at a rate of 0.8 ml/min was carried out with 30% acetonitrile and 0.1% phosphoric acid, and the eluant was monitored by UV detection at 254 nm to detect anthranilate, benzoate, and cis,cis-muconate and at 282 nm to detect catechol. Under these conditions, the retention times for benzoate, anthranilate, catechol, and cis,cis-muconate were 7.7, 6.0, 4.6, and 3.5 min, respectively. Peak areas corresponding to standards and experimental samples were integrated by using the ValuChrom software package (Bio-Rad). To assess the metabolism of benzoate by mutants blocked in its degradation, cultures were grown in LB broth with 2.5 to 3 mM benzoate added. Culture samples were analyzed by HPLC before and after 24 h of growth to determine the benzoate concentration. The concentration determinations of duplicate samples varied no more than 100 µM.

Metabolic observation. Metabolic observation-NMR (MO-NMR) was carried out as described previously (7). Briefly, Acinetobacter strains were deuterated by overnight growth in a complex ²H-IG medium prepared (by Isogenetics, Inc.) from algae grown photoautotrophically in deuterated water (7). The Acinetobacter cells were used to inoculate a tube containing 6 ml of a defined deuterated minimal medium, ²H-MM (7), and 600 µl of ²H-IG medium. The addition of benzoate or a mixture of benzoate and p-hydroxybenzoate, dissolved in ²H₂O, marked the start of each experiment (time zero). The culture tubes were incubated at 34°C with aeration. Culture samples were taken periodically and analyzed by NMR spectroscopy. Concentrations of compounds were determined by integrating the appropriate spectral peaks.

Northern hybridization analysis. Total RNA was isolated from 20-ml Acinetobacter cultures by using a Snap-O-Sol kit (Biotecx Laboratories). RNA species were electrophoretically separated on agarose-formaldehyde gels with 2 to 8 µg of RNA per lane (32). A rapid downward transfer system was used to transfer RNA to Nytran nylon membranes (TurboBlotter; Schleicher & Schuell). RNA was cross-linked to the membranes by exposure to a total dose of UV light (254 nm) of 120 mJ cm². Sizes of the mRNAs were estimated by using RNA standards of sizes 9.4, 6.2, 3.9, 2.8, 1.9, 0.9, 0.6, and 0.4 kb (Promega) that were stained with methylene blue dye following transfer to the nylon membranes (32).

Nucleotide sequence accession number. The nucleotide sequence of benM was deposited in the GenBank database (accession no. AF009224).

	RESULTS

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Identification of the benM gene. In previous studies, a deletion encompassing the benABC genes and a region upstream of benA was shown to eliminate inducible catA and catB expression in a catM mutant (30). As presented here, the DNA sequence upstream of benA revealed a divergently transcribed 912-bp open reading frame, designated benM. The deduced 304-amino-acid sequence of BenM is homologous to sequences of LysR-type transcriptional activators including CatM, to which it is 59% identical and 75% similar. Similarities are greatest within a subset of this family containing proteins that regulate the degradation of catechol, such as CatM and CatR, or that regulate the degradation of chlorocatechols, such as ClcR, TcbR, and TfdR (Fig. 3). In pairwise sequence comparisons of BenM with Pseudomonas regulators CatR (12, 31), ClcR (3), and TcbR (36) or TfdR from Ralstonia eutropha (15), identity between aligned residues ranges from 29 to 41% and similarity ranges from 50 to 61%.

View larger version (79K): [in this window] [in a new window]   FIG. 3.   Protein sequence alignment of homologous LysR-type transcriptional activators: ClcR (3), TcbR (36), TfdR (15), BenM, CatM (30), and CatR (31). Aligned residues identical or similar to those of BenM are in black or gray boxes, respectively. The underlined region, BenM residues 109 to 162, may be involved in inducer recognition.

Disruption of benM and isolation of suppressor mutations. Strain ISA36 was created by interposon mutagenesis (29) of benM with the S cassette (Fig. 2 and Table 1). The benM disruption did not prevent the catabolism of cis,cis-muconate, catechol, or anthranilate, and with each of these as the sole carbon source, ADP1 and ISA36 grew at comparable rates (data not shown). The degradation of these substrates requires the gene products of catA and catBCIJFD but not those of benABCD. In contrast, the benM disruption prevented benzoate degradation. Growth of ISA36 on catechol but not benzoate suggested that BenM regulates ben gene expression.

5

Regulation of benA expression. A benA::lacZ transcriptional fusion, on plasmid pBAC54 (Table 1), was introduced into the wild-type chromosome by allelic replacement, forming ACN32. In this strain, the promoterless lacZ cassette (18) lies 1.1 kbp downstream of the ATG initiation codon of benA, and -galactosidase (LacZ) activity reflects benA expression. HPLC confirmed that the benA disruption had blocked further degradation of benzoate (data not shown). The addition of benzoate to the growth medium increased -galactosidase activity in ACN32 approximately 10-fold compared to additive-free medium (Fig. 4), establishing benzoate as an inducer of benA expression.

View larger version (43K): [in this window] [in a new window]   FIG. 4.   -Galactosidase (LacZ) activity resulting from expression of a chromosomal benA::lacZ fusion in strains ACN32, ACN39, ACN41, ACN47, ACN47(pBAC14), and ACN42(pBAC14) (Table 1). Genotypes are noted above bars. Strains were cultured in LB with the inducers indicated. Activities are averages of at least three independent repetitions, and the corresponding standard deviations were <20% of the average value.

benM is required for benA expression. ACN47 carries a chromosomal benM disruption as well as the chromosomal benA::lacZ fusion (Table 1). In ACN47, expression of the benA::lacZ fusion was not inducible (Fig. 4). The wild-type pattern of induction was restored by benM in trans on pBAC14, although the induced levels of -galactosidase were all approximately 40% lower than in ACN32 (Fig. 4). This same pattern of expression was observed with benM in trans in strain ACN42. Since ACN42 additionally carries a chromosomal deletion of the catMBC genes (Fig. 4), CatM is not required for the synergistic effect of benzoate and cis,cis-muconate on benA expression.

BenM-mediated regulation of catA expression. CatM has been shown to activate catA expression in response to cis,cis-muconate but not benzoate (30). Since benzoate induces catechol 1,2-dioxygenase (CatA) independently of cis,cis-muconate (23) and CatM, the possibility that BenM regulates catA expression was tested. CatA activity was measured in the wild-type and benM mutants grown on LB to which no inducer, cis,cis-muconate, or benzoate was added. In the benM mutant ISA36, cis,cis-muconate, but not benzoate, induced CatA to wild-type levels (Fig. 5). These results, consistent with CatM regulating catA expression in ISA36, suggested that the loss of benzoate induction was due to the absence of BenM-mediated catA expression.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   CatA (catechol 1,2-dioxygenase) activity in strains ADP1, ISA36, ISA35, and ISA35(pBAC14) (Table 1). Genotypes are noted above bars. Strains were cultured in LB with the inducers indicated. Activities are the averages of at least three independent repetitions, and the corresponding standard deviations were <20% of the average value. In ADP1, but not the other strains, benzoate can be metabolized to cis,cis-muconate.

Coexpression of the benABCDE genes. Northern hybridization studies assessed coexpression of the benABCDE genes, which form an approximately 5-kbp region with no more than 50 nt separating each coding region (GenBank accession no. M76990; Fig. 2). Labeled probes were hybridized to RNA from cells grown with or without benzoate. Probe A, from the 5' region of benA, between BsaAI and NsiI restriction sites (Fig. 2), detected a 5.5-kb transcript in RNA from the wild type grown with benzoate (Fig. 6A, lane 2) but not in those grown without benzoate (data not shown). Hybridization signals, of variable relative intensities, corresponding to RNA sizes of approximately 4 and 2 kb were typically observed (Fig. 6A, lane 2).

View larger version (41K): [in this window] [in a new window]   FIG. 6.   Northern hybridization analysis of ben gene transcripts. Regions of benA and benE were labeled to make probes A and E, respectively, as described in Materials and Methods. Arrows indicate transcripts detected in RNA from the wild-type ADP1 grown with benzoate (A) or from ACN32, with a transcriptional terminator following the benA::lacZ fusion, grown with benzoate and cis,cis-muconate (B). Total wild-type RNA in one sample (A, lane 1) was stained with methylene blue to demonstrate the positions of the rRNA species.

Metabolic flow in regulatory mutants. To assess the physiological significance of the individual CatM and BenM activators in ben and cat gene expression, metabolite flow was analyzed in a strain lacking a functional benM (ISA36) and a strain lacking both benM and catM (ACN9) (Table 1). Comparisons could then be made with results of previous studies in which identical methods were used to analyze the wild type and a catM mutant (ISA13) (7). Using MO-NMR techniques (7), either 3 mM [¹H]benzoate or a mixture of 3 mM [¹H]benzoate and 3 mM [¹H]p-hydroxybenzoate was provided to deuterated bacterial cultures. ¹H compounds were detected by NMR analyses of samples taken during growth of the batch culture.

View larger version (23K): [in this window] [in a new window]   FIG. 7.   Metabolites detected by MO-NMR following the addition, at time zero, of 3 mM benzoate (A and B) or 3 mM benzoate and 3 mM p-hydroxybenzoate (A and C) to deuterated cultures of ISA36 or ACN9. Concentrations of benzoate (ben), p-hydroxybenzoate (pob), catechol (cat), and cis,cis-muconate (ccm) were calculated by integration of NMR spectral peaks.

	DISCUSSION

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BenM activates ben gene expression. The dependence on benM for inducible expression of a benA::lacZ fusion identified BenM as a transcriptional activator of the benABCDE operon needed for the conversion of benzoate to catechol (Fig. 4). This metabolic conversion has been studied primarily in pseudomonads, where transcriptional control can be mediated by the XylS regulator encoded on the TOL plasmid pWWO. XylS, an AraC-type (8) rather than a LysR-type (33) activator, controls expression of the xylXYZ genes encoding broad-substrate-specific homologs of the BenABCD enzymes (10, 28). The benR locus of Pseudomonas putida has been suggested to encode a XylS-like transcriptional regulator of chromosomal ben genes (13). The lack of benR-xylS DNA hybridization, however, and differences between BenR- and XylS-mediated regulation (17) raise the possibility that BenR is not a XylS homolog and may be a BenM homolog.

Interaction of BenM with effectors. The ability of BenM to respond to multiple effectors raises questions about ligand binding. BenM probably binds cis,cis-muconate since its putative inducer binding region is approximately 60% identical in sequence to that of CatR and CatM, activators that also respond to this compound (Fig. 3, positions 109 to 162) (2, 33). The inducer binding regions of LysR-type proteins that recognize dissimilar ligands usually differ significantly from each other (33). For example, identity in sequence alignments of this region is only 15% between BenM and the N-acetylserine-responsive CysB, the only LysR-type protein for which the structure of the effector binding domain is known (35a). Therefore, the approximately 30% sequence identity in pairwise comparisons of this region of BenM with those of the chloromuconate-responsive ClcR, TcbR, and TfdR is significant. As expected, the central regions of ClcR, TcbR, and TfdR, are more similar to each other than each is to BenM.

Transcription initiation of the ben operon. Amino acids in BenM likely to be involved in DNA recognition are nearly identical to those in CatM, in the helix-turn-helix regions of the N-terminal domains (20). Therefore, CatM and BenM operator sequences would be expected to be similar, leading to the predication of a BenM binding site (Fig. 2) (30). Although protein binding to the proposed BenM operator depicted in Fig. 2 remains to be demonstrated, the ATACN₇GTAT sequence is not only identical to that involved in CatM recognition and binding but also identical or nearly so to sequences that bind its most closely related homologs (discussed in reference 30). Binding of BenM to this site could have a negative autoregulatory function characteristic of LysR-type regulators (33). The spacing of this BenM binding site relative to a region in which two mutations affected benA expression corresponds to that between the operator and promoter regions upstream of catB (Fig. 2). The mutation causing constitutive benA expression, a G-to-A transition, may affect RNA polymerase interactions in the 10 promoter region, although confirmation requires mapping the transcription initiation site.

Benzoate catabolism by benM-disrupted mutants. As would be expected from the coexpression of benD with benABC that was indicated by Northern hybridization studies (Fig. 6), benzoate cis-diol was never detected by MO-NMR (Fig. 7). In strains lacking BenM, it was most likely basal levels of ben gene expression that enabled the slow degradation of benzoate (Fig. 7). These basal expression levels, as much as 80-fold lower than in fully induced cultures, were not negligible: LacZ levels were approximately 200 to 400 Miller units in strains with a benA::lacZ transcriptional fusion that lacked BenM (Fig. 4). Similarly, in the absence of BenM and CatM, basal levels of cat gene expression probably enabled catechol degradation. A comparison of metabolite accumulations in strains with different regulatory mutations helped to assess the individual roles of BenM and CatM in ben and cat gene expression in vivo.