genes coding for a new pathway of aerobic benzoate ... · homology to an open reading frame (orf)...

JOURNAL OF BACTERIOLOGY, Nov. 2002, p. 6301–6315 Vol. 184, No. 220021-9193/02/$04.00�0 DOI: 10.1128/JB.184.22.6301–6315.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Genes Coding for a New Pathway of Aerobic Benzoate Metabolism inAzoarcus evansii

Johannes Gescher,1 Annette Zaar,1 Magdy Mohamed,1 Hermann Schagger,2 and Georg Fuchs1*Mikrobiologie, Institut Biologie II, Universitat Freiburg,1 and Zentrum der Biologischen Chemie, Klinikum der Johann

Wolfgang Goethe-Universitat Frankfurt, Frankfurt,2 Germany

Received 23 May 2002/Accepted 16 August 2002

A new pathway for aerobic benzoate oxidation has been postulated for Azoarcus evansii and for a Bacillusstearothermophilus-like strain. Benzoate is first transformed into benzoyl coenzyme A (benzoyl-CoA), whichsubsequently is oxidized to 3-hydroxyadipyl-CoA and then to 3-ketoadipyl-CoA; all intermediates are CoAthioesters. The genes coding for this benzoate-induced pathway were investigated in the �-proteobacterium A.evansii. They were identified on the basis of N-terminal amino acid sequences of purified benzoate metabolicenzymes and of benzoate-induced proteins identified on two-dimensional gels. Fifteen genes probably codingfor the benzoate pathway were found to be clustered on the chromosome. These genes code for the followingfunctions: a putative ATP-dependent benzoate transport system, benzoate-CoA ligase, a putative benzoyl-CoAoxygenase, a putative isomerizing enzyme, a putative ring-opening enzyme, enzymes for �-oxidation of CoA-activated intermediates, thioesterase, and lactone hydrolase, as well as completely unknown enzymes belongingto new protein families. An unusual putative regulator protein consists of a regulator protein and a shikimatekinase I-type domain. A deletion mutant with a deletion in one gene (boxA) was unable to grow with benzoateas the sole organic substrate, but it was able to grow with 3-hydroxybenzoate and adipate. The data supportthe proposed pathway, which postulates operation of a new type of ring-hydroxylating dioxygenase acting onbenzoyl-CoA and nonoxygenolytic ring cleavage. A �-oxidation-like metabolism of the ring cleavage product isthought to lead to 3-ketoadipyl-CoA, which finally is cleaved into succinyl-CoA and acetyl-CoA.

Aerobic metabolism of aromatic compounds, such as ben-zoate, has been studied in considerable detail in various mi-croorganisms (45a; for a recent review, see reference 23). Cat-echol (1,2-dihydroxybenzene) and protocatechuate (3,4-dihydroxybenzoate) were identified as early intermediates,depending on the initial oxygenases catalyzing benzoate hy-droxylation. Benzoate metabolism via 4-hydroxybenzoate andprotocatechuate is common in fungi, whereas in bacteria thecatechol pathway has been established and the protocat-echuate route (45a) is uncertain. Both compounds serve assubstrates for ring-cleaving dioxygenases, which in the case ofthe ortho-cleavage pathway cleave the aromatic ring betweenthe hydroxyl groups. Catechol and protocatechuate orthocleavage and the subsequent reactions lead to 3-ketoadipate,which is converted into succinyl coenzyme A (succinyl-CoA)and acetyl-CoA via 3-ketoadipyl-CoA (Fig. 1).

However, some observations could not be explained by theestablished mechanisms. Thus, cell extracts of some Bacillusspp. grown on benzoate or 3-hydroxybenzoate utilized genti-sate but not catechol or protocatechuate. In an attempt toexplain these findings, hydroxylation of benzoate resulting in3-hydroxybenzoate and gentisate (2,5-dihydroxybenzoate) wasproposed (9, 10, 12). However, direct evidence for the sug-gested intermediates and reactions has not been obtained sofar.

More recently, it was shown that a gram-positive Bacillusstearothermophilus-like strain (26) and the facultatively deni-

trifying gram-negative bacterium Azoarcus evansii, belongingto the �-group of the Proteobacteria (2, 6), are able to utilizebenzoate, 3-hydroxybenzoate, and gentisate aerobically as solesources of carbon and energy (1, 26, 32). 2-Hydroxy- and 4-hy-droxybenzoates, protocatechuate, catechol, and 2,3-dihydroxy-benzoate did not support aerobic growth (1, 26). In conjunc-tion with the presence of an aerobically inducible benzoate-CoA ligase (AMP forming) and gentisate 1,2-dioxygenase (1,42), the degradation of benzoate was proposed to proceed viabenzoyl-CoA and either 2-hydroxybenzoyl-CoA or 3-hydroxy-benzoyl-CoA as intermediates (1, 26, 32). Further hydroxyla-tion of either compound hypothetically could yield gentisyl-CoA, which might undergo thioester hydrolysis to gentisate (1,26). However, the enzymatic reactions catalyzing the proposedpathway to gentisate have remained elusive. 3-Hydroxybenzo-ate was shown to be metabolized via 6-hydroxylation of 3-hy-droxybenzoate to gentisate, and gentisate is cleaved by genti-sate 1,2-dioxygenase to maleylpyruvate. Maleylpyruvate isisomerized to fumarylpyruvate, which is cleaved into fumarateand pyruvate (1).

A study of the conversion of 13C-labeled benzoyl-CoA bycell extracts of A. evansii and the B. stearothermophilus-likestrain under aerobic conditions revealed unexpected interme-diates. In contrast to earlier proposals, benzoate was not con-verted into hydroxybenzoate or gentisate. Under aerobic con-ditions benzoyl-CoA was an in vivo product of benzoatecatabolism in both microbial species and was converted intovarious CoA thioesters by cell extracts in oxygen- andNADPH-dependent reactions. By using [13C]benzoyl-CoA as asubstrate, cis-3,4-dehydroadipyl-CoA, trans-2,3-dehydroadipyl-CoA, the 3,6-lactone of 3-hydroxyadipyl-CoA, and 3-hydroxy-adipyl-CoA were identified as products by nuclear magnetic

* Corresponding author. Mailing address: Mikrobiologie, InstitutBiologie II, Schanzlestr. 1, D-79104 Freiburg, Germany. Phone: 49-761-2032649. Fax: 49-761-2032626. E-mail: [email protected].

6301

on April 10, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

resonance spectroscopy. A protein mixture from A. evansiitransformed benzoyl-CoA in an NADPH- and oxygen-depen-dent reaction into 6-hydroxy-3-hexenoyl-CoA (48). The datasuggested that there is a novel aerobic pathway of benzoatecatabolism via CoA intermediates leading to �-ketoadipyl-CoA, an intermediate of the known �-ketoadipate pathway(23) (Fig. 2).

Hence, the committed first step in the new benzoate path-way is the activation of benzoate to benzoyl-CoA by a specif-ically induced benzoate-CoA ligase (AMP forming) that cata-lyzes the following reaction: benzoate � CoA � Mg-ATP 3benzoyl-CoA � Mg-AMP � pyrophosphate. This enzyme waspurified and was shown to differ from an isoenzyme that cat-alyzes the same reaction under anaerobic conditions (29). Thesecond step is postulated to involve the hydroxylation of ben-zoyl-CoA to an unknown product by a novel benzoyl-CoAoxygenase, presumably a multicomponent enzyme system. Abenzoate-induced iron-sulfur flavoprotein, BoxA, which maybe a component of this system, was purified and characterized(29). This protein has a native molecular mass of 98 kDa(homodimer of 50-kDa subunits) and contains (per mole ofnative protein) 0.72 mol of flavin adenine dinucleotide (FAD),10.4 to 18.4 mol of Fe, and 13.3 to 17.9 mol of acid-labile

sulfur, depending on the method of protein determination.This enzyme catalyzes a benzoyl-CoA-, FAD-, and O2-depen-dent NADPH oxidation, surprisingly without hydroxylation ofthe aromatic ring. However, H2O2 is formed as follows:NADPH � H� � O2 3 NADP� � H2O2.

The gene coding for this enzyme (boxA, for benzoyl-CoAoxidizing) was cloned and sequenced (29). This gene codes fora 46-kDa protein (414 amino acids) with two consensus aminoacid sequences for two [4Fe-4S] centers at the N terminus. Thededuced amino acid sequence shows homology with subunitsof ferredoxin-NADP� oxidoreductase, nitric oxide synthase,NADPH-cytochrome P450 oxidoreductase, and phenol hy-droxylase. Upstream of the boxA gene another benzoate-in-duced gene, boxB, encoding a 55-kDa protein (473 amino ac-ids), was found. The boxB gene exhibits the highest level of

FIG. 1. Conventional routes of microbial benzoate oxidation viaortho cleavage of catechol (in bacteria) or protocatechuate (in fungi)by the �-ketoadipate pathway (23, 45a). 1, benzoate; 2, protocat-echuate; 3, catechol; 4, 3-oxoadipate (�-ketoadipate); 5, 3-oxoadipyl-CoA; 6, succinyl-CoA; 7, acetyl-CoA. Not all intermediates are shown.

FIG. 2. Proposed new aerobic benzoate oxidation pathway in A.evansii and a B. stearothermophilus-like strain. Experimentally docu-mented compounds are enclosed in boxes. The suggested pathwayleads to �-ketoadipyl-CoA, a known intermediate of the �-ketoadipatepathway (see Fig. 1).

6302 GESCHER ET AL. J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

homology to an open reading frame (ORF) in Sulfolobus sol-fataricus (44) which probably codes for a component of a pu-tative aerobic phenylacetyl-CoA-oxidizing system (15). In thepresent work we tried to find and study the missing genesinvolved in this novel aerobic benzoate oxidation pathway in A.evansii. Here we show that up to 15 genes are involved, someof which belong to new enzyme families.

MATERIALS AND METHODS

Materials and bacterial strains. Chemicals were obtained from Sigma-Aldrich(Deisenhofen, Germany), Merck (Darmstadt, Germany), Biomol (Hamburg,Germany), or Roth (Karlsruhe, Germany); biochemicals were obtained fromRoche Diagnostics (Mannheim, Germany) or Gerbu (Craiberg, Germany).High-pressure liquid chromatography equipment was obtained from Waters(Eschborn, Germany), Grom (Herrenberg-Kayh, Germany), and Raytest(Straubenhardt, Germany). Enzymes used for cloning experiments were pur-chased from MBI Fermentas (St.Leon-Rot, Germany), Roche Diagnostics, andAmersham Pharmacia Biotech (Freiburg, Germany).

A. evansii KB 740 (� DSMZ6869) (2) (formerly designated Pseudomonas sp.strain KB 740) (6) has been deposited in the Deutsche Sammlung fur Mikroor-ganismen und Zellkulturen (Braunschweig, Germany). Escherichia coli strainsXL1-blue, MRF� {�(mcrA)183 �(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1recA1 gyrA96 relA1 lac [F� proAB lac IqZ�M15 Tn10 (Tetr)]}, and SURE {e14�

(mcrA) �(mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB recJsbcC umuC::Tn5 (kan) uvrC [F� proAB lacIqZ�M15 Tn10 (Tetr)]} from Strat-agene (Heidelberg, Germany) and strain S17-1 (45) were used for boxA genereplacement experiments with A. evansii. E. coli strains XL1-blue, SURE, andXLOLR {�(mcrA)183 �(mcrCB-hsdSMR-mrr)173 endA1 thi-1 recA1 gyrA96relA1 lac [F� proAB lac IqZ�M15 Tn10 (Tetr)] Su� �r} were used for cloning andsubcloning experiments. The DNA clones and vectors used are summarized inTable 1.

Bacterial cultures. A. evansii was grown aerobically at 37°C with benzoate asthe sole source of carbon and energy (32, 46) in a 200-liter fermentor (airflow,100 liter/min; 200 rpm). Benzoate was added continuously when the substrateadded initially (5 mM) was almost consumed. Cells were harvested in the expo-nential growth phase at an optical density at 578 nm of 2.3, which correspondedto 0.6 g (dry weight) of cells/liter. The culture was cooled to 15°C, and cells wereharvested by continuous-flow centrifugation. The yield was 200 g (wet weight) ofcells/mol of benzoate. For comparative experiments with the wild type and theboxA mutant, cells were grown in shaker flasks containing 400 ml of medium.Cells were harvested by centrifugation in the exponential growth phase at anoptical density at 578 nm of 0.3 to 0.4. The substrates used were benzoate (5mM), 3-hydroxybenzoate (5 mM), phenylacetate (5 mM), adipate (10 mM), andacetate (5 mM). The combinations of substrates used were benzoate (5 mM) plus3-hydroxybenzoate (5 mM), benzoate (5 mM) plus phenylacetate (5 mM), ben-zoate (5 mM) plus adipate (10 mM), and benzoate (5 mM) plus acetate (5 mM).For a negative control of immunodetection of BoxA, A. evansii was grown onmalate (5 mM).

E. coli strains were grown at 37°C in Luria-Bertani medium (3, 40). Antibioticswere added to E. coli cultures as follows (final concentrations): ampicillin, 50 �gml�1; kanamycin, 50 �g ml�1; and gentamicin, 15 �g ml�1.

Preparation of cell extracts. All steps used for preparation of cell extracts wereperformed at 4°C. Frozen cells were suspended in an equal volume of watercontaining 0.1 mg of DNase I ml�1. The suspensions were passed through aFrench pressure cell at 132 MPa and then centrifuged (100,000 g). For

comparative experiments with the wild type and the boxA mutant, 0.6 ml of coldcell suspension was disrupted by grinding with 1.2 g of glass beads (diameter, 0.1to 0.25 mm) in a mixer mill (MM 200; Retsch, Haan, Germany) for 7 min at 30Hz.

Construction of A. evansii gene banks. A cosmid library of A. evansii wasconstructed as described by Redenbach et al. (39). A �ZAP Express gene librarywas constructed as described in the ZAP Express cloning kit instruction manual(Stratagene).

Screening of cosmid and �ZAP Express gene banks. PCR products used asprobes in screening analyses were labeled with digoxigenin-11-dUTP by PCR.They were detected by using anti-digoxigenin-AP Fab fragments, nitroblue tet-razolium chloride, and 5-chloro-4-bromo-3-indoylphosphate (toluidine salt) (Bi-omol, Hamburg, Germany). Probes were amplified with primers MoI and MorI,Probelilyfor and Probelilyrev, ABC-probefor and ABC-proberev, and hbahforand hbahrev (Table 2).

RT-PCR. Total RNA from A. evansii cells grown aerobically on benzoate andfrom cells grown aerobically on malate was used for reverse transcription-PCR(RT-PCR). RNA was isolated with an RNeasy total RNA kit (Qiagen, Hilden,Germany) and was separated from contaminating DNA by treatment with fastprotein liquid chromatography-purified DNase I (1 U per �g of total RNA;Amersham Pharmacia Biotech) for 30 min at 37°C. Complete removal of DNAfrom RNA preparations was verified by amplifying the intergenic region betweentwo benzoate catabolic genes coding in different directions with cDNA as thetemplate. One microgram of purified total RNA was used to prepare cDNA byusing avian myeloblastosis virus reverse transcriptase (20 U/�g of RNA; Amer-sham Pharmacia Biotech) and a mixture of completely random hexanucleotides(3.2 �g/20 �l) for random priming. Gene expression was studied by amplificationof intergenic regions between ORFs. The sequences of the primers used areshown in Table 2.

DNA techniques and purification of nucleic acids. Standard protocols wereused for DNA cloning, transformation, amplification, purification, and sequenc-ing (3, 40). Plasmid DNA was purified by the method of Birnboim and Doly (5).Both strands of the cloned chromosomal DNA containing the gene cluster weresequenced.

Computer analysis. DNA and amino acid sequences were analyzed by usingthe BLAST network service at the National Center for Biotechnology Informa-tion. Alignments were generated by using the CLUSTLW program contained inthe DNAman software package (Lynnon, Montreal, Canada).

Synthesis of CoA esters. Benzoyl-CoA was prepared by previously describedprocedures (19, 41). The yield was 65%.

Purification of component A of benzoyl-CoA oxygenase (BoxA). BoxA waspurified by the method of Mohamed et al. (29).

Enzyme assay for BoxA. BoxA enzyme activity was monitored spectrophoto-metrically at 365 nm by determining benzoyl-CoA-, oxygen-, and FAD-depen-dent oxidation of NADPH at 37°C. The standard assay mixture (0.5 ml) con-tained 100 mM Tris-HCl (pH 8.0), 0.1 mM FAD, 0.3 mM NADPH, and cellextract (10 �l of a 100,000--g supernatant). The test was started by adding 0.1mM benzoyl-CoA.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide(11.5%) gel electrophoresis was performed by the Laemmli method (11). Pro-teins were visualized by Coomassie blue staining (49).

Two-dimensional gel electrophoresis. For two-dimensional gel electrophoresisthe first dimension (isoelectric focusing) was performed with cell extract (120 �gof protein) as described by Gorg et al. (17) by using the Immobiline Dry Stripsystem (linear gradient from pH 3 to 10; Amersham Pharmacia Biotech) accord-ing to the manufacturer’s protocol. The second dimension (sodium dodecylsulfate-polyacrylamide gel electrophoresis) was performed as described above. A

TABLE 1. DNA clones and vectors used

Clone or vector Genes Source or reference

pGEX 6P-1 amp lacIq Amersham Pharmacia Biotech, Freiburg, GermanySupercos1 amp neo Stratagene, Amsterdam, The NetherlandspJQ200SK Gm, sacB traJ 38pUC4-KSAC vector kan ampR Amersham Pharmacia Biotech, Freiburg, GermanypBK-CMV kan Stratagene, Amsterdam, The NetherlandspBK-CMVe5 Includes bp 1 to 4370 of benzoate gene cluster This studypBK-CMVw17 Includes bp 2028 to 6862 of benzoate gene cluster This studypBK-CMVcII Includes bp 4115 to 9506 of benzoate gene cluster This studySupercosIGD11 Includes bp 7131 to 19189 of benzoate gene cluster This study

VOL. 184, 2002 NEW AEROBIC BENZOATE PATHWAY 6303


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

comparison of extracts of cells grown on benzoate and 3-hydroxybenzoate al-lowed identification of benzoate-induced proteins.

Electrophoretic transfer of protein and determination of N-terminal aminoacid sequences. Proteins of benzoate-grown cells were separated by two-dimen-sional gel electrophoresis and transblotted onto an Immobilon-Psq transfer mem-brane (Millipore, Bedford, Mass.) by using the Nova Blot system (Multiphor II;Pharmacia LKB, Freiburg, Germany) (11). Transblotted proteins were detectedby Commassie blue staining (49). Benzoate-induced proteins were excised andsequenced by using an Applied Biosystems 473A sequencer.

Immunodetection. Cell extracts of benzoate-grown and malate-grown cellswere separated by two-dimensional gel electrophoresis and transblotted ontonitrocellulose filters (pore size, 0.45 �m) (11). The transblotted proteins weredetected by Ponceau S staining (11). Polyclonal antibodies raised against BoxAof A. evansii were used to test for the presence of BoxA. BoxA was detected byusing the Amersham enhanced chemoluminescence system.

Construction of boxA mutant. The boxA mutant was constructed by replace-ment of previously cloned boxA that had been manipulated by insertion of akanamycin resistance Geneblock (Amersham Pharmacia Biotech). Two primerswere amplified upstream and downstream of the gene coding for BoxA (ORF13); these primers carried restriction sites for BamHI and NotI, respectively(BoxABamHI-forw and BoxANotI-rev [Table 2]). The boxA gene was amplifiedby PCR and cloned into the pGex vector (Amersham Pharmacia Biotech) (Table1) by using E. coli XL1-blue as the host. The kanamycin resistance cassette wascut out of the pUC4-KSAC vector (Amersham Pharmacia Biotech) with SacIand inserted into a SacI restriction site of the boxA gene in the recombinantplasmid. A DNA fragment carrying the boxA gene with the kanamycin cassettewas cut out with NotI and BamHI and cloned into the sacB-containing suicidevector pJQ200SK (38) (Table 1). The resulting plasmid was transformed into E.coli S17-1 and transferred by conjugation into A. evansii (35). The mating mixturewas plated on Gelrite (0.8%, wt/vol) minimal medium containing sucrose (5 mM)and kanamycin (50 �g ml�1). Exconjugants which had lost the sacB-containingvector due to double recombination were selected by screening for sucroseresistance. The presence of the desired boxA mutation was confirmed by colony

PCR performed with the primers mentioned above and by Western immuno-blotting, which showed that the mutant failed to produce BoxA.

Protein contents. Protein contents were determined by the method of Brad-ford (11) by using bovine serum albumin as the standard.

Nucleotide sequence accession number. The sequence data reported here havebeen deposited in the EMBL database under accession no. AF548005.

RESULTS AND DISCUSSION

Proteins induced by benzoate. The ability to metabolize ben-zoate aerobically is induced by the substrate benzoate. Benzo-ate-grown cells contained 20-fold-higher specific activities ofbenzoate-CoA ligase and BoxA enzyme activity than 3-hy-droxybenzoate-grown cells, and in acetate- or malate-growncells these enzyme activities were hardly detectable. In con-trast, the 3-hydroxybenzoate pathway was induced to similarextents in 3-hydroxybenzoate-grown cells and in benzoate-grown cells. This is consistent with similar specific activities ofcharacteristic enzymes of 3-hydroxybenzoate metabolism inthe two types of cells (i.e., 3-hydroxybenzoate 6-hydroxylaseand gentisate 1,2-dioxygenase activities) (32). We thereforeused 3-hydroxybenzoate-grown cells as a reference for com-parison of the protein patterns. Benzoate-grown cells shoulddiffer only by additional, benzoate-induced proteins when theyare compared to 3-hydroxybenzoate-grown cells.

Two-dimensional gel electrophoresis of soluble cell extracts(100,000--g supernatant) revealed at least seven strongly ben-zoate-induced proteins in benzoate-grown cells, five of which

TABLE 2. Primers used

Primer Sequence Usage

RT5� Enoyl-for GACACGGCGGGATCGCGGTC RT-PCR, fragment 0Thio-for CGACTTTTTCGCACCGAGCATC RT-PCR, fragment 0RT-Thioesterase-for CTCATCGCCGTGCATGCTCG RT-PCR, fragment 1Hydro-rev GATCCAGGGCGAGGACGAG RT-PCR, fragment 1RT-Hydro-for CTGCGGGAAGTCGCGCCAC RT-PCR, fragment 2hbah-rev GCCACATCCTGCACGGCG RT-PCR, fragment 2RTligase-rev GCGTTGCCGGAAGACATCGG RT-PCR, fragment 3bindeprot-for CTGCTCCTGCACGTACTGCTTG RT-PCR, fragment 3RT-ligase-for CGTGGGCGGACTCGTGCTG RT-PCR, fragment 4ORF3-rev TGCGCGCGCTGGCGATGTATC RT-PCR, fragment 4paaZ-for GTTTCGCGGCGCGCTCCTCG RT-PCR, fragment 5Regulator-rev CGCTTGCCGAGCGTCGGCAG RT-PCR, fragment 5Shiki-Fus-for GCGACAACCCGCGCGCCGTG RT-PCR, fragment 6Shiki-Fus-rev GTTGAGCTTCAGCTTGTAGC RT-PCR, fragment 6Fus-boxB-for CTGGATCTTCAACCGCCCCA RT-PCR, fragment 7Fus-boxB-rev GATGATGCGGCGCAGGGTCG RT-PCR, fragment 7box b-boxA-for GTTCGGCTTCGACTTCCGCTTC RT-PCR, fragment 8box b-boxA-rev GCTCGGTGGTGTCGGGCAGG RT-PCR, fragment 8boxA-Thio-for GCTACATCTACATCTGCGGC RT-PCR, fragment 9NMO4 GCGCCAGCCGATGGTGGT RT-PCR, fragment 9Thio-Hypo-for GCCAGGGCATCGCGCTCGCG RT-PCR, fragment 10Thio-Hypo-rev CTGATCGCGAGGTGCTCGTC RT-PCR, fragment 10MoI ATGAACGCSCCSGCSGARCA Screening probe 1MorI CCGAAGTASGGSAGYTCYTC Screening probe 1Probelilyfor CGCCGCGAGTTCATCGTAGC Screening probe 2Probelilyrev ACGGCTTCAGCGTCGACGTC Screening probe 2ABC-probefor CAGCAGCACCTTGCCGAAGG Screening probe 3ABC-proberev GATCCAGAAGACGCGCCTC Screening probe 3hbahfor GGATCAGGTTCTCCCGGAC Screening probe 4hbahfor GCCACATCCTGCACGGCG Screening probe 4BoxABamHI-forw CAGCGGATCCCGCCATCACCCGCTC boxA mutantBoxANotI-rev ATAAGAATGCGGCCGCCGACGCATTGCGCGCGACG boxA mutant



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

were N terminally sequenced (Fig. 3). The induced proteinspots were labeled protein spots 1 to 7. The proteins of threeof the spots, protein spots 1, 3, and 5, could be sequenced, andtwo others (protein spots 2 and 4) gave no sequence, possibly

due to an N-terminal block. In addition, the N-terminal se-quences of two purified proteins, benzoate-CoA ligase andBoxA, were known (see above). All N-terminal amino acidsequences obtained, as well as the estimated sizes and isoelec-tric points of benzoate-induced proteins, are summarized inTable 3. Antibodies raised against BoxA were used to deter-mine by Western blotting whether one of the benzoate-in-duced proteins was BoxA. Cells grown on malate were used asa negative control. Malate-grown cells instead of 3-hydroxy-benzoate-grown cells were used in this experiment sincemalate-grown cells contained virtually no BoxA activity,whereas 3-hydroxybenzoate-grown cells contained approxi-mately 5% of the fully benzoate-induced enzyme activity. Thislow level was still detected by the sensitive immunoassay. Afaint protein spot next to benzoate-induced protein spot 1,which was not identical to protein spot 1, specifically reactedwith the serum. This protein spot did not appear when cellswere grown on malate. This confirmed that BoxA is anotherbenzoate-induced protein which is present at only a low con-centration.

Cloning and sequencing of the genes coding for benzoate-induced proteins. Before this study, only the genes for BoxAand BoxB had been sequenced; the order of these genes isboxBA. As determined by inference from the high number ofbenzoate-induced soluble proteins, the complexity of the path-way is much greater, and several genes must be considered stillmissing. A cosmid gene library of chromosomal DNA wasgenerated, and DNA probes derived from boxA were used forscreening. The cloning strategy is summarized in Fig. 4.

One positive clone was obtained, which contained a 37-kbinsert carrying boxBA. Upstream of boxBA the DNA sequencecontained two additional ORFs which were oriented in thesame direction as boxBA. The N-terminal sequence deducedfrom the 5� end of one of these ORFs (ORF 11) was identicalto the N-terminal sequence of induced protein 3. Further up-stream, three ORFs oriented in the opposite direction werefound, and the last of these ORFs was incomplete. The N-terminal amino acid sequence deduced from the 5� end of this

FIG. 3. Patterns after two-dimensional gel electrophoresis of solu-ble proteins of benzoate-grown cells (A) and 3-hydroxybenzoate-grown cells (B). Strongly benzoate-induced spots are indicated bynumbers. See Table 3 for properties of proteins.

TABLE 3. Properties of purified and/or benzoate-induced proteins thought to play a role in benzoate oxidationa

Purified and/or inducedprotein (PRF)

Estimatedmolecular

mass(kDa)

Estimatedisoelectric

pointN-terminal amino acid sequenceb Deduced N-terminal amino acid sequence

Benzoate-CoA ligase(ORF 7)

56 5.7 MTTLSAADHSTSPPTItLPRQYNAAD MTTLSAADHSTSPPTITLPRQYNAAD

BoxA (ORF 13) 46 5.7 MNAPAEHANLARQHLIDPE MNAPAEHANLARQHLIDPEICIRCNTProtein 1 (BoxB; ORF 12) 50 5.8 XINYSERIPN MINYSERIPNNVNLProtein 2 55 6.2 No sequenceProtein 3 (ORF 11) 60 5.6 XqAVANKPVAELvDYRtEPs MQAVANKPVAELVDY RTEPSProtein 4 57 5.7 No sequenceProtein 5 (ORF 6) 40 8.7 AEKIKVGLMLPYTGTYAALG MKNARMNRRTLMQAMLGVIAGALVPLGAAQA

AEKIKVGLMLPYTGTYAALGProtein 6

(possibly ORF 3)28 8.6 NDc

Protein 7 (possiblyORF 1 or ORF 8)

19 5.5 ND

a The experimentally determined N-terminal amino acid sequences are compared to the N-terminal amino acid sequences deduced from the presumed genes of thebenzoate oxidation pathway. X indicates an unidentified amino acid, lowercase letters indicate uncertain amino acids, and underlining indicates a leader peptide.

b The N-terminal amino acid sequences of the ORF 7 and 13 products were reported in references 1 and 29 and are included for comparison.c ND, not determined.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

incomplete ORF was identical to the N-terminal amino acidsequence of benzoate-CoA ligase. This suggested that all theseORFs are likely to be involved in benzoate metabolism. Se-quencing of the ORFs downstream of boxBA revealed twoother ORFs oriented in the same direction as boxBA, whichwere separated from each other by only short intergenic re-gions. Then 74 bp farther downstream there was a putativeORF without a ribosome-binding site, which exhibited similar-ity only to putative ORFs in the database. Hence, the whole11.8-kbp DNA sequence contained one incomplete and eightcomplete putative genes.

Further screening of a �ZAP Express gene bank led to thediscovery of a total of 15 ORFs which were found to be clus-tered and which are likely to be involved in benzoate metab-olism; 126 bp downstream of ORF 1 an incomplete ORFcoding for another putative enoyl-CoA hydratase was found.The order of these ORFs, their orientations, their sizes, andtheir secondary DNA structures are shown in Fig. 4. Table 4summarizes the properties of these ORFs.

Gene organization and induction. The orientation and or-ganization of the 15 ORFs indicate that the benzoate meta-bolic genes are organized in at least two different operons.Several DNA duplex structures appear to be present (Fig. 4);the function of these secondary structures is unknown. Thelengths of the intergenic regions vary (Table 4); the largestintergenic region is between ORFs 9 and 10 and is the pre-sumed promoter and operator region of two divergently tran-scribed operons. This region contains several direct and in-verted repeats that are more than 7 bp long. All ORFs butORF 1 contain ribosome-binding sites which are very similar tothe consensus sequence (AGGAGG).

Induction of ORFs during growth on benzoate was studiedby performing RT-PCR experiments with mRNA from benzo-ate-grown cells and comparing the results to the results ob-

tained with cells grown on malate (Fig. 5 and Table 5). Theamplified DNA fragments contained the intergenic regionsbetween adjacent genes. The data indicate that there was in-duction of all ORFs but ORF 1. Clearly, as expected, notranscript between ORFs 9 and 10 could be obtained. ORFs 2to 9 and 11 to 13 were cotranscribed and detected even inhighly (100- to 1,000-fold) diluted samples. At high dilutionsthere was no amplification of the intergenic region upstream ofORF 1 and of the regions between ORFs 1 and 2, ORFs 10 and11, ORFs 13 and 14, and ORFs 14 and 15. Basal expression ofORFs 2 to 9 and ORFs 11 to 13 could be part of a globalregulation strategy because ORFs 3 to 6 code for putativebenzoate transport and ORFs 7, 11, 12, and 13 code for theproven or putative first steps in benzoate metabolism (seebelow). A basal level of expression of these genes could help ifthere were a change in carbon source. Cells grown on malatedid not show any BoxA in immunodetection experiments, al-though RT-PCR detected boxA transcription. This may indi-cate that BoxA production is also regulated at the translationstage. In summary, these data suggest that ORFs 2 to 13function in benzoate metabolism. The putative regulator geneORF 10 may be transcribed separately. The role of ORFs 1and 15 is questionable.

Of the products of the 15 ORFs of the putative benzoatedegradation gene cluster, 5 proteins were directly identified asbeing part of this functional unit (Table 3). The products ofORFs 7 and 13 were purified as benzoate-induced enzymes orproteins, and their N-terminal amino acid sequences agreedwell with the deduced sequences. The products of ORFs 6, 11,and 12 were identified as benzoate-induced proteins by N-terminal sequencing. In addition, the product of ORF 13 wasidentified as a benzoate-induced protein by Western blottingperformed with antibodies raised against BoxA.

Characterization of the genes of benzoate metabolism and

FIG. 4. Structure of benzoate metabolism gene cluster. ORF 7 encodes benzoate-CoA ligase, ORF 12 encodes BoxA, and ORF 13 encodesBoxB. Genes are indicated by arrows. The numbers in the arrows are ORF numbers. The lengths of ORFs are indicated under the arrows. Thelines at the top indicate the positions and lengths of inserts in �ZAP and cosmid clones. The genomic DNA of A. evansii used for construction ofgene libraries was cut either by Sau3AI or EcoRI. Possible secondary structures are indicated by hairpin symbols. The numbers under these symbolsindicate the positions of secondary structures within the gene cluster. The double-ended arrows under the ORFs show the regions which wereamplified by RT-PCR (see Fig. 5 and Table 5). Note that intergenic regions between ORFs are always parts of the fragments.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

their putative functions. The putative functions of the se-quenced genes and deduced gene products are summarized inTable 4. The first gene cluster consists of the following nineORFs.

ORF 1 encodes a protein that has similarity to thioesterases,notably 4-hydroxybenzoyl-CoA thioesterase involved in theconversion of 4-chlorobenzoate to 4-hydroxybenzoate (4). Theenzyme may function in benzoate metabolism as a securityvalve, cleaving CoA thioesters (or derived dead-end products)that accumulate and trap CoA if subsequent steps (e.g., oxy-gen-dependent steps) become limiting. This may easily occurin bacteria that profit from their ability to change quickly froman oxic life style to an anoxic, denitrifying life style, which ischaracteristic of A. evansii. Otherwise, CoA trapping would belife threatening. The missing ribosome-binding site could thenbe part of a downregulation strategy for this ORF, with the aimof securing just a low level of the corresponding protein.

The ORF 2 product has similarity to lactone hydrolases. Itssecondary structure is similar to the conserved structure of/�-hydrolase fold enzymes (31), and the amino acid sequence103GHSDGG108 fits well with the consensus motif (Sm-X-Nu-X-Sm-Sm, where Sm is a small amino acid, X is any amino, andNu is a nucleophilic amino acid) for the nucleophile member(the nucleophile elbow) of the catalytic triad (34). Since thelactone of 3-hydroxyadipyl-CoA was observed to be product ofbenzoate transformation by cell extracts, this enzyme mayfunction in hydrolyzing this lactone.

The product of ORF 3 has similarity to a putative ATP-binding subunit of an ABC transporter system. It has the typ-ical Walker motifs of ATP-binding proteins (Walker A,61GRNGMGKTT69; Walker B, 179LLILDE184) (24). The ORF4 product has similarity to a membrane-spanning subunit of anABC transporter system (24). A prediction for transmembranehelices indicates that there are nine possible membrane-span-ning -helices. Furthermore, this protein has Walker consen-sus motifs for an ATP-binding site (Walker A, 358GPN-GAGKST366; Walker B, 488VLLLDE494). Therefore, thisprotein seems to be a two-domain protein. The protein en-coded by ORF 5 has similarity to a putative membrane-span-ning protein of an ABC transporter system. It seems to containsix transmembrane helices. The ORF 6 product has similarityto a putative substrate-binding protein of an ATP transportersystem. This protein corresponds to benzoate-induced protein5 (Table 3). It contains a 31-amino-acid leader peptide. Thesequence of this leader peptide is typical of the sequencesfound in the Sec transport system (37). It contains an N do-main with a net positive charge (amino acids 1 to 10) and ahydrophobic H domain (amino acids 11 to 24). In summary,the products of ORFs 3 to 6 are likely to represent the fourcomponents of an ATP transporter system responsible for thehighly efficient uptake of benzoate.

ORF 7 codes for the aerobically induced benzoate-CoAligase, which differs from the corresponding isoenzyme in-duced during anoxic growth on aromatic substrates. The de-duced N-terminal amino acid sequence is identical to the 20-amino-acid N-terminal sequence determined for the purifiedenzyme. The purified enzyme is a homodimer which has anative molecular mass of 130 kDa and subunits with molecularmasses of approximately 56 kDa (deduced molecular mass, 58

kDa). The enzyme acts on benzoate but not on 3-hydroxyben-zoate (1).

The protein encoded by ORF 8 shows low similarity to theenzyme 4-hydroxylaminobenzoate lyase, which catalyzes anodd reaction, the hydrolytic transformation of 4-hydroxylami-nobenzoate to protocatechuate (3,4-dihydroxybenzoate) andammonia during 4-nitrobenzoate degradation in Pseudomonasputida TW3 (25). There are similar entries in the database (18)which show only very low similarity to other gene products,indicating that these proteins may form a new family. The roleof the ORF 8 product in benzoate metabolism is enigmatic. Inany case, the substrate of this enzyme seems to be a benzoatederivative in both types of metabolism (i.e., 4-nitrobenzoatedegradation and benzoate degradation).

The ORF 9 product has similarity to proteins which areassumed to play a role in aerobic phenylacetate metabolism inE. coli (PaaZ) (14, 15), P. putida (PhaL) (33), A. evansii (PaaZ)(30), and presumably other bacteria (30). Phenylacetate me-tabolism also proceeds via CoA thioesters, and the products ofthe ORFs related to ORF 9 are thought to be involved inhydrolytic or acyloin (or aldol) cleavage of the ring and/or inaldehyde oxidation. The hypothetical substrate for this kind ofring cleavage is a nonaromatic product (a cis-dihydrodiol)formed by a dioxygenase/reductase acting on phenylacetyl-CoA and benzoyl-CoA. Ring cleavage may be preceded byisomerization of the double bonds (see below). The proteinencoded by ORF 9 may be involved in further oxidation of theintermediate formed after COC bond cleavage (see below).The N-terminal amino acid sequence of the ORF 9 product(amino acids 10 to 434) shows similarity to the sequences ofaldehyde dehydrogenases. The conserved domains254IEADSVN260 and 291GQKCTAIR298 probably contain theglutamate-268 and cysteine-302 homologues (underlined),which in mammal aldehyde dehydrogenases are involved in theactive center (14).

The second gene cluster, which is oriented in a differentdirection, consists of six genes with the following properties.The product of ORF 10 seems to represent a two-domainprotein. A similar ORF was found in a gene cluster of the�-proteobacterium Thauera aromatica, which contains genesfor BoxA, BoxB, and benzoate-CoA ligase (K. Schuhle and G.Fuchs, unpublished data). The N-terminal domain (amino ac-ids 32 to 87) has similarity to various regulatory proteins of theHTH family and seems to have the typical primary and sec-ondary structures of helix-turn-helix proteins (7, 47). The firstputative helix is between amino acids 44 and 51; it is separatedfrom the second helix by a typical turn sequence (52GVS54).The second helix is between amino acids 55 and 62. The C-terminal domain (amino acids 137 to 285) has similarity toshikimate kinase I of E. coli. Shikimate kinase I does notfunction in shikimate phosphorylation and in the biosynthesisof aromatic amino acids, although it phosphorylates shikimatein vitro, but with very low affinity for its substrate (Km, 20 mM).The actual shikimate kinase II has a 100-fold-lower Km (13).The ORF 10 product may play a role as an unprecedentedregulator protein that becomes activated or inactivated byATP-dependent phosphorylation in response to benzoate.

The protein encoded by ORF 11 corresponds to benzoate-induced protein spot 3. It has a mosaic structure. An N-termi-nal domain (amino acids 47 to 162) and a C-terminal domain



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

TA

BL

E4.

Prop

ertie

sof

gene

san

dge

nepr

oduc

tsas

sum

edor

prov

ento

bein

volv

edin

benz

oate

oxid

atio

na

Gen

e

G�

Cco

nten

t(m

ol%

)

Len

gth

ofin

terg

enic

regi

onto

next

OR

F(b

p)

Puta

tive

func

tion

ofge

nepr

oduc

t

Mol

ecul

arm

ass

(kD

a)

Isoe

lect

ric

poin

t(p

H)

Puta

tive

cellu

lar

loca

lizat

ion

Sim

ilar

prot

eins

inda

taba

ses

%Id

entit

y%

Sim

ilari

tyE

valu

eA

cces

sion

no.

OR

F1

65.1

6T

hioe

ster

ase

165.

72C

ytos

olic

Puta

tive

4-hy

drox

yben

zoyl

-C

oAth

ioes

tera

se(R

alst

onia

sola

nace

arum

)

4160

7e-2

0N

C_0

0329

6

4-H

ydro

xy-b

enzo

yl-C

oAth

ioes

tera

se(P

seud

omon

assp

.str

ain

Cbs

-3)

4377

7.7

P566

53

OR

F2

68.5

1L

acto

nase

285.

22C

ytos

olic

Puta

tive

hydr

olas

e-re

late

dpr

otei

n(R

alst

onia

sola

nace

arum

)

4659

6e-4

8N

C_0

0329

5

Hyd

rola

se-r

elat

edpr

otei

n(D

eino

cocc

usra

diod

uran

s)

4260

2e-4

1N

C_0

0126

3

OR

F3

66.9

2A

BC

tran

spor

ter,

AT

Pbi

ndin

g28

9.56

Cyt

osol

icPu

tativ

eA

BC

tran

spor

ter

subu

nit

Hba

H(R

hodo

pseu

dom

onas

palu

stris

)

5268

1e-6

0A

AC

1336

3

Prob

able

AB

Ctr

ansp

orte

rsu

buni

t(P

seud

omon

assp

.st

rain

CA

10)

4358

3e-4

5B

AB

3246

2

OR

F4

67.2

17A

BC

tran

spor

ter,

AT

P-bi

ndin

gm

embr

ane-

span

ning

prot

ein

626.

88M

embr

ane

Puta

tive

AB

Ctr

ansp

orte

rsu

buni

tH

baG

(Rho

dops

eudo

mon

aspa

lust

ris)

3954

1e-1

04A

AC

1336

4

Bra

nche

d-ch

ain

amin

oac

idA

BC

tran

spor

ter,

AT

P-bi

ndin

gpr

otei

n(D

eino

cocc

usra

diod

uran

s)

2741

2e-4

3N

P_28

5584

OR

F5

65.1

77A

BC

tran

spor

ter,

mem

bran

e-sp

anni

ngpr

otei

n31

6.18

Mem

bran

ePu

tativ

eA

BC

tran

spor

ter

subu

nit

Hba

F(R

hodo

pseu

dom

onas

palu

stris

)

4559

1e-5

3A

AC

1336

5

Puta

tive

mem

bran

e-sp

anni

ngpr

otei

n(P

seud

omon

asae

rugi

nosa

)

3751

1e-2

9A

AC

6948

7

OR

F6

65.2

140

AB

Ctr

ansp

orte

r,su

bstr

ate-

bind

ing

prot

ein

41(w

ithle

ader

pept

ide)

,39 (w

ithou

tle

ader

pept

ide)

9.03 (w

ithle

ader

pept

ide)

,8.

52(w

ithou

tle

ader

pept

ide)

Peri

plas

mPu

tativ

esu

bstr

ate-

bind

ing

prot

ein

(Azo

arcu

sev

ansi

i)80

861e

-171

AA

L02

070

Puta

tive

AB

Ctr

ansp

orte

rsu

buni

t(A

zoar

cus

evan

sii)

7181

1e-1

50C

AD

2164

1

OR

F7

66.6

218

Ben

zoat

e-C

oAlig

ase

585.

61C

ytos

olic

Ben

zoat

e-C

oAlig

ase

(Tha

uera

arom

atic

a)76

84�

1e-1

70C

AD

2168

3



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Ben

zoat

e-C

oAlig

ase

(Azo

arcu

sev

ansi

i)69

79�

le-1

70C

AD

2164

0

OR

F8

66.0

98U

nkno

wn

174.

97C

ytos

olic

p-H

ydro

xyla

min

oben

zoat

ely

ase

(Pse

udom

onas

sp.

stra

inY

H10

2)

3144

1e-1

2A

AF

0144

8

PnbB

(Pse

udom

onas

putid

a)30

431e

-11

AA

G01

543

OR

F9

70.4

327

Ald

ehyd

ede

hydr

ogen

ase

548.

62C

ytos

olic

Mao

C-li

kepr

otei

n(p

heny

lace

ticac

idde

grad

atio

npr

otei

nPa

aZ)

(Esc

heric

hia

coli)

4159

2e-9

7P7

7455

PhaL

(Pse

udom

onas

putid

a)42

521e

-95

AA

C24

340

OR

F10

66.5

40U

nkno

wn

regu

lato

rytw

o-do

mai

npr

otei

n34

6.37

Cyt

osol

icH

elix

-tur

n-he

lixpr

otei

n(P

yrob

acul

umae

roph

ilum

)

3761

3e-4

A65

134

Shik

imat

eki

nase

I(E

C2.

7.1.

71)

(Esc

heric

hia

coli)

3152

3e-1

2N

P_56

0573

OR

F11

68.1

159

Eno

yl-C

oA-h

ydra

tase

/isom

eras

ean

dpo

ssib

leri

ng-c

leav

ing

enzy

me

615.

44C

ytos

olic

Eno

yl-C

oAhy

drat

ase

(Fad

-1)

(Arc

haeo

glob

usfu

lgid

us)

3448

9e-6

NP_

0692

71

Eno

yl-C

oAhy

drat

ase

(Bac

illus

halo

dura

ns)

3648

2e-5

NP_

2420

01

OR

F12

64.3

163

Ben

zoyl

-CoA

oxyg

enas

eco

mpo

nent

B55

5.62

Cyt

osol

icR

ing

oxid

atio

nco

mpl

ex,

phen

ylac

etic

acid

degr

adat

ion-

rela

ted

prot

ein

(Sul

folo

bus

solfa

taric

us)

3047

4e-1

4N

P_34

2758

OR

F13

66.9

245

Ben

zoyl

-CoA

oxyg

enas

eco

mpo

nent

A46

5.59

Cyt

osol

icF

erre

doxi

n-N

AD

P�

redu

ctas

e(E

C1.

18.1

.2)

prec

urso

r(C

yano

phor

apa

rado

xa)

4256

8e-5

4A

5666

4

Fer

redo

xin-

NA

DP�

oxid

ored

ucta

se(S

ynec

hoco

ccus

elon

gatu

s)

3853

6e-4

8B

AB

6106

0

OR

F14

71.0

179

�-K

etoa

dipy

l-CoA

thio

lase

425.

68C

ytos

olic

Bet

a-ke

toad

ipyl

-CoA

thio

lase

(Bur

khol

deria

pseu

dom

alle

i)

7585

1e-1

57A

AG

1215

9

Bet

a-ke

toad

ipyl

-CoA

thio

lase

(Aci

neto

bact

erca

lcoa

cetic

us)

6882

1e-1

54Q

4397

4

OR

F15

67.2

Unk

now

n21

9.51

Cyt

osol

icH

ypot

hetic

alpr

otei

n(T

haue

raar

omat

ica)

4652

4e-4

0Sc

huhl

eran

dF

uchs

,un

publ

ishe

dH

ypot

hetic

alpr

otei

n(V

ibrio

chol

erae

)35

420.

016

NP_

2319

88

aSi

mila

rity

sear

ches

wer

edo

new

ithth

eth

epr

ogra

mbl

astp

(htt

p://w

ww

.ncb

i.nlm

.nih

.gov

/BL

AST

/).

The

perc

enta

geof

iden

tity

was

defin

edas

the

perc

enta

geof

amin

oac

ids

that

are

iden

tical

intw

opr

otei

ns.

The

perc

enta

geof

sim

ilari

tyw

asde

fined

asth

epe

rcen

tage

ofam

ino

acid

sth

atar

eid

entic

alor

cons

erve

din

two

prot

eins

.The

Eva

lue

isan

estim

ate

ofth

est

atis

tical

sign

ifica

nce

ofth

em

atch

,whi

chsp

ecifi

esth

enu

mbe

rof

mat

ches

with

agi

ven

scor

eth

atis

expe

cted

ina

sear

chof

ada

taba

seof

this

size

abso

lute

lyby

chan

ce.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

(amino acids 387 to 471) together form a 199-amino-acid do-main found in the enoyl-CoA hydratase/isomerase (crotonase)protein family. Members of this enzyme family catalyze a va-riety of different reactions, including enoyl-CoA hydration,enoyl-CoA isomerization, and COC bond cleavage (20), all

based on abstraction or addition of the alpha-proton of thecarboxylic acid. The majority of the rest of the ORF 11 productshows no similarity to other proteins in the databases. This mayindicate that the protein encoded by ORF 11 catalyzes a com-plex reaction in which enoyl-CoA hydration and/or isomeriza-tion or even COC bond cleavage is involved. The ORF 11product seems to be a novel type of enzyme whose catalyzedreaction will be interesting to understand.

The product of ORF 12 is identical to BoxB and correspondsto benzoate-induced protein spot 1. This protein has similari-ties to a component of a putative multicomponent phenylac-etyl-CoA oxygenase in E. coli (PaaA) (15) and P. putida (PhaF)(33). The phenylacetyl-CoA oxygenase is thought to consist offour subunits (PaaABCD) and to require a one-subunitNAD(P)H oxidoreductase component (PaaE). Similar ORFsare found in phenylacetate metabolism gene clusters of otherbacteria, including A. evansii (30) and S. solfataricus (44). Theprimary structure of BoxB shows the two repeats of residuesEX2H separated by 86 amino acids (150EGRH153 and239EEAH242) that characterize the dinuclear iron-binding siteof the large oxygenase subunit of methane, phenol, and tolu-ene diiron monooxygenase (16, 36). We postulate that BoxBfunctions as the oxygenase part of benzoyl-CoA oxygenase inconjunction with BoxA, the reductase component.

The ORF 13 product is identical to the benzoate-inducedenzyme BoxA. A similar ORF was found in the related organ-ism T. aromatica next to the gene for BoxB (Schuhle andFuchs, unpublished). The similarity of BoxA to ferredoxin-NADP� oxidoreductases and the benzoyl-CoA-dependent ox-idation of NADPH catalyzed by the enzyme suggest that BoxAfunctions as the reducing component of benzoyl-CoA oxygen-ase, which, upon binding of benzoyl-CoA, transfers two elec-trons to the ring in the course of dioxygenation.

The protein encoded by ORF 14 has strong similarity to3-ketoadipyl-CoA thiolase. This is probably also the functionof the protein in benzoate metabolism, which is assumed tolead to 3-ketoadipyl-CoA. This implies that acetyl-CoA, succi-nyl-CoA, and CO2 are the products of the benzoate oxidationpathway.

FIG. 5. Study of the organization and induction of genes of thebenzoate degradation gene cluster performed with RT-PCR and aga-rose gel electrophoresis. cDNA from A. evansii cells grown on benzo-ate was compared with cDNA from cells grown on malate. StandardPCRs were carried out with these cDNAs and with genomic DNA ofA. evansii as a positive control. The cDNA templates used in thesereactions were not diluted and were diluted 10-, 100-, and 1,000-fold.The numbers of the amplified fragments are indicated by numbers incircles. The positions of these fragments are indicated in Fig. 4. Theagarose gel shows the results of the experiments with fragments 5 to 10(experiments with the first cluster are not shown). Lanes 1 and 20contained a 1-kb ladder; lanes 2, 5, 8, 11, 14, and 17 contained cDNAfrom benzoate-grown cells; lanes 3, 6, 9, 12, 15, and 18 containedcDNA from malate-grown cells; and lanes 4, 7, 10, 13, 16, and 19contained genomic DNA (positive control). Lanes 2, 3, 5, 6, 14, 15,undiluted cDNA; lanes 17 and 18, cDNA diluted 10-fold; lanes 8, 9, 11,and 12, cDNA diluted 100-fold.

TABLE 5. Induction of genes of the benzoate degradation gene cluster, as determined by using cDNA from cells grown on benzoate andfrom cells grown on malate

Amplifiedfragmenta

Fragmentlength (bp)

Templates

Undiluted cDNA cDNA diluted 10-fold cDNA diluted 100-fold cDNA diluted 1,000-fold

Benzoate-growncells

Malate-growncells

Benzoate-growncells

Malate-growncells

Benzoate-growncells

Malate-growncells

Benzoate-growncells

Malate-growncells

0 500 �b � � � � � � �1 402 � � � � � � � �2 801 � � � � � � � �3 1,008 � � � � � � � �4 862 � � � � � � � �5 618 � � � � � � � �6 500 � � � � � � � �7 593 � � � � � � � �8 721 � � � � � � � �9 836 � � � � � � � �

10 259 � � � � � � � �

a See Fig. 5.b �, DNA fragment detected; �, no fragment detected.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

ORF 15 is the last ORF of the cluster. Its product has nosimilarity to proteins in the database. Interestingly, a similarORF was found in the gene cluster of T. aromatica mentionedabove, which may code for benzoate oxidation in this relatedbacterium. The occurrence in similar gene clusters is an argu-ment for a role for the ORF 15 product in the benzoatepathway, although no function can be ascribed to this proteinyet.

Genes encoding a homologue of �-hydroxyacyl-CoA dehy-drogenase and possibly another enoyl-CoA hydratase/isomer-ase are missing in the gene cluster sequenced. A �-oxidationdehydrogenase is needed to catalyze dehydrogenation of thedetected intermediate 3-hydroxyadipyl-CoA to 3-oxoadipyl-CoA. Possibly the benzoyl-CoA pathway enzyme(s) is supple-mented through the homologous protein(s) of primary metab-olism.

Two-dimensional gel electrophoresis revealed at least sevenbenzoate-induced soluble proteins, and Western blotting re-vealed another protein. Of the proteins encoded by the 15ORFs, 2 are likely to be membrane-bound components of anABC transport system and therefore are not soluble; only smallamounts of the regulator protein and the thioesterase are likelyto be present. Hence, one would expect there to be 11 solublebenzoate-induced proteins. Thus, the complex protein induc-tion pattern corresponds to the complex set of benzoate met-abolic genes.

BoxA� mutant and expression of BoxA under different

growth conditions. A boxA mutant was constructed by homo-logous recombination between the wild-type chromosome andan insertionally inactivated version of the gene carried on plas-mid pJQ200MK. The boxA mutant failed to grow on benzoateas a sole carbon source. This shows that BoxA is essential forgrowth on benzoate.

To find a growth-supporting substrate (for further mutantanalysis and biochemical studies of the pathway) which allowedexpression of the benzoate pathway, regulation of boxA expres-sion and therefore induction of the benzoate genes were testedwith different substrates. Wild-type cells were grown on differ-ent substrates and combinations of substrates along with ben-zoate as an inducer for the benzoate genes. Benzoate geneexpression was assessed by spectrophotometric measurementof BoxA activity (i.e., NADPH oxidation in the presence ofbenzoyl-CoA, FAD, and O2 under H2O2 production condi-tions). The substrates used, alone and in combination, includedbenzoate, 3-hydroxybenzoate, phenylacetate, adipate, malate,and acetate. No BoxA activity was observed with extracts ofcells grown on acetate, malate, and adipate, while some activitywas detected with cells grown on phenylacetate (10%) and3-hydroxybenzoate (5%). Aromatic compounds resembling thearomatic substrate benzoate, like phenylacetate and 3-hydroxy-benzoate, may act as poor gratuitous inducers. A combinationof benzoate and 3-hydroxybenzoate resulted in 12% BoxAactivity, a combination of benzoate and phenylactetate resultedin 50% BoxA activity, and a combination of benzoate and

FIG. 6. Proposed benzoate pathway and putative function of gene products. Experimentally documented compounds are enclosed in boxes. Thenumbers in boxes indicate the ORFs that encode proteins which might catalyze the individual steps.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

adipate resulted in 82% BoxA activity. No activity was ob-served with benzoate and acetate. This suggests that acetateacts as a strong catabolite repressor, whereas adipate does not.Also, 3-hydroxybenzoate and phenylacetate seemed to repressactivity.

Proposal for the benzoate pathway and putative role ofORFs. We propose a benzoate pathway (Fig. 6) which is in-duced by benzoate and may be regulated by the product ofORF 10, a two-domain protein. An ABC transporter systemconsisting of the products of ORFs 3 to 6 may be responsiblefor effective benzoate uptake. The ORF 7 product activatesbenzoate to benzoyl-CoA. A protein mixture from A. evansiitransformed benzoyl-CoA in an NADPH- and oxygen-depen-dent reaction into 6-hydroxy-3-hexenoyl-CoA. The proteinsencoded by ORFs 9, 11, 12, and 13 may be involved in thiscomplex reaction. Benzoyl-CoA is attacked by a putative 2,3-dioxygenase to obtain the cis-dihydrodiol product (unpub-lished results); BoxA (encoded by ORF 13) is thought to de-liver electrons from NADPH to BoxB (encoded by ORF 12),which interacts with substrate and oxygen. BoxA has errone-ously been reported to be benzoyl-CoA 3-monooxygenase (32),but a detailed study of the purified protein revealed that FAD-dependent oxidation of NADPH occurs in the presence ofbenzoyl-CoA without hydroxylase activity (29). The ORF 1product may act as a thioesterase. This protein may be re-quired to release CoA from the intermediates of the pathwayif enzymes downstream of benzoyl-CoA become limiting,which would lead to trapping of CoA.

The next steps are different from what one would expectfrom conventional pathways involving a non-CoA-activatedfree cis-dihydrodiol intermediate. In conventional pathways,the cis-diol undergoes oxidation and rearomatization to a di-hydroxy aromatic product (Fig. 1). Yet a putative cis-diol de-hydrogenase gene could not be found in the gene cluster. Wepropose that the CoA thioester grouping of the activated diolallows isomerization of the conjugated double-bond system ofthe cis-diol, leading to an enol which tautomerizes to the morestable unsaturated cyclic 3-ketoacyl-CoA. The postulated in-termediate formed contains an -hydroxycarbonyl (acyloin)group. Two types of hydrolytic COC cleavage reactions can beenvisaged (Fig. 6), followed by decarboxylation. These reac-tions may be catalyzed by the ORF 11 product, a complexprotein which contains an enoyl-CoA hydratase/isomerase do-main.

Oxidation of the alcohol group of 6-hydroxy-3-hexenoyl-CoA to the carboxyl group results in cis-3,4-dehydroadipyl-CoA, which was also detected. The protein encoded by ORF 9may be involved in this four-electron oxidation. The order ofevents involved in transformation of cis-3,4-dehydroadipyl-CoA, possibly via trans-2,3-dehydroadipyl-CoA and/or the 3,6-lactone of 3-hydroxyadipyl-CoA, to 3-hydroxyadipyl-CoA can-not be determined on theoretical grounds; all theseintermediates were observed, but the order of formation couldnot be inferred. The lactone may form spontaneously and/or bea preparation artifact. The product of ORF 2 may be involvedin lactone hydrolysis. In any case, it seems logical that 3-hy-

FIG. 7. Gene cluster for aerobic benzoate metabolism in A. evansii and similar genes or gene clusters in other organisms. Corresponding ORFsare indicated by the same color. Putative functions: dark blue, benzoate-CoA ligase; medium blue, regulatory protein; green, enoyl-CoAhydratase/isomerase; light blue, BoxB; yellow, BoxA; white, aldehyde dehydrogenase; red, lactonase; green stripes, PnbB-type protein; grey stripes,thioesterase; black, ORFs which show no similarity to ORFs 1 to 15 of A. evansii; grey, ORFs which are present only in the A. evansii gene clusterso far. The numbers in the arrows indicate the ORF numbers in the A. evansii gene cluster and the deduced numbers of amino acids in the geneproducts. The percentages under the ORFs indicate the percentages of similar amino acids in an ORF product and the corresponding ORFproduct in A. evansii. An asterisk indicates that an ORF could not be fully translated into an amino acid sequence, either because of possibleframeshifts in sequence or because of unsequenced regions.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

droxyadipyl-CoA is oxidized to 3-ketoadipyl-CoA, which isthiolytically cleaved by the ORF 14 product into succinyl-CoAand acetyl-CoA. Hence, the pathway converges with the clas-sical �-ketoadipate pathway of benzoate oxidation at the lastintermediate, 3-ketoadipyl-CoA. So far, the roles of the pro-teins encoded by ORF 8 and ORF 15 are completely unknown.

Similar genes in other bacteria. Genes similar to those de-scribed here are found in very recent database entries forproteobacterial genomes, but their roles are unknown (Fig. 7).The bacteria examined include Burkholderia fungorum LB400,which contains two very similar sets of genes, Ralstonia metal-lidurans CH34, Magnetospirillum magnetotacticum MS-1, andRhodopseudomonas palustris CGA009. Similar genes have alsobeen found in T. aromatica K172 (Schuhle and Fuchs, unpub-lished).

Interestingly, in most cases homologs of ORFs 11 to 13occur in the same order as in A. evansii. In T. aromatica se-

quencing is incomplete, and the ORF 11 homologue is ex-pected to be upstream of the ORF 12 homologue. In R. palus-tris, ORF 13 is still missing. We postulate that the products ofORFs 11 to 13 in these cases represent the core enzymes thatact on benzoyl-CoA and its first product, the 2,3-dihydrodiol.In most cases the regulator protein (encoded by ORF 10) andthe putative lactone hydrolase (encoded by ORF 2) are alsopresent. The ABC transporter (encoded by ORFs 3 to 6) isgenerally missing. The putative thioesterase (encoded by ORF1) could be found only in M. magnetotacticum. The product ofORF 15, whose function is not known, could be found only inT. aromatica. This may suggest that ORFs 1, 3 to 6, and 15 arenot crucial for the new benzoate pathway. The thiolase (en-coded by ORF 14) is missing in all other organisms. Thefunction of this enzyme may be performed by common �-ke-tothiolases; this argument also holds true for 3-hydroxyacyl-CoA dehydrogenase.

FIG. 8. Working hypothesis showing the proposed initial steps in the pathways of metabolism of 2-aminobenzoate, benzoate, and phenylacetatein A. evansii. The reactions involved in further metabolism of the CoA-activated aromatic acids remain to be identified. The compounds in bracketsare assumed to be enzyme bound or rapidly transformed to more stable nonaromatic intermediates. The COC bonds which are thought to becleaved nonoxygenolytically are indicated. The dead-end products of rearomatization reactions are indicated in side reactions. These dead-endproducts are formed if the intermediates (in brackets) accumulate in the cell.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

The R. palustris strain which has been sequenced may growaerobically on benzoate, although other strains reportedly donot do this (22). In this case the benzoate-CoA ligase of theanaerobic benzoate degradation cluster could function in bothanaerobic and aerobic metabolism of benzoate, although therewould be two completely different strategies for dearomatiza-tion under the two conditions. There are indications that thebenzoate-CoA ligase (encoded by an ORF 7 homologue) in T.aromatica not only is involved in benzoate transformation tobenzoyl-CoA under aerobic conditions but also is involved inactivation of benzoate and 2-aminobenzoate under anoxic,denitrifying conditions (Schuhle and Fuchs, unpublished).

Comparison with other aromatic degradation pathways thatproceed via CoA thioesters and working hypothesis for futureexperiments. In A. evansii, aerobic metabolism of 2-aminoben-zoate, benzoate, and phenylacetate proceeds via the CoA es-ters of the individual substrates. While the pathway for 2-ami-nobenzoate has been studied so far only in A. evansii (8, 21, 27,28, 43, 50), the new benzoate pathway has also been found inthe related organism T. aromatica (unpublished results) and ina gram-positive thermophilic B. stearothermophilus-like strain(48). The phenylacetate pathway is widely distributed (re-viewed in reference 30). The three metabolic pathways seem touse a novel principle. Our working hypothesis is schematicallypresented in Fig. 8. The assumptions are as follows. (i) Thepathways start with activation of the substrates to CoA thio-esters, and the intermediates are further processed in thisform. (ii) The aryl-CoA thioesters are attacked by dioxygen-ases and reductases (benzoate, phenylacetate) to obtain thecorresponding cis-dihydrodiol, which is not rearomatized byoxidation but is isomerized to a dihydroxylated nonaromaticproduct with an acyloin structure. In the case of anthranilate,a monooxygenase/reductase forms a monohydroxylated non-aromatic product. The CoA thioester allows reactions thatwould not be feasible in the case of nonactivated aromaticacids and their intermediates. (iii) The early reactive interme-diates are labile and may rearomatize spontaneously, in thecase of 2-aminobenzoate to 5-hydroxy-2-aminobenzoyl-CoA(by isomerization), in the case of benzoyl-CoA to 3-hydroxy-benzoyl-CoA (by water elimination), and in the case of phe-nylacetyl-CoA to 2-hydroxyphenylacetyl-CoA (by water elimi-nation). Thioesterases cleave the thioesters of these dead-endproducts, and the corresponding acids (5-hydroxy-2-aminoben-zoate, 3-hydroxybenzoate, 2-hydroxyphenylacetate) appear inthe medium; they cannot be metabolized further by the in-duced pathways. (iv) The ring is opened nonoxygenolytically,and the resulting noncyclic product is further oxidized via �-ox-idation to presumably �-ketoadipyl-CoA. Thiolytic cleavageyields succinyl-CoA and acetyl-CoA.

ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsgemeinschaft,Bonn, Germany, and by the Fonds der chemischen Industrie, Frank-furt/Main, Germany.

We thank Caroline S. Harwood (University of Iowa) for the kind giftof the suicide vector and J. Alt-Morbe (Labor fur DNA-Analytik,Freiburg, Germany) for DNA sequencing. We especially thank J. Hei-der (Freiburg, Germany) for helpful suggestions and critical com-ments.

REFERENCES

1. Altenschmidt, U., B. Oswald, E. Steiner, H. Herrmann, and G. Fuchs. 1993.New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hy-droxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp. J. Bacteriol.175:4851–4858.

2. Anders, J. H., A. Kaetzke, P. Kampfer, W. Ludwig, and G. Fuchs. 1995.Taxonomic position of aromatic-degrading denitrifying pseudomonad strainsK172 and KB740 and their description as new members of genera Thauera,as Thauera aromatica sp. nov., and Azoarcus, as Azoarcus evansii sp. nov.,respectively, members of the beta subclass of Proteobacteria. Int. J. Syst.Bacteriol. 45:327–333.

3. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.Smith, and K. Struhl. 1987. Current protocols in molecular biology. JohnWiley and Sons, New York, N.Y.

4. Benning, M. M., G. Wesenberg, R. Liu, K. L. Taylor, D. Dunaway-Mariano,and H. M. Holden. 1998. The three-dimensional structure of 4-hydroxyben-zoyl-CoA thioesterase from Pseudomonas sp. strain CBS-3. J. Biol. Chem.273:33572–33579.

5. Birnboim, H., and J. Doly. 1979. A rapid alkaline extraction procedure forscreening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523.

6. Braun, K., and D. T. Gibson. 1984. Anaerobic degradation of 2-aminoben-zoate (anthranilic acid) by denitrifying bacteria. Appl. Environ. Microbiol.48:102–107.

7. Brennan, R. G., and B. W. Matthews. 1989. The helix-turn-helix DNA bind-ing motif. J. Biol. Chem. 264:1903–1906.

8. Buder, R., K. Ziegler, G. Fuchs, B. Langkau, and S. Ghisla. 1989. 2-Ami-nobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme.Studies on the stoichiometry and the course of reaction. Eur. J. Biochem.185:637–643.

9. Buswell, J. A., and J. S. Clark. 1976. Oxidation of aromatic acids by afacultative thermophilic Bacillus sp. J. Gen. Microbiol. 96:209–213.

10. Clark, J. S., and J. A. Buswell. 1979. Catabolism of gentisic acid by twostrains of Bacillus stearothermophilus. J. Gen. Microbiol. 112:191–195.

11. Coligan, J. E., B. M. Dunn, H. L. Ploegh, D. W. Speicher, and P. T. Wing-field. 1995. Current protocols in protein science. John Wiley & Sons, Inc.,New York, N.Y.

12. Crawford, R. L. 1976. Pathways of 4-hydroxybenzoate degradation amongspecies of Bacillus. J. Bacteriol. 127:204–210.

13. DeFeyter, R. C., and J. Pittard. 1986. Genetic and molecular analysis ofaroL, the gene for shikimate kinase II in Escherichia coli K-12. J. Bacteriol.165:226–232.

14. Ferrandez, A., M. A. Prieto, J. L. Garcia, and E. Diaz. 1997. Molecularcharacterization of PadA, a phenylacetaldehyde dehydrogenase from Esch-erichia coli, FEBS Lett. 406:23–27.

15. Ferrandez, A., B. Minambres, B. Garcia, E. R. Olivera, J. M. Luengo, J. L.Garcia, and E. Diaz. 1998. Catabolism of phenylacetic acid in Escherichiacoli. Characterization of a new aerobic hybrid pathway. J. Biol. Chem. 273:25974–25986.

16. Fox, B. G., J. Shanklin, J. Ai, T. M. Loehr, and J. Sanders-Loehr. 1994.Resonance Raman evidence for an Fe-O-Fe center in stearoyl-ACP desatu-rase. Primary sequence identity with other diiron-oxo proteins. Biochemistry.33:12776–12786.

17. Gorg, A., W. Postel, and S. Gunther. 1988. The current state of two-dimen-sional electrophoresis with immobilized pH gradients. Electrophoresis9:531–546.

18. Groenewegen, P. E., P. Breeuwer, J. M. van Helvoort, A. A. Langenhoff, F. P.de Vries, and J. A. de Bont. 1992. Novel degradative pathway of 4-nitroben-zoate in Comamonas acidovorans NBA-10. J. Gen. Microbiol. 138:1599–1605.

19. Gross, G. G., and M. H. Zenk. 1966. Darstellung und Eigenschaften vonCoenzym A-Thioestern substituierter Zimtsauren. Z. Naturforsch. Teil B21:683–690.

20. Haller, T., T. Buckel, J. Retey, and J. A. Gerlt. 2000. Discovering newenzymes and metabolic pathways: conversion of succinate to propionate byEscherichia coli. Biochemistry 39:4622–4629.

21. Hartmann, S., C. Hultschig, W. Eisenreich, A. Bacher, and S. Ghisla. 1999.NIH shift in flavin-dependent monooxygenation: mechanistic studies with2-aminobenzoyl-CoA monooxygenase/reductase. Proc. Natl. Acad. Sci. USA96:7831–7836.

22. Harwood, C. S., and J. Gibson. 1988. Anaerobic and aerobic metabolism ofdiverse aromatic compounds by the photosynthetic bacterium Rhodopseudo-monas palustris. Appl. Environ. Microbiol. 54:712–717.

23. Harwood, C. S., and R. E. Parales. 1996. The beta-ketoadipate pathway andthe biology of self-identity. Annu. Rev. Microbiol. 50:553–590.

24. Higgins, C. F. 1992. ABC transporters: from microorganisms to man. Annu.Rev. Cell Biol. 8:67–113.

25. Hughes, M. A., and P. A. Williams. 2001. Cloning and characterization of thepnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomo-nas putida TW3. J. Bacteriol. 183:1225–1232.

26. Kiemer, P., B. Tshisuaka, S. Fetzner, and F. Lingens. 1996. Degradation ofbenzoate via benzoyl-coenzyme A and gentisate by Bacillus stearothermophi-



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

lus PK1, and purification of gentisate 1,2-dioxygenase. Biol. Fertil. Soils23:307–313.

27. Langkau, B., S. Ghisla, R. Buder, K. Ziegler, and G. Fuchs. 1990. 2-Ami-nobenzoyl-CoA monoxygenase/reductase, a novel type of flavoenzyme. Iden-tification of the products. Eur. J. Biochem. 191:365–371.

28. Langkau, B., P. Vock, V. Massey, G. Fuchs, and S. Ghisla. 1995. 2-Amino-benzoyl-CoA monooxygenase/reductase. Evidence for two distinct loci cat-alyzing substrate monooxygenation and hydrogenation. Eur. J. Biochem.230:676–685.

29. Mohamed, M. E., C. Ebenau-Jehle, A. Zaar, and G. Fuchs. 2001. Reinves-tigation of a new type of aerobic benzoate metabolism in the proteobacte-rium Azoarcus evansii. J. Bacteriol. 183:1899–1908.

30. Mohamed, M. E., W. Ismail, J. Heider, and G. Fuchs. 2002. Aerobic metab-olism of phenylacetic acids in Azoarcus evansii. Arch. Microbiol. 178:180–192.

31. Nardini, M., and B. W. Dijkstra. 1999. Alpha/beta hydrolase fold enzymes:the family keeps growing. Curr. Opin. Struct. Biol. 9:732–737.

32. Niemetz, R., U. Altenschmidt, S. Brucker, and G. Fuchs. 1995. Benzoyl-coenzyme A 3-monooxygenase, a flavin-dependent hydroxylase. Purification,some properties and its role in aerobic benzoate oxidation via gentisate in adenitrifying bacterium. Eur. J. Biochem. 227:161–168.

33. Olivera, E. R., B. Minambres, B. Garcia, C. Muniz, M. A. Moreno, A.Ferrandez, E. Diaz, J. L. Garcia, and J. M. Luengo. 1998. Molecular char-acterization of the phenylacetic catabolic pathway in Pseudomonas putida U:the phenylacetyl-CoA catabolon. Proc. Natl. Acad. Sci. USA 95:6419–6424.

34. Ollis, D. L., E. Cheah, M. Cygler, B. Dijkstra, F. Frolow, S. M. Franken, M.Harel, S. J. Remington, I. Silman, and J. Schrag. 1992. The alpha/betahydrolase fold. Protein Eng. 5:197–211.

35. Parales, R. E., and C. S. Harwood. 1993. Construction and use of a newbroad-host-range lacZ transcriptional fusion vector, pHRP309, for gram�bacteria. Gene 133:23–30.

36. Pikus, J. D., J. M. Studts, C. Achim, K. E. Kauffmann, E. Munck, R. J.Steffan, K. McClay, and B. G. Fox. 1996. Recombinant toluene-4-monoox-ygenase: catalytic and Moessbauer studies of the purified diiron and Rieskecomponents of a four-protein complex. Biochemistry 35:9106–9119.

37. Pugsley, A. P. 1993. The complete general secretory pathway in gram-neg-ative bacteria. Microbiol. Rev. 57:50–108.

38. Quandt, J., and M. F. Hynes. 1993. Versatile suicide vectors which allowdirect selection for gene replacement in gram-negative bacteria. Gene 127:15–21.

39. Redenbach, M., H. M. Kieser, D. Denapaite, A. Eichner, J. Cullum, H.Kinashi, and D. A. Hopwood. 1996. A set of ordered cosmids and a detailed

genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chro-mosome. Mol. Microbiol. 21:77–96

40. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

41. Schachter, D., and J. V. Taggart. 1976. Benzoyl coenzyme A and hippuratesynthesis. J. Biol. Chem. 203:925–933.

42. Schennen, U., K. Braun, and H. J. Knackmuss. 1985. Anaerobic degradationof 2-fluorobenzoate by benzoate-degrading, denitrifying bacteria. J. Bacte-riol. 161:321–325.

43. Schuhle, K., M. Jahn, S. Ghisla, and G. Fuchs. 2001. Two similar geneclusters coding for enzymes of a new type of aerobic 2-aminobenzoate(anthranilate) metabolism in the bacterium Azoarcus evansii. J. Bacteriol.183:5268–5278.

44. She, Q., R. K., Singh, F. Confalonieri, Y. Zivanovic, G. Allard, M. J. Awayez,C. C. Chan-Weiher, I. G. Clausen, B. A. Curtis, A. De Moors, G. Erauso, C.Fletcher, P. M. Gordon, I. Heikamp-de Jong, A. C. Jeffries, C. J. Kozera, N.Medina, X. Peng, H. P. Thi-Ngoc, P. Redder, M. E. Schenk, C. Theriault, N.Tolstrup, R. L. Charlebois, W. F. Doolittle, M. Duguet, T. Gaasterland, R. A.Garrett, M. A. Ragan, C. W. Sensen, and J. Van der Oost. 2001. Thecomplete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl.Acad. Sci. USA 98:7835–7840.

45. Simon, R., U., Priefer, and A. Puhler. 1983. A broad host range mobilizationsystem for in vivo genetic engineering. Transposon mutagenesis in gramnegative bacteria. Bio/Technology 1:784–791.

45a.Stanier, R. Y., and L. N. Ornston. 1973. The �-ketoadipate pathway. Adv.Microb. Physiol. 9:89–151.

46. Tschech, A., and G. Fuchs. 1987. Anaerobic degradation of phenol by purecultures of newly isolated denitrifying pseudomonads. Arch. Microbiol. 148:213–217.

47. Wintjens, R., and M. Rooman. 1996. Structural classification of HTH DNA-binding domains and protein-DNA interaction modes. J. Mol. Biol. 262:294–313.

48. Zaar, A., W. Eisenreich, A. Bacher, and G. Fuchs. 2001. Intermediates of anew aerobic benzoate metabolic pathway in the bacteria Azoarcus evansii andBacillus stearothermophilus. J. Biol. Chem. 276:24997–25004.

49. Zehr, B. D., T. J. Savin, and R. E. Hall. 1989. A one-step, low backgroundCoomassie staining procedure for polyacrylamide gels. Anal. Biochem. 182:157–159.

50. Ziegler, K., R. Buder, J. Winter, and G. Fuchs. 1989. Activation of aromaticacids and aerobic 2-aminobenzoate metabolism in a denitrifying Pseudomo-nas strain. Arch. Microbiol. 151:171–176.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

genes coding for a new pathway of aerobic benzoate ... · homology to an open reading frame (orf)...

Documents