INTRODUCTION

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Acinetobacter sp. strain ADP1 is capable of utilizing a range of aromatic compounds as sole sources of carbon. These compounds are dissimilated via the -ketoadipate pathway either through benzoate and catechol or, alternatively, through 4-hydroxybenzoate and 3,4-dihydroxybenzoate (protocatechuate) (15, 22). Although the two branches have three terminal reactions in common, from -ketoadipate enol lactone to the coenzyme A esters of succinate and acetate, each branch has its own genes and enzymes. The genes for the two branches are located in two supraoperonic clusters, the ben-cat cluster (7, 8, 20) (GenBank accession no. AF009224 and AF150928) and the pob-qui-pca cluster (9, 10) (GenBank accession no. L05770), which both extend for >20 kbp but are separated on the chromosome by approximately 270 kbp (13). Recently we reported that at one end of the ben-cat cluster is a group of four genes, areA, -B, -C, and -R, encoding an esterase, two dehydrogenases, and a regulator protein, that are responsible for the catabolism of alkanoate esters of benzyl alcohol, 2-hydroxybenzyl (salicyl) alcohol, and 4-hydroxybenzyl alcohol to benzoate, salicylate, and 4-hydroxybenzoate, respectively, and thus feeding these substrates into one of the two branches of the -ketoadipate pathway (17). In this paper, we report a further extension, by about 5 kbp, of the ben-cat supraoperonic cluster beyond the are genes to include previously unreported genes responsible for the catabolism of alkyl salicylates through salicylic acid to catechol and thus channeling new substrates into the catechol branch of the pathway.

	MATERIALS AND METHODS

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Strains and plasmids. The plasmids and bacterial strains used in this study are listed in Table 1. Isolates ADPW1 and ADPW38 were spontaneous mutants selected by the procedure outlined in reference 31.

Chemicals and media. Aromatic substrates were obtained from Sigma-Aldrich Co. Ethyl salicylate was redistilled under reduced pressure to remove small amounts of contaminating ethanol. Luria-Bertani (LB) medium (23) was used for the cultivation of bacteria unless otherwise noted. For growth on defined carbon sources in liquid medium, the substrates were added to minimal salts medium (4) at the following concentrations: ethyl salicylate, sodium salicylate, benzyl acetate, benzoate, and 4-hydroxybenzoate at 2.5 mM and succinate at 10 mM. For growth on solid medium, a single nonvolatile carbon source (succinate, benzoate, salicylate, or 4-hydroxybenzoate) was added to minimal agar at the same concentrations, but volatile compounds (i.e., all of the esters) were presented in small tubes in the lids of inverted petri dishes containing minimal medium. When appropriate, ampicillin was incorporated at 100 µg/ml and kanamycin was added at 50 µg/ml for Escherichia coli and at 10 µg/ml for Acinetobacter.

DNA manipulations. Standard methods were used for DNA manipulations (23). Total DNA was prepared from Acinetobacter sp. strain ADP1 by the cetyltrimethylammonium bromide method (3). Plasmids carrying insertions of Acinetobacter DNA were isolated from and maintained in E. coli host strain XL1-Blue MRF' or DH5 (Table 1) unless otherwise noted. Plasmid DNA was prepared from E. coli by the alkaline lysis miniprep method (23) or by using Qiaprep columns (Qiagen). DNA fragments were recovered from agarose gels by using Qiaquick columns (Qiagen). Southern blots were prepared as described by Sambrook et al. (23), and hybridizations were carried out with an ECL direct labeling kit (Amersham) in accordance with the manufacturer's instructions.

PCR amplification. PCR amplifications were carried out in 50-µl volumes of reaction buffer (New England Biolabs) containing 10 ng of template DNA, 100 pmol of each primer, 2.5 nmol of each deoxynucleoside triphosphate, 300 nmol of MgSO₄, and 1 U of Vent polymerase (New England Biolabs). In some reactions, 200 nmol of MgCl₂ and 1 U of Taq polymerase were used in place of the MgSO₄ and Vent polymerase. The mixtures were subjected to a 4-min hot start at 94°C and then to 30 cycles of 1 min at 94°C, 1 min at 56°C, and 2 min at 74°C.

RT-PCR. Cells were grown on minimal medium containing either ethyl salicylate, salicylate, or succinate until they reached a density of about 10⁸/ml. Total RNA was prepared from 10 ml of the culture by using RNeasy Mini columns (Qiagen), with elution in 50 µl of water. To remove any contaminating genomic DNA, the RNA was incubated with 1 U of RNase-free DNase (Promega) and 1 U of RNasin (Promega) in 40 mM Tris-HCl (pH 7.9) containing 10 mM NaCl (10 mM), CaCl₂ (10 mM), and MgSO₄ (6 mM) for 30 min at 37°C. The RNA was cleaned by passage through an RNeasy Mini column prior to use in reverse transcription (RT)-PCR. RT-PCR was carried out with an Access RT-PCR kit (Promega). Amplifications were carried out across the salD-salE and salA-salR intergenic regions by using primer pair EDf (5'-AGATTTAGGTATTCAGCAATTCAGGGCAAAAGGTG-3')-EDr (5'-AAGGCTCAGGCGTAAGCATCTTGTAAGTTTCCTC-3') and ARf (5'-CCATGGACACGTGCGGTAGAC-3')-ARr (5'-TTTTTGGTGCATGTGCTCGTAAGT-3'), respectively. PCRs were carried out in 50-µl volumes of reaction buffer (Promega) containing 0.5 µg of total RNA, 50 pmol of each primer, 50 µM (each) deoxynucleoside triphosphate, 1 mM MgSO₄, 5 U of avian myeloblastosis virus reverse transcriptase, and 5 U of Tfl DNA polymerase. After RT at 48°C for 1 h, the reaction mixtures were heated to 94°C for 2 min and subjected to 40 cycles of 30 s at 94°C, 1 min at 55°C, and 2 min at 68°C. Negative-control reactions, designed to ensure that residual genomic DNA was not amplified, were performed in the same way, except that the reverse transcriptase was omitted from the reaction mixtures.

Cloning of Acinetobacter sp. strain ADP1 DNA. DNA adjacent to areABC was isolated by using the chromosomal drug resistance cassette in areB of strain ADPW57 (17). A plasmid library in E. coli (pUC18) was made from chromosomal DNA of ADPW57, from which plasmid pADPW32 with an 8.0-kbp SacI insert (Fig. 1) was selected by screening for Km^r Ap^r colonies. pADPW34 was constructed as a SacI-to-XbaI subclone of pADPW32 (Fig. 1) so as to have sequence overlap with the previously sequenced plasmid pADPW33 (17). pADPW34 was used as the DNA sequencing template. Sequence alignment confirmed its overlap with pADPW33.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Physical map of the salA, salR, salE, and salD genes and their location relative to areABC at the left-hand end (as drawn) of the supraoperonic ben-cat cluster. The inserts of the plasmids produced from cloning genomic DNA into vectors are specified in Table 1. pADPW32 was cloned directly from genomic DNA. All other plasmids were produced by PCR from genomic DNA (denoted by asterisks) or by subcloning from plasmids containing genomic DNA. Sites at the termini of the inserts marked with asterisks were incorporated via PCR primers. The Kmr cassette insertions are not to scale. The abbreviations for the restriction sites are as follows: Bc, BclI; C, ClaI; E, EcoRI; H, HindIII; N, NsiI; Nd, NdeI; S, SacI; and X, XbaI.

Expression of salE in E. coli. Oligonucleotide primers were designed to produce a PCR fragment of the salE gene with (i) an NdeI site introduced at the putative start site of the reading frame, (ii) a constructed EcoRI site upstream of the NdeI site, and (iii) an EcoRI site downstream of the gene. The PCR fragment generated from pADPW34 was cut with EcoRI and first ligated into EcoRI-cut pUC18 to create pADPW49 (Table 1). The insert was sequenced on one strand to ensure that mutations had not been incorporated during the PCR. A fragment was excised with NdeI and EcoRI, religated into the expression vector pET5a, and transformed into E. coli BL21(DE3)pLysS to produce plasmid pADPW70 (Fig. 1). Since salE contained a NdeI restriction site from bp 8 to 14, the forward primer was designed with a mutation in the ninth base pair (AG) that destroyed the native NdeI site but did not change the amino acid encoded (Thr). The primers used were 5'-AGGAGAATTCATATGATAACGTATGTACTTGTTC-3' (forward) and 5'-AGCGAATTCCTCGGATATGGTTGATTCAAAC-3' (reverse) (the NdeI site is italicized, the EcoRI restriction sites used for cloning into pUC18 are underlined, and the bases which differ from the wild-type sequence are in boldface type). The SalE protein encoded on the expression vector pADPW70 was expressed in E. coli BL21(DE3)pLysS by growth of the bacterium in LB medium to an optical density at 600 nm of 0.6 and subsequent induction for 4 h by addition of 0.4 mM isopropyl--D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in a discontinuous gel in a Mini-PROTEAN II electrophoresis cell (Bio-Rad) in accordance with the manufacturer's instructions.

Chromosomal disruption of salA, salR, salE, and salD in Acinetobacter sp. strain ADP1. As a first step in the construction of gene knockouts, pUC18-derived plasmids carrying part or all of gene salA, salR, salE, or salD disrupted by a Km^r cassette were constructed. For salA, a SacI-HindIII fragment of pADPW34 was cloned to create pADPW41 (Fig. 1). The Km^r cassette of pUI1637 (11) was cloned into a unique ClaI site of pADPW41, creating pADPW44. The salE gene was disrupted by the insertion of the cassette from pUI1637 into the unique ClaI site of pADPW49 (see above) to form pADPW76. Plasmid-borne salR disruption was achieved after first creating pADPW78, which has a PCR-generated EcoRI insert in pUC18. This 1.0-kbp fragment, internal to salR and flanking its ClaI site, was amplified from pADPW34. Primer sequences (with the EcoRI sites underlined and the altered bases in boldface) were as follows: 5'-TGGAATTCATGAACAGATCCGAAAAGAACG-3' (forward) and 5'-CATGAATTCCCTGAGTATGCCCGGTA-3' (reverse). The central ClaI site in the pADPW78 was used as the insertion site for the Km^r cassette from pUI1637 (11) to create pADPW79. Disruption of plasmid-borne salD was performed after first creating pADPW82, which has a PCR-generated EcoRI insert in pUC18. This 1.2-kbp fragment, flanking the NsiI site in salD, was amplified from pADPW32. Primer sequences (with the EcoRI sites underlined and the altered bases in boldface) were as follows: 5'-AGGGGGAATTCTGGCAGCAATCACTG-3' (forward) and 5'-GGGCTGGAATTCCCAAGTACTACCTAT-3' (reverse). The central NsiI site in pADPW82 was used as the insertion site for a Km^r cassette from pUC4K (28) to create pADPW86. Plasmids pADPW44, pADPW76, pADPW79, and pADPW86 were linearized by digestion with an appropriate restriction enzyme, and each was used to transform ADP1 via natural transformation. Southern hybridization confirmed that in strains ADPW67 (salA::Km^r), ADPW70 (salE::Km^r), ADPW72 (salR::Km^r), and ADPW78 (salD::Km^r) the altered plasmid-borne allele had replaced the corresponding chromosomal wild-type region (data not shown).

Preparation of cell extracts. Cells were harvested by centrifugation, washed with 100 mM phosphate buffer (pH 7.4), and stored as pellets at 20°C. Cell extracts were prepared by disrupting frozen pellets, suspended in ice-cold 100 mM phosphate buffer (pH 7.4), with a French pressure cell (SLM Instruments, Inc., Urbana, Ill.) and centrifuging the broken cells at 120,000 × g for 30 min at 4°C. The supernatant was stored frozen as 1-ml portions at 20°C.

Transformation of metabolites. Transformation of ethyl salicylate into salicylate by cell extracts of SalE was monitored spectrophotometrically. The measuring cell contained 100 µM substrate, 100 µM Tris (pH 7.5), and 10 µl of cell extract, while the reference cell contained only buffer and enzyme.

Enzyme assays. Salicylate hydroxylase (SalA) activity was measured in 3-ml reaction mixtures containing 50 mM Tris (pH 7.5), 100 µM NADH, and 100 µM salicylate. The reaction was initiated by addition of 20 µl of cell extract, and the rate of oxidation of NADH was determined spectrophotometrically at 340 nm (extinction coefficient, 6,220 mol¹ cm¹). Salicylate esterase (SalE) activity was assayed by spectrophotometric monitoring (405 nm) of the hydrolysis of 4-nitrophenyl ester substrates in 1-ml reaction mixtures containing 50 mM phosphate buffer (pH 8) and 2 mM 4-nitrophenyl ester. The 4-nitrophenyl esters with an aliphatic moiety of six or less carbon atoms were dissolved in methanol, and an aliquot was added to the assay mixture such that the final concentration of the ester was 2 mM. The 4-nitrophenyl esters with an aliphatic moiety of eight carbon atoms or longer were first dissolved in 2-propanol at 60°C and then added dropwise to 50 mM Tris-HCl (pH 8.0), prewarmed to 60°C, to a final ester concentration of 2 mM. The assay reactions were initiated by the addition of 10 µl of enzyme. The molar extinction coefficient of 4-nitrophenol was taken as 14,800 mol¹ cm¹. The activity of the esterase with ethyl salicylate as the substrate was determined in a linked assay. The rate of increase of absorbance at 340 nm was measured in 1-ml reaction mixtures containing 100 mM phosphate buffer (pH 8), 2 mM NAD⁺, 100 µM substrate, and 10 U of yeast alcohol dehydrogenase (Sigma-Aldrich Co.). The reaction was initiated by the addition of esterase. A linear response of rate to added esterase verified that the esterase-catalyzed reaction was the rate-limiting step. The assay produced 1 mol of NADH per mol of ethyl salicylate utilized.

Determination of kinetic parameters for salicylate esterase. To obtain K_m and maximum velocity (V_max) values, initial velocities were measured at several nonsaturating concentrations of each compound. Preliminary experiments determined the approximate value of K_m, and accurate rate determinations were then performed with from 7 to 10 different substrate concentrations spanning the approximate K_m value. Initial velocities were analyzed by direct linear analysis using the program EnzPack, which calculates the most probable values for the kinetic parameters with their 68% confidence limits (30). Each reaction velocity was determined in triplicate with two separate extract preparations. The concentration of the substrate stock solution was accurately determined enzymatically by making the substrate limiting in the assay while other components were in excess, and the change in absorbance at 405 nm, corresponding to the total conversion of added substrate, was determined.

Nucleotide sequencing and sequence analysis. DNA sequences were determined by primer walking of fragments cloned in pUC18 by MWG-Biotech Ltd. (Ebersberg, Germany). Searches of the GenBank database were carried out with the BLASTN and BLASTP programs from the National Center for Biotechnology Information, Bethesda, Md. (2). Sequence data were aligned and edited by using the Lasergene software package (DNAStar, Inc., Madison, Wis.). Amino acid sequence alignments were performed with the program ClustalW (PAM350 matrix) (27).

Nucleotide sequence accession number. The DNA sequence obtained in this study has been added to the GenBank database (accession no. AF150928).

	RESULTS

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Analysis of nucleotide sequences of protein products. Analysis of the nucleotide sequence downstream of areA revealed the presence of four open reading frames (Table 2). Immediately downstream of areA, but separated by 195 bp and an inverted repeat, which could serve as a termination loop for areA expression (17), is a gene designated here as salD. SalD shows 13 to 17% amino acid sequence similarity to putative proteins encoded by other operons of aromatic catabolism in different bacteria, which include TodX (29), XylN (GenBank accession no. D63341), and TbuX (H.-Y. Kahng, A. M. Byrne, R. H. Olsen, and J. J. Kukor, submitted for publication), which have been suggested to be membrane proteins involved in the transport of hydrocarbons (J. J. Kukor, personal communication). Downstream of salD is a gene, which we have called salE, whose product shows homology to serine esterases of the / hydrolase family of enzymes (21, 26) but which is not closely related to the benzyl esterase AreA-encoding gene lying upstream of it (26% similar, 15% identical). Transcribed convergently toward salE is a gene, which we have called salR, apparently encoding a regulatory protein of the LysR family. The closest matches to SalR are two NahR proteins involved in naphthalene catabolism, one from Pseudomonas stutzeri (6) and one from the Pseudomonas putida NAH7 plasmid (25). The latter protein has a dual role as a positive regulator of two functionally related operons, for the conversion of naphthalene to salicylate and for the further conversion of salicylate to central metabolites via catechol and the subsequent extradiol (meta) cleavage pathway (24, 25, 33). The final gene in this cluster is salA, whose putative product's closest relatives have both been named NahG. These enzymes are salicylate hydroxylases, converting the salicylate produced from naphthalene to catechol. The genes encoding both enzymes head the salicylate catabolic operon on the NAH7 plasmid (34) and in P. stutzeri (6), respectively. However, it has been noted that ADP1 does not grow on naphthalene as a sole carbon source. The alignments of both salA and salR with their Pseudomonas homologues strongly indicate that unlike the Pseudomonas genes, they both have a GTG start codon.

Insertional inactivation of single genes. The chromosomal copies of salA, salE, salR, and salD were specifically disrupted, individually, by the insertion of a Km^r cassette into each of the genes in plasmid constructs (pADPW44, pADPW76, pADPW79, and pADPW86, respectively [Fig. 1]). The disrupted genes were introduced into ADP1, using the high frequency of natural transformation of which this strain is capable. Two of the resulting strains, ADPW67 (salA::Km^r) and ADPW72 (salR::Km^r), failed to grow on salicylate, unlike ADP1, which grows vigorously on salicylate overnight. By contrast, ADPW70 (salE::Km^r) grew on salicylate as well as did ADP1. ADPW78 (salD::Km^r) was a very unhealthy strain; it grew slowly compared with ADP1 even on succinate minimal medium and rich (LB) medium and lost viability when maintained on agar after 2 to 3 days. However, despite these limitations it, too, like ADPW70, grew on salicylate. Because SalE showed amino acid sequence homologies with other esterases, we compared the abilities of ADPW70 and ADP1 to grow on a range of esters containing aromatic components both as the alcohol and as the acid moiety, as well as a number of exclusively aliphatic esters. Thirteen esters (n-propyl acetate, benzyl acetate, n-butyl propionate, ethyl propionate, benzyl propionate, ethyl valerate, ethyl butyrate, benzyl butyrate, ethyl caproate, ethyl benzoate, n-propyl benzoate, n-butyl benzoate, and ethyl benzoylacetate) were growth substrates for both strains, and 6 (ethyl acetate, n-butyl acetate, n-butyl butyrate, benzyl benzoate, n-propyl cinnamate, and benzyl salicylate) were substrates for neither strain. However, ethyl salicylate and methyl salicylate supported growth of ADP1 but not ADPW70, suggesting that SalE is a hydrolase specific for these esters. Neither ADPW67, ADPW72, nor ADPW78 was able to grow on either of the two alkyl salicylates.

Expression of cloned salE. The salE gene was cloned into expression vector pET5a as plasmid pADPW70 with its start codon (ATG) located in the optimal position for expression. SDS-PAGE of the induced E. coli BL21(DE3)pLysS containing pADPW70 revealed a strong protein band with an expected molecular mass of 27 kDa (Fig. 2). The esterase activity in extracts of induced cells against a range of 4-nitrophenyl esters was measured and compared with the activity of the upstream benzyl esterase AreA (17) (Table 3). Whereas AreA shows a broad specificity for the alkanoate side chain, going up to C₁₆, SalE shows a much more restricted range, up to only C₆. The K_m values of SalE for 4-nitrophenyl acetate and 4-nitrophenyl butyrate were 106 and 77 µM, respectively, whereas the relative V_max dropped 10-fold, from 28 to 2.8 µmol/min/mg, for the same two substrates.

View larger version (73K): [in this window] [in a new window]   FIG. 2.   SDS-PAGE of overexpressed SalE and AreA proteins. The lanes contain lysates from E. coli BL21(DE3)pLysS carrying the following plasmids, induced with IPTG (I) or uninduced (U): lane 2, pADPW70 (I); lane 3, pADPW70 (U); lane 4, pADPW40 (I); lane 5, pADPW40 (U); and lane 6, pET5a. Lane 1 contained molecular mass standards (A, 97.4 kDa; B, 66.2 kDa; C, 45.0 kDa; D, 31.0 kDa; and E, 21.5 kDa). The estimated molecular masses for the overexpressed bands are 27 kDa for SalE (lane 2) and 37 kDa for AreA (lane 4).

Expression of salicylate hydroxylase. Using the standard NADH-linked assay procedure, we were able to detect salicylate hydroxylase activity in a number of strains (Table 5). For ADP1, activity was not detectable in succinate-grown cells but was induced by growth on both salicylate and ethyl salicylate, and when grown on succinate in the presence of both salicylate and ethyl salicylate the activity was significantly induced, although at a lower level than in the absence of succinate. For both ADPW67 (salA::Km^r) and ADPW72 (salR::Km^r), no activity was detected when the cells were grown on succinate plus salicylate or, for the latter, succinate in the presence of ethyl salicylate. However, for ADPW70 (salE::Km^r) there was high-level induction when grown on salicylate and a low, but significant, level of induction when grown in the presence of ethyl salicylate, which it is unable to transform.

Phenotypes of mutants. To demonstrate that the aromatic moiety arising from salicylate and ethyl salicylate is channeled down the -ketoadipate pathway, we checked the phenotypes of mutants blocked both in the sal genes and at three different points in the -ketoadipate pathway (Table 7). The three -ketoadipate pathway mutants were ADP6 (pcaG), which does not grow on 4-hydroxybenzoate or any substrate that feeds into the protocatechuate branch of the pathway; ADPW1 (catA), with a functionless catechol 1,2-dioxygenase which blocks the utilization of benzoate and catechol and which accumulates catechol as demonstrated by the brown coloration on agar plates from any substrate that feeds into the catechol branch; and ADPW38 (ben), which has an uncharacterized lesion in benABC and is unable to convert benzoate to catechol. The Km^r cassette insertion mutation in salE was also individually introduced into ADP6, ADPW1, and ADPW38 by natural transformation to produce double mutants, all of which were tested for growth on the appropriate carbon sources (Table 7). Only ADPW1 of the single -ketoadipate pathway mutants failed to grow on salicylate or ethyl salicylate, with catechol accumulating in both media. The growth phenotypes of the double mutants were consistent with the proposed pathway (Fig. 3) in which the salicylate nucleus is fed into the benzoate branch at the level of catechol.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Proposed pathway for the catabolism of alkyl salicylates linked to the ben-cat branch of the -ketoadipate pathway in Acinetobacter sp. strain ADP1.

RT-PCR analysis of ADP1 transcripts. To confirm that the sal genes are transcribed during both ethyl salicylate and salicylate catabolism and that the operon structure is as implied by the gene organization (Fig. 1), transcripts from cells grown on both of these substrates and on succinate as a noninducing negative control were examined. Two primer sets, spanning from salD through to salE and from salA through to salR, were constructed (Fig. 4A). The expected RT-PCR product sizes for the salD-salE and salA-salR amplicons were 1,195 and 988 bp, respectively. The PCR products obtained, together with restriction digests chosen to confirm the presence of expected restriction sites, were analyzed by agarose gel electrophoresis. The salAR products obtained from the total RNA of cells grown on both ethyl salicylate and salicylate were of the expected sizes (Fig. 4B). Also, a salED product of the expected size was obtained from the total RNA of cells grown on ethyl salicylate. The presence of restriction sites in the expected positions within the fragments was confirmed by digestion with ClaI (salED) and HindIII (salAR). No products were obtained from total RNA of succinate-grown cells or from reaction mixtures from which the reverse transcriptase had been omitted (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   RT-PCR of sal genes. (A) Positions of the genes relative to the SacI site (at bp 1), the primers used for the RT-PCR, and the HindIII and ClaI restriction sites. (B) Agarose gel electrophoresis of RT-PCR products amplified from ADP1 grown on ethyl salicylate and salicylate. The sizes of molecular size markers (in base pairs) in lanes S (HyperLadder I; Bioline, London, United Kingdom) are indicated on the right. Lanes: 1, salAR, salicylate-grown cells (expected size, 988 bp); 2, salAR, salicylate-grown cells digested with HindIII (582 and 406 bp); 3, salAR, ethyl salicylate-grown cells (expected size, 988 bp); 4, salAR, salicylate-grown cells digested with HindIII (582 and 406 bp); 5, salED, ethyl salicylate-grown cells (expected size, 1195 bp); 6, salED, ethyl salicylate- grown cells digested with ClaI (860 and 335 bp). No detectable products were obtained in control reactions, with each pair of primers, from which reverse transcriptase had been omitted or in reactions carried out on succinate-grown cells (data not shown).

	DISCUSSION

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Catabolic role of sal genes and proteins. The two enzymes salicylate esterase (SalE) and salicylate hydroxylase (SalA) reported in this paper are involved in the sequential catabolism of alkyl salicylates via salicylate to catechol. They represent another route into the -ketoadipate pathway for substrates which are likely to be found as natural products, either directly of plant origin or as microbial breakdown products of plant compounds. In this study, both enzymes were expressed from cloned genes, SalE from the expression vector pET5a, from which it is expressed to a very high specific activity, and SalA from a pUC18 clone carrying salERA. The SalA salicylate hydroxylase activity shows the same relative substrate preferences as does the activity found in wild-type ADP1 grown on salicylate alone. In addition, insertional salE and salA knockout mutants, whose construction was facilitated by the natural transformation of ADP1, show the phenotype expected from the proposed pathway (Fig. 3). The further catabolism of the aromatic moiety into the ben-cat branch of the -ketoadipate pathway at the level of catechol was also confirmed by the fact that only mutations in catA (for catechol 1,2-dioxygenase), and not those in the ben or the pca genes, eliminated the ability to utilize either the ester or the free salt of salicylate.

Comparisons of Sal proteins. Examination of the deduced amino acid sequence of SalE in the PROSITE database (16) shows that from residues 68 to 77 (IVLLGHSYGG) it has the signature characteristic of serine lipases, [LIV]-x-[LIVFY]-[LIVMST]-G-[HYWV]-S-x-G-[GSTAC], in which the serine is the active-site nucleophile. Its primary sequence does not align closely (<26% similarity) with other reported Acinetobacter esterases, one from Acinetobacter lwoffii RAG-1 (1) and two (a carboxylesterase [19] and the adjacent benzyl esterase, AreA [17]) from strain ADP1, all of which are longer and have their putative active site serines further from the N terminus than SalE.

Regulation of sal genes. SalA activity is induced by growth of ADP1 on salicylate or on ethyl salicylate (Table 5). It is probable that SalR is the protein that regulates expression of SalA, since (i) insertion of a Km^r cassette into salR in strain ADPW72 stops induction of salicylate hydroxylase activity by salicylate (Table 5); (ii) salicylate hydroxylase is also salicylate inducible in E. coli from pADPW34, which carries only salARE (Table 5); and (iii) there are close amino acid sequence homologies between SalR, SalA, and the Pseudomonas homologues NahR and NahG. A further point of comparison with the Pseudomonas genes is that there is also homology with the regions upstream of salA identified by Schell (24) as being the promoter sites at which NahR interacts; upstream of nahAa is sequence TCA-N₆-TGA, and upstream of nahG is sequence TCA-N₃-TGATGA (24) (GenBank accession no. M11863). The sequence upstream of salA is TCA-N₃-TGATGG. Just downstream of this there is also a 35 sequence, TAGGCAATT, which has 5 bases that correspond to those of the 35 sequence identified for nahAa, TGGTGTATT (24) (GenBank accession no. M11863), but the putative 10 region shows no similarity. A major difference between the sal and nah genes is that whereas nahR is transcribed divergently from its adjacent catabolic gene, nahAa, salA, and salR appear to be cotranscribed as a single regulatory unit, as shown by the RT-PCR results. This implies that SalR controls its own expression.