The TOL Plasmid pWW0 xylN Gene Product fromPseudomonas putida Is Involved inm-Xylene Uptake

Bacterial strains, plasmids, and culture conditions.The bacterial strains and plasmids used in this study are listed in Table1. pWW0-161 is a Tn401insertion derivative of TOL plasmid pWW0 and confers resistance to ampicillin on Escherichia coli hosts (6). The Tn401 insertion occurs outside the catabolic genes in pWW0-161. In this study, anxylN::Km^r mutant of pWW0-161 was isolated and named pWW0-161-xylN. E. coli JM110 was used for routine genetic manipulation. E. coli cells containing recombinant plasmids were maintained on Luria-Bertani plates (29) supplemented with appropriate antibiotics. The concentrations of the antibiotics used were 50 μg/ml for ampicillin and streptomycin and 20 μg/ml for chloramphenicol, while the kanamycin concentration was either 50 μg/ml for E. coliharboring a high-copy-number kanamycin resistance plasmid or 20 μg/ml for E. coli harboring pWW0-161-xylN. P. putida PaW94 (42) harboring pWW0-161 was maintained on M9 minimal medium (29) containing 5 mMm-toluate, while P. putida PaW94 harboring pWW0-161-xylN was maintained on M9 medium containing 5 mMm-toluate and 50 μg of kanamycin/ml. To induce the upper and meta operons on pWW0-161, cells of PaW94 harboring pWW0-116 (hereafter referred to as the wild type) or those harboring pWW0-116-xylN (hereafter referred to as the xylNmutant) were grown for 16 h at 30°C with shaking in 100 ml of M9 minimal medium containing 5 mM benzyl alcohol. m-Xylene vapor was supplemented for the cultures when required. The simultaneous addition of benzyl alcohol and m-xylene vapor to the culture assured the reproducible induction of TOL catabolic enzymes.

DNA sequence analysis.DNA sequencing with double-stranded DNA was carried out by using a dye terminator cycle-sequencing kit (Applied Biosystems) according to the manufacturer's instructions, and the products were analyzed with a model 377 DNA sequencer (Applied Biosystems). The computer-assisted sequence analysis was made with the PC/GENE software package (IntelliGenetics).

Fractionation of membranes.Cells of the wild-type strain were induced as described above, harvested by centrifugation, washed with a 50 mM sodium phosphate buffer (pH 8.6), and resuspended in the same buffer. The cells were disrupted on ice using a sonifier (model 250; Branson) at 100-W output by three 30-s bursts of sonication interspersed with a 30-s cooling. Nondisrupted bacteria and large cell debris were removed by centrifugation at 8,000 × g for 5 min. The supernatant was removed and centrifuged at 138,000 ×g for 1 h. After this step, the supernatant was used as the soluble cytoplasmic fraction. The inner and outer membranes were prepared as described by Feilmeier et al. (5) with slight modifications. Cells were suspended in 20% (wt/vol) sucrose in 10 mM Tris (pH 8.0) and disrupted by passing the cell suspension through a French pressure cell (Ohtake) at 137 MPa. After nonlysed bacteria and cell debris were removed, the clarified lysate was layered onto a 60 to 70% (wt/vol) discontinuous sucrose gradient and centrifuged at 247,000 × g for 18 h. The inner membrane fraction was recovered at the top of the 60% (wt/vol) sucrose layer, while the outer membrane fraction was collected at the top of the 70% (wt/vol) sucrose layer. The recovered samples were diluted threefold with distilled water. Each fraction was layered on a second 60 to 70% (wt/vol) discontinuous sucrose gradient and centrifuged as described above. The membrane fractions were again diluted as described above and centrifuged at 100,000 × g for 2 h to pellet the membranes. The pellets were washed once with 1 M KCl followed by an additional centrifugation step at 100,000 × g for 2 h to recover the membranes. The protein concentration was estimated with a DC protein assay kit (Bio-Rad Laboratories).

Western blot analysis.An xylN gene fragment was amplified by PCR using a set of PCR primers, pXN1 (5′-GCGGATCCATGAAAATAAAAAATTTA) and pXN2 (5′-GCAGATCTGAATGAATAATTATAGGC) (the introducedBamHI and BglII restriction sites are underlined). The 1,411-bp PCR product was separated by electrophoresis on a 0.8% (wt/vol) low-melting-point agarose gel and purified with a QIAquick gel extraction kit (Qiagen). The amplified DNA was subsequently digested with BamHI and BglII and cloned into pQE-60 (Qiagen). The constructed plasmid was transferred toE. coli M15(pREP4). The recombinant XylN with a tail of six histidine residues at the N-terminal end was expressed in the transformant and purified using an Ni-nitrilotriacetic acid spin column (Qiagen) according to the manufacturer's protocol.

For Western blot analysis, proteins in the soluble, inner membrane, and outer membrane fractions were separated by 10% (wt/vol) polyacrylamide gel electrophoresis by the method of Laemmli (20) and transferred to a polyvinylidene difluoride membrane by electroblotting using a Hoefer electroblotter according to the manufacturer's instructions. Prestained protein molecular mass standards (New England BioLabs) were used for molecular mass estimation. Blots were incubated for 1 h with 10,000-fold-diluted rabbit anti-XylN serum and then with horseradish peroxidase-conjugated anti-rabbit donkey immunoglobulin G (Amersham Pharmacia Biotech) for 1 h. Detection of the XylN protein was carried out using ECL Plus Western blotting detection reagents (Amersham Pharmacia Biotech) according to the manufacturer's protocol.

Inactivation of xylN.Plasmid pFC121 is a pUC18 derivative containing a 4-kbSacI-PstI fragment that incorporates the intact xylN gene. This plasmid was digested withPvuII and ClaI, and the resulting 2.8-kbPvuII-ClaI DNA fragment was extracted after electrophoretic separation in a 0.8% (wt/vol) agarose gel to be subcloned into the SmaI site of pHSG398 (35) after its sticky end was converted to a blunt end with a DNA-blunting kit (Takara Shuzo). The constructed plasmid was called pXN960. The xylN gene was inactivated by excising the 1.3-kb fragment containing a kanamycin resistance gene from plasmid pUC4K (36) with BamHI and inserting it into the DraIII site of xylN on pXN960 to yield pXN961.

The pWW0-161 plasmid in P. putida KT2440 (11) was transferred to E. coli JM110 by conjugation as previously described (18), and ampicillin-resistant derivatives of JM110 were selected on Luria-Bertani plates containing ampicillin and streptomycin. JM110(pWW0-161) thus constructed was subsequently transformed with pXN961, and those transformants resistant to kanamycin and chloramphenicol were selected. One of the transformants, JM110(pWW0-161, pXN961), was conjugated with P. putida PaW94, and kanamycin-resistant transconjugants capable of growing on m-toluate were selected on M9 plates containing 5 mM m-toluate and 50 μg of kanamycin/ml.

PCR for the detection ofxylN::Km^r.PCR for amplifying the DNA fragment containing xylN orxylN::Km^r was carried out in a volume of 50 μl with a GeneAmp kit (Perkin-Elmer). The oligonucleotides used as PCR primers were XYLU7191 (5′-GTTCACTTGATGCCAAGTGGAC-3′) and XYLU3 (5′-CCGCTGTAACAGTCCCCTTC-3′).

Growth inhibition by m-xylene.TOL catabolic enzymes were induced in cells of the wild-type strain and thexylN mutant as already described, harvested by centrifugation, washed twice with a 50 mM potassium phosphate buffer (pH 7.4), and resuspended in the same buffer. Four-hundred microliters of the suspension containing 6 × 10⁶ cells was then used to inoculate 100-ml bottles containing 20 ml of the M9 medium supplemented with 5 mM benzyl alcohol with or without 2.5 mMm-xylene. The bottles were tightly sealed with a Teflon-lined rubber septum and a crimped aluminum seal to prevent the evaporation of m-xylene. The cultures were grown at 30°C with shaking, and the turbidities of the cultures at 600 nm were periodically measured.

Enzyme assays.Cells grown under inducing conditions were harvested at the late exponential growth phase by centrifugation, resuspended in 1/10 volume of a 10 mM ethylene diamine buffer (pH 7.4) containing 10% (vol/vol) isopropanol, and disrupted by sonication. After centrifugation at 20,000 × g for 30 min, the supernatants were used as cell-free crude extracts. The protein concentration of each crude extract was estimated with a protein assay kit (Bio-Rad Laboratories). Assays for benzyl alcohol dehydrogenase were carried out at 25°C in a 100 mM glycine-NaOH buffer (pH 9.4) containing 1 mM NAD⁺, 0.1 mg of bovine serum albumin/ml, and 200 μM benzyl alcohol as a substrate. NADH formation was measured spectrophotometrically at 340 nm (15). Catechol 2,3-dioxygenase was assayed at 25°C in a 100 mM potassium phosphate buffer (pH 7.5) with 33 μM catechol as the substrate; the amount of the reaction product (2-hydroxymunoic semialdehyde) was determined spectrophotometrically at 375 nm (26).

m-Xylene-dependent oxygen consumption by P. putida cells.Cells of the wild-type strain and thexylN mutant were cultivated in the presence of benzyl alcohol and m-xylene vapor as described previously, harvested, washed twice with a 50 mM potassium phosphate buffer (pH 7.4), and resuspended in the same buffer. The cells were adjusted to a turbidity of 1.0 at 600 nm and incubated for 5 h at 30°C with shaking. For oxygen consumption assays, cells were placed in a Clark-type oxygen electrode (model 5/6 Oxygraph; Gilson), and the oxygen consumption rates in response to the addition of various concentrations of m-xylene were determined. The apparent kinetic constants, K_s andV_max, were determined by reciprocal plots.

Chemicals and reagents.All the chemicals used in this study were of the highest purity commercially available. m-Xylene was purchased from Tokyo Kasei Kogyo; the other chemicals were purchased from Wako Pure Chemical Industries, Difco, Aldrich Chemical, and Gibco BRL. The enzymes and reagents used for nucleic acid manipulation were purchased from Takara Shuzo.

Nucleotide sequence accession number.The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank libraries under accession number D63341 .