INTRODUCTION

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Defined sulfonated aromatic compounds are synthesized only rarely in nature (24) but are produced in large amounts as surfactants (e.g., 2.5 × 10⁶ metric tons of linear alkylbenzenesulfonates per annum [38]), dyestuffs (e.g., 3 × 10⁵ metric tons per annum [2]), and dyestuff precursors and additives in oils and inks (13). The major individual compound is probably p-toluenesulfonate (TSA) (about 2.7 × 10⁴ metric tons per annum in Europe), which is used, for example, in household detergent formulations, preparation of foundry molds, and syntheses of pharmaceuticals (7). Degradation of TSA was established in 1957 (37), and three dissimilatory pathways have been detected in aerobic microorganisms: 2,3-dioxygenation (14); 1,2-dioxygenation, which is frequently plasmid encoded (5, 21); and the best-characterized system, methyl monooxygenation, in Comamonas testosteroni T-2 (Fig. 1), where it is encoded on plasmid pTSA (11).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Degradation of TSA and TCA by C. testosteroni T-2. The four regulons are shown as R1, R2, R3, and R4. Reactions that are catalyzed by chromosomal gene products are framed. Regulatory units R1 and R3 are plasmid encoded (pTSA, IncP1). Abbreviations: TSA, p-toluenesulfonate; SOL, p-sulfobenzylalcohol; SYD, p-sulfobenzaldehyde; PSB, p-sulfobenzoate; PCA, protocatechuate; CHS, 4-carboxy-2-hydroxymuconate semialdehyde; TCA, p-toluenecarboxylate; COL, carboxybenzylalcohol; CYD, carboxybenzaldehyde; TER, terephthalate; DCD, 1,2-dihydroxy-3,5-cyclohexadien-1,4-dicarboxylic acid. Enzymes: TsaMB, toluenesulfonate methylmonooxygenase; TsaC, p-sulfobenzylalcohol dehydrogenase; TsaD, p-sulfobenzaldehyde dehydrogenase; PsbA(C), p-sulfobenzoate-3,4-dioxygenase; PmdAB, protocatechuate-4,5-oxygenase; TphA231, terephthalate dioxygenase (49); TphB, 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid dehydrogenase (49).

Junker and Cook (19) concluded that there are two plasmids in C. testosteroni T-2, pTSA and pT2T. The function of plasmid pT2T is unknown. A deletion mutant, strain TER-1, lacks plasmid pTSA, on which regulatory groups R1 (at least tsaMBCD and tsaR) and R3 (at least some genes for the p-sulfobenzoate [PSB]-dioxygenase system) and, presumably, genes encoding TSA transport are believed to be located (11, 19; J. Mampel, unpublished results). Plasmid pTSA belongs to the IncP1 group and is conjugative (19). Regulatory group R4 (encoding ring cleavage and the meta pathway enzymes) is chromosomally located (19).

IncP1 plasmids can be highly promiscuous as either resistance or degradative plasmids (6, 16, 17, 46), and the degradative genes are often found between the oriV and trfA genes (9, 44), although few IncP1 plasmids have been mapped to show this.

Junker and Cook (19) found two copies of the insertion element IS1071 (29, 50) associated with plasmid pTSA and postulated that this could be associated with a transposon involved with regulatory group R3. A similar element was postulated in C. testosteroni PSB-4 associated with plasmid pPSB, where active transposition was observed (19).

We have now mapped the known tsa genes as well as the two known IS1071 elements on pTSA, the first degradative IncP1 plasmid to be mapped. The data indicated a tsa transposon with one functional version of the tsa operon and one cryptic version. The latter contained mutations. In addition, a complete mercury resistance (mer) operon was found. The backbone of the plasmid, where sequenced, showed >99% identity to the sequenced (44) IncP1 resistance plasmid, suggesting a common ancestor. Comparison of pTSA to plasmid R751 showed significant differences concerning the genetic load (12) at the two integration sites. The established tsa genes were found to be widespread and highly conserved in nature.

The research was funded by the Deutsche Forschungsgemeinschaft (T. Tralau), the University of Konstanz, and the Fonds der Chemischen Industrie.

	MATERIALS AND METHODS

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Bacteria and growth conditions. C. testosteroni T-2 (DSM 6577) (10, 45) and its pTSA-negative (pTSA) mutant strain TER-1 (22) were grown in minimal medium as described previously (22, 45). Enrichment cultures (5 ml) were done with 6 mM TSA as the sole source of carbon and energy for aerobic growth (45). Purity of substrates used as sources of carbon and energy was >99%.

Preparation and restriction analysis of plasmid DNA. Large plasmids were prepared with the Plasmid Midi Kit (Qiagen). The manufacturer's protocol for low-copy-number plasmids was optimized for high yields of plasmids larger than 50 kb from C. testosteroni T-2 and found to be effective in other organisms. Lysis time was reduced to the time needed for gentle tube inversion (three times), when it was stopped by addition of the supplemented neutralization buffer. The additional isopropanol precipitation step prior to application to the column was eliminated. Our preparations contained plasmids of up to 80 kb.

PCR, quantification of DNA, cycle sequencing, and Southern hybridization. PCR was conducted in reaction mixtures with final volumes of 20 to 50 µl. All reactions contained 10% (vol/vol) dimethyl sulfoxide. DNA (180 to 200 ng) prepared by cetyltrimethylammonium bromide precipitation (4), or a bacterial culture (3 µl), was used as a template. Templates of 5 kb were amplified with Taq polymerase (MWG) and buffer system 2 of Roche's Expand system; templates of >5 kb and <15 kb were amplified with the Expand Long PCR-System (Roche); and templates of >15 kb were amplified with the Expand 20 kb Plus System (Roche). Reactions were conducted according to the manufacturers' recommendations. DNA was quantified fluorimetrically (DyNA Quant 200; Hoefer) according to the manufacturer's instructions.

DNA size markers. The DNA size markers used were  DNA cut with EcoRI and HindIII from New England BioLabs and the 1-kb ladder of MBI-Fermentas with a range from 0.25 kb to 10 kb.

PFGE. For pulsed-field gel electrophoresis (PFGE), DNA was prepared from whole cells embedded in low-melting-point agarose (SeaPlaque GTG, FMC, Biozym) as described elsewhere (41). Electrophoresis at 14°C for 66 to 70 h was done with a Rotaphor V (Biometra). For the gel, 1% agarose (UltraPure, electrophoresis grade; Gibco) and 0.025 M Tris-borate buffer (36) were used. A linear gradient from 24 s to 147 s was used with a switching angle of 120° and a field strength of 10 V/cm.

Nucleotide sequence accession numbers. Nucleotide sequences are available in the NCBI GenBank library under accession numbers AF311820 (tsaMBCD₂), AF305549 (oriTpTSA), AF303942 (oriVpTSA), and AF311437 (trfAupf).

	RESULTS

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Physical map of pTSA. We planned to purify pTSA from C. testosteroni T-2 and generate a restriction map directly. Conjugation experiments failed to yield strains with pTSA as the sole episomal element (19). Electroporation of single plasmids, separated by PFGE, into the plasmid-free C. testosteroni type strain (DSM 50244^T) also failed. Preparative PFGE yielded insufficient material to elute it from the gel, and the quality of the DNA was too low for in-gel restriction. The new experiments did, however, establish the presence of a third, larger, plasmid in strain T-2, and we termed it pT2L (Fig. 2). The pTSA mutant, strain TER-1, contained two of the three plasmids (Fig. 2), pT2T and pT2L, the latter of which was recovered in low yield in plasmid preparations.

View larger version (72K): [in this window] [in a new window]   FIG. 2.   Electropherogram of the PFGE of C. testosteroni T-2 (lane 1), its pTSA derivative TER-1 (lane 2) (Table 2), and of the isolates D. acidovorans Mu4 (lane 3) and S. maltophilia MuF (lane 4). The arrow points to the weak band representing pT2T.

View larger version (52K): [in this window] [in a new window]   FIG. 3.   Electropherogram and Southern hybridization of the double digestion of the plasmids in C. testosteroni T-2 and its derivative TER-1. Restriction enzymes used were BamHI and SmaI. (A) Agarose gel electrophoresis. Lanes: 1 and 2,  marker and 1-kb ladder; 3 and 4, digested plasmid DNA from strains T-2 (plasmids pT2L, pTSA, and pT2T) and TER-1 (plasmids pT2L and pT2T), respectively. Fragments present in lane 3 but not in lane 4 belong to pTSA. Fragments present in both lanes are marked with bold lines and belong to pT2T. (B) Southern hybridization with a tsaM1 probe; signals occur only in lane 3 (T-2).

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Map of plasmid pTSA. The resolution is about 1%; fragments of <0.8 kb are not resolved. Sites with an asterisk (SmaI) are sequenced but not cut by SmaI in these mapping assays. The tsa region is shown in enlarged form at the top of the diagram. I to IV refer to PCR fragments; the map positions and genes are indicated in kilobases. Triangles indicate the direction of transcription.

General structure of pTSA. The long-distance-PCR products (Fig. 4) confirmed the estimate of the size of the plasmid (72 ± 4 kb) and enabled us to refine the genetic map. The known tsa region (tsaR and tsaMBCD [20]) was located, and the 3' end of the tsaR gene was selected as the starting position for mapping.

View this table: [in this window] [in a new window]   TABLE 1.   Genes identified on pTSA and comparison with sequences from plasmids R751 (IncP1) and RK2 (IncP1)a

A second tsa operon as part of a composite transposon. We sequenced downstream of the tsaMBCD₁ operon defined by Junker et al. (20) and found an open reading frame termed tsaQ₁ (which will be described elsewhere [T. Tralau, unpublished data]) and then a region with no significant homologies in BLAST searches and an IS1071 element (Fig. 4).

Backbone of pTSA. IncP plasmids are routinely classified by amplifying and sequencing a 241-bp segment of the trfA2 gene (12, 15). Junker and Cook (19) reported 97% identity with plasmid R751 on the DNA level for pTSA. We have now sequenced this and another 18 backbone genes (Table 1) and found only two genes altered in comparison to the backbone of R751. The trfA and trfA2 genes were 99% identical to R751 on the DNA sequence level and 96% identical on the amino acid level. The lower value is due to frameshifts and a deletion at four positions. Three different frameshifts result in three altered amino acids; a gap is offset after 16 bp by an additional base in pTSA, resulting in five altered amino acids. The second altered backbone gene in pTSA was trbM, which showed a deletion; three gaps resulted in seven altered amino acids and one amino acid fewer in the gene product from pTSA. The other identified genes are 99 or 100% identical to R751 on the DNA and the amino acid sequence levels (Table 1), which shows that this part of the plasmid backbone is even more closely related to the corresponding R751 region than the sequence comparison of trfA2 (12, 19) indicated. On the other hand, we found much lower identity (65 to 87%) for those genes which are comparable on the IncP1 plasmids (32) on the amino acid level (76 to 94% similarity) (Table 1). In contrast to R751, there is no upf31.0 gene at 61.5 kb, upstream of the putative tsa transposon.

Distribution of the tsa genes. Samples (n = 98) from 84 sites on four continents (France, Germany, Iceland, Ireland, Norway, Spain, Switzerland; Argentina, United States, Greenland; India, Taiwan; Tahiti) were used as inocula for enrichment cultures to detect utilization of TSA as the sole source of carbon and energy for aerobic growth. The samples were largely (n = 72) from pristine soils, lakes, and rivers; the others were from sewage works, landfills, and contaminated rivers. The 16 positive enrichment samples, all prokaryotic, were largely (n = 15) from wastewater or sites of industrial pollution; only 1 enrichment sample (from Tahiti) was from a pristine site. Most enrichments (n = 13) yielded a pure culture, and all were examined (Table 2).

View this table: [in this window] [in a new window]   TABLE 2.   Comparison of 16 isolates and mixed cultures of TSA-utilizing bacteria with C. testosteroni T-2 and its pTSA mutant, strain TER-1

	DISCUSSION

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This is the first degradative plasmid to be mapped by subtractive analysis of restriction digests. Large plasmids are usually mapped by restriction analysis (e.g., see reference 51), which requires separation of the plasmid, if several are present in the organism under study. We had to use an alternative strategy, because we could not isolate pTSA. Our data (72 kb [Fig. 4]) show the plasmid to be smaller than our original estimate of 85 kb but within the error range (19); our new data with several enzymes and improved markers are supported by the sequenced tsa region and the lengths of the long-distance-PCR fragments. With hindsight, the mer operon might serve as a selective marker, but mercury resistance is apparently weak in this strain (19).

The use of long-distance PCR was a key method in establishing the structure of pTSA. Much work was done before the map of plasmid R751 was available (44), but the latter data showed the near identity of backbone genes in both plasmids (Table 1) and allowed us to choose primers for suitably sized amplification products (Fig. 4 and Materials and Methods).

IncP1 plasmids are considered to form a structurally more diverse subgroup than IncP1 plasmids (12, 40). Here, however, we present an IncP1 plasmid, whose backbone, where sequenced, is almost identical to that of the IncP1 plasmid R751. The overall structure is also highly homologous to that of plasmid R751, though it confers degradative capabilities rather than resistance factors. Degradative IncP plasmids were shown to have their degradative genes integrated between the oriV and trfA regions (9). In contrast, the degradative genes of pTSA are integrated within a transposon-like structure adjacent to the upf30.5 gene, a region where R751 carries a transposon conferring trimethoprim resistance (Tn402/5090 [18, 44]). No trimethoprim resistance is found in C. testosteroni T-2 (19). The Tn501-related mer operon found between oriV and trfA, a common feature of IncP plasmids (40), is complete on pTSA but only a remnant on R751. One characteristic feature of R751 is the inverted repeats found in the oriV/trfA and tra-1/tra-2 gaps (44). These repeats are discussed as hot spots of recombination. On the sequenced backbone of pTSA we found seven of eight possible inverted repeats. Between the mer operon and the trf region all five repeats could be located, two of them with minor mutationsa point mutation changing C to G in one repeat and a 1-bp shift of a second repeat. The other two repeats are positioned at the insertion site of the tsa transposon. Compared to R751, one repeat is missing (44). It seems that these repeats are linked to the insertion sites on the plasmidon pTSA as well as on R751.

The major difference between the structure of the plasmid and the predictions is that genes encoding regulatory unit R3 (Fig. 1) are not present. Junker and Cook (19) predicted a transposon carrying the R3 genes between two IS1071 elements. There is indeed a transposon of this kind, which has been characterized in C. testosteroni PSB-4 (J. Ruff et al., unpublished data). Furthermore, a copy of this transposon, with a deleted psbC, is present in C. testosteroni T-2 (11), but it is encoded on pT2L (J. Ruff et al., unpublished data). The IS1071 elements, which Junker and Cook (19) detected on pTSA, can now be seen to belong to a tsa transposon-like structure (Fig. 4). The separation of the regulons R1 and R3 on two plasmids, pTSA and pT2L, explains why pTSA was not separable by conjugation to, e.g., C. testosteroni^T, which encodes neither R1 nor R3 (19).

The tsa operon discovered by Junker et al. (20) can now be seen to be part of a composite transposon (Fig. 4). The transposon has been found on three continents (Table 2), sometimes plasmid encoded and sometimes chromosomally encoded (Table 2), and so we can assume that the transposon has an active transposase.

We now have a total of eight bacteria that carry this transposon with essentially the same structure (except that strain TA12 lacks tsaT) (Table 2). Each organism from among seven genera and three continents thus contains a second, silent, and almost perfect duplicate set of genes (tsaMBCD₂ with tsaQ₂ and junction to the insertion element) (Fig. 4). Duplication of degradative genes is not uncommon, as in 3-chlorobenzoate metabolism in Pseudomonas sp. strain B13 (34) or on the TOL plasmid pWW53, where two homologous and functional, but not identical, meta pathway operons were characterized (31), and these duplications are discussed as an important mechanism for the genetic adaptation of microorganisms to xenobiotics (47).

The duplication within the tsa transposon (Fig. 4) may allow the development of novel degradative pathways in the future, but the plasmid location should enhance this (43) as well as the location within a transposon. The evolution of the silent operon is coupled to the spread of the active tsa genes and therefore dependent on the release of TSA. To our knowledge this is the first description of a genetic constellation where the possible evolution of a duplicated catabolic operon may be enhanced by the release of a xenobiotic substance. Furthermore, the nature of pTSA, one of the highly promiscuous IncP plasmids (33), coupled to the obviously active transposase of the tsa transposon, has presumably contributed to its widespread occurrence. Götz et al. (15) have linked IncP1 plasmids to contaminated sites, and we have found degradation of TSA almost exclusively in contaminated areas. In corroboration of the results of Götz et al. (15), IncP1 genes occur in 6 of our 19 isolates (Table 2).

In contrast to the Group I isolates (Table 2), with their genetic and physiological similarities to C. testosteroni T-2 (Fig. 1 and Table 4), we had trouble obtaining isolates of Group II, in which our three mixed cultures are found in these six cultures (Table 2). Group II organisms can grow on BS and TSA and show a 4-methylcatechol 2,3-dioxygenase activity, properties which indicate the presence of a desulfonative TSA dioxygenase, as in Alcaligenes sp. strain O-1 (11, 21). This pathway is frequently plasmid encoded (5), but two cultures (mixture KNP1 and isolate EW13 [Table 2]) contain no detectable plasmid. We consider it possible that the Group III organisms encode the TSA 2,3-dioxygenase pathway enzymes (14), but we have no proof as yet. Further analysis may show whether there are pathways for the degradation of TSA that have not been described up to now.

Strain TA12 seemed to adapt to the utilization of TSA during enrichment, because the growth rate increased steadily during the enrichment (T. Tralau, unpublished data). This could have been the result of mutations in regulatory genes as described by Arai et al. (3) for the utilization of phenol by C. testosteroni TA441.

Two of the newly isolated TSA degraders (EW13 and OrL1) are strains of C. testosteroni but do not have tsa genes and belong to Groups II and III, respectively. Similarly, strains of Stenotrophomonas maltophilia are found in each group (Table 2). S. maltophilia is of clinical relevance and is well known for its multiple antibiotic resistance (e.g., see reference 53). Strains of this genus reveal a high intraspecies diversity and do not show any resistance patterns correlating specifically with the source of isolation, clinical or environmental (8). We presume that Stenotrophomonas is a highly adaptive genus that reacts rapidly towards clinical and metabolic selection.

We conclude that there was independent evolution of several pathways for the degradation of TSA and that there was an intergeneric distribution of the genetic modules necessary for TSA utilization.