Financial support: This work was supported by grants 1960262 and 8990004 from FONDECYT-Chile.

Keywords: chloroaromatics, chlorocatechols, metabolic complementation, Ralstonia eutropha, ortho ring cleavage, tfd genes.

Present addresses: # Laboratorio de Microbiología Industrial, Departamento de Ingeniería, Facultad de Ingeniería, Pontificia Universidad Católica de Chile, Av Libertador Bernardo O'Higgins Nº 340, piso 3, Santiago, Chile. Tel: 56 2 686 4053. § Environmental Biotechnology (DGR), Swiss Federal Institute of Technology Laussane (EPFL), CH-1015 Laussane, Switzerland. Tel: 41 21 693 6231. Fax: 41 21 693 4722.

Chloroaromatic pollutants from bleached Kraft pulp mill effluents (BKME) are difficult to degrade, because bacterial strains present in BKME aerobic treatments, only partially degrade these compounds, accumulating the corresponding chlorocatechol intermediates. To improve the catabolic performance of chlorocatechol-accumulating strains, we introduced, by chromosomal insertion, the tfd_ICDEF gene cluster from Ralstonia eutropha JMP134 (pJP4). This gene cluster allows dechlorination and channelling of chlorocatechols into the intermediate metabolism. Two bacterial strains, R. eutropha JMP222 and Pseudomonas putida KT2442, able to produce chlorocatechols from 3-chlorobenzoate (3-CB) were used. Acinetobacter lwoffii RB2 isolated from BKME by its ability to grow on guaiacol as sole carbon source and shown to be able to produce the corresponding chlorocatechols from the BKME pollutants 4-, and 5-chloroguaiacol, was also used. The tfd_ICDEF gene cluster was inserted in the chromosome of these strains using miniTn5-derived vectors that allow expression of the Tfd enzymes driven by the lacI^q/P_trc or tfdR/P_tfd-I regulatory systems, and therefore, responding to the inducers isopropyl-ß-D-thiogalactopyranoside (IPTG) or 3-CB, respectively. Crude extracts of cells from strains JMP222, KT2442 or RB2 engineered with the tfd genes, grown on benzoate and induced with IPTG or 3-CB showed Tfd specific activities of about 15% - 80% of that of the strain JMP134. Dechlorination rates for 3-CB or chloroguaiacols correlated with levels of Tfd enzymes. However, none of the strains containing the chromosomal copy of the tfd_ICDEF cluster grew on monochloroaromatics as sole carbon source. Experiments with BKME aerobic treatment microcosms showed that the catabolic performance of the engineered bacteria was also lower than the wild-type R. eutropha strain JMP134.

Bleached Kraft pulp mill effluents (BKME) are complex wastes containing organic matter and chloroorganic compounds generated in the chlorine bleaching process (Leuenberger et al. 1985). Low molecular weight compounds such as chlorophenols, chlorobenzoates, chlorocatechols and chloroguaiacols, among others, are generated in small but significant quantities (Kringstad and Lindström, 1984). BKME are usually treated in aerobic systems where the microbial activity substantially decreases the organic matter content, but is less effective degrading low, and specially, high molecular weight chlorinated material (Graves et al. 1995). The presence and adaptation of chloroorganic degrading microorganisms, especially bacteria, in such polluted systems, makes BKME a suitable place to search for new strains with catabolic activity towards chloroaromatic pollutants (Fulthorpe et al. 1993; González et al. 1993; Acevedo et al. 1995).

Different metabolic pathways have been described for bacterial chloroaromatic degradation (Reineke and Knackmuss, 1988). In most cases, a specific intermediate is the bottleneck in the metabolic pathway due to impaired enzyme activity and/or toxic effects. This is the case for chlorocatechols, which are key intermediates in the biodegradation of several chloroaromatics. These compounds can be accumulated or, otherwise, further metabolised through the ortho, modified ring-cleavage pathway (Reineke and Knackmuss, 1988). One extensively studied example of an efficient chloroaromatic degrading bacteria that use such a chlorocatechol degradation pathway is Ralstonia eutropha JMP134 (pJP4), a strain able to grow on 2,4-dichlorophenoxyacetate (2,4-D), and 3-chlorobenzoate (3-CB), as well as other chloroaromatics (Don and Pemberton, 1981; Pieper et al. 1988; Clément et al. 1995). Most of its catabolic abilities are encoded on the tfd genes from the plasmid pJP4 (Figure 1a). The enzymes of the catabolism of chloroaromatics in pJP4 have been studied (Pieper et al. 1988; Fukumori and Hausinger, 1993; Kasberg et al. 1995; Vollmer et al. 1999). Catabolism of 2,4-D is started by the 2,4-D/a-ketoglutarate dioxygenase (tfdA) and the 2,4-dichlorophenol hydroxylase (tfdB) encoded in pJP4, to form 3,5-dichlorocatechol. Metabolism of 3-CB is initiated by a chromosomally encoded-, low specificity- enzyme benzoate dioxygenase and 1,2-dihydro-1,2-dihydroxybenzoate dioxygenase to form 3-, and 4-chlorocatechol (Pieper et al. 1993). Chlorocatechol metabolism is performed by the enzymes encoded on the pJP4 gene clusters: tfd_ICDEF and tfd_IIDCEF (Figure 1a). tfdC, tfdD, tfdE, and tfdF genes encode for chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase, respectively (Figure 1b). The first three enzymes encoded in tfd_ICDEF are more active than in tfd_IIDCEF, whereas the TfdF activity of the latter is more active that in tfd_ICDEF (Pérez-Pantoja et al. 2000). The regulation of the tfd genes is not well understood. Recent works indicate that tfdR, a LysR- type transcriptional activator, may play the main role in expression of both chlorocatechol-catabolism tfd clusters (Matrubutham and Harker, 1994; Leveau and Van der Meer, 1996; Leveau et al. 1999).

In this work, we explored the improvement of the ability to degrade, and eventually, grow on chloroaromatics in bacterial strains that accumulate chlorocatechols, by engineering them with the chlorocatechol degradation tfd_ICDEF gene cluster from R. eutropha JMP134. We chose R. eutropha JMP222, a pJP4 plasmid-free derivative of strain JMP134, and Pseudomonas putida KT2442, both accumulating monochlorocatechols from 3-CB. We also included in this study a not previously reported bacterial strain, Acinetobacter lwoffii RB2, isolated from a BKME aerobic treatment system by its ability to grow on guaiacol. This strain accumulates monochlorocatechols from chloroguaiacols. In derivatives of these strains containing a chromosomal insertion of the tfd_ICDEF gene cluster, we assessed the expression of Tfd enzymes, the dechlorination of monochloroaromatic compounds, and the ability to grow on them. With some of these derivatives we studied the ability to remove 3-CB in microcosms of BKME aerobic treatment.

Bacterial strains, plasmids and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. R. eutropha JMP134 (pJP4), R. eutropha JMP222 (cured of pJP4) and P. putida KT2442 were grown at 30ºC in a chlorine-free minimal media (Kröckel and Focht, 1987) with 2 mM 2,4-D, 3 mM benzoate plus 1,000 mg/ml streptomycin, or 3 mM benzoate plus 50 mg/ml rifampycin, respectively. A. lwoffii RB2 was isolated by enrichment culture with 1 mM guaiacol, using BKME as inoculant. The source of BKME has been described elsewhere (Céspedes et al. 1996; Valenzuela et al. 1997). 16S RNA sequencing analysis of strain RB2 was performed in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), resulting in a 97.0% overlap with the species Acinetobacter lwoffii. Escherichia coli strains were maintained in Luria Bertani (LB) medium plus the appropriate antibiotic: ampicillin: 50 µg/ml, kanamycin: 50 µg/ml, rifampycin: 50 µg/ml, chloramphenicol: 20 µg/ml, or streptomycin: 1,000 µg/ml.

DNA manipulation. Restriction, ligation, and dephosphorylation reactions, purification of DNA, and Southern analysis were performed by standard procedures (Ausubel et al. 1992). Detection by PCR was performed using primers 5'-TCTAGAATTCATGACGGAGGCAAAGTGAAC-3' and 5'-GCCGTGGAATTCGCCAGTGGGAACCTGCAG-3' for a 1.8 kb fragment of the tfd_ICD sequence and 5'- ACTCGGGATCCGGGATTACGAACTGCAGGT-3', and 5'-CCCGGGATCCATTATTTGAAATCCGGTCTT-3' for a 1.9 kb fragment of the tfd_IEF sequence (Pérez-Pantoja et al. 2000). The program was: initiation 95ºC x 120 s, 35 cycles of melting 95ºC x 45 s, annealing 55ºC x 30 s, and extension 72ºC x 180 s, final extension 72ºC x 10 min. Competent cells for the introduction of DNA by thermal shock (42ºC x 1 min) were prepared following a described procedure (Nishimura et al. 1990). Triparental mating was performed on LB agar plates inoculated with cells grown overnight on LB medium plus antibiotic (Table 1), centrifuged and washed twice with 50 mM MgCl₂. E. coli CC118 lpir containing the derivatives of pCNB5 (pITFD), or pUT (pTFD, pDTFD) was used as donor, E. coli HB101 (pRK600) as helper, and A. lwoffii RB2, R. eutropha JMP222, or P. putida KT2442 as recipient. A donor to helper to recipient ratio of 1:1:2 was used. After overnight incubation, cells were streaked on a nitrocellulose filter, resuspended, and plated on 2 mM guaiacol or 3 mM benzoate, plus kanamycin.

Construction of tfd_ICDEF gene clusters. Construction of the lacI^q/P_trctfd_ICDEF (pITFD) gene cluster: The tfd_ICDEF genes were cloned into pJRC42, as previously described (Pérez-Pantoja et al. 2000). The 4.2 kb insert of pJRC42 was digested with NotI, and introduced into the same restriction site in miniTn5 vector pCNB5 (de Lorenzo et al. 1993), to form pITFD. The proper orientation of the insertion was checked by digestion with EcoRI, which produced a fragment of 1.0 kb. Construction of the tfdR/P_tfd-Itfd_ICDEF (pDTFD) and the P_tfd-Itfd_ICDEF (pTFD) gene clusters: The tfdR/P_tfd-Itfd_ICDEF gene cluster was cloned in pUCLG2, as described elsewhere (Pérez-Pantoja et al. 2000); P_tfd-Itfd_ICDEF was cloned in pUCLG1 during the construction of pUCLG2. Plasmids pUCLG1 and pUCLG2 were digested with NotI and the insertions introduced into pUT (de Lorenzo et al. 1990), to give pTFD and pDTFD, respectively.

Dechlorination and enzyme activity assays. Dechlorination studies were performed with cell suspensions (O.D._{660 nm}=1.0) prepared from cells grown on 2 mM guaiacol or LB, in the absence or presence of 1 mM of isopropyl-ß-D-thiogalactopyranoside (IPTG) or 1 mM 3-CB. Cells were incubated for up to 6 h with 5 ml of free-chlorine minimal medium, containing 1 mM of a monochloroaromatic compound. Chloride released was determined by the mercury thiocyanate method (Florence and Farrar, 1971). For enzyme activity assays, cultures grown on minimal medium containing 3 mM benzoate, with or without inducer, were obtained. About 100 ml of each culture was centrifuged, and cells were washed and resuspended in 5 ml of 50 mM Tris-acetate (pH 7.5), supplemented with 1 mM MnCl₂. Cells were disrupted by sonication with a Vibracell apparatus (Sonics & Materials Inc.). The soluble protein fraction was obtained after ultracentrifugation at 130,000 x g, for 60 min, in a Beckman ultracentrifuge L-80. Crude extracts (0.1 - 5.3 mg of proteins per ml) were used without further purification. Enzyme activities were determined in spectrophotometrical assays as described previously (Pérez-Pantoja et al. 2000).

BKME microcosms experiments. BKME microcosms consisted on BKME or sterile BKME 10-ml batch cultures (Valenzuela et al. 1997). The microcosms were aerated at a constant rate (1 ml air/min/ml), and separately inoculated (50% v/v) with late log cultures of strains JMP134, JMP222, RB2 or their tfd genes containing derivatives, and incubated at room temperature (20-25ºC), with or without addition of 1 mM 3-CB. Survival of each introduced strain was determined by viable counts plating samples taken after 6 days of incubation in the corresponding selective medium. Removal of 3-CB was followed by UV spectrophotometry, after 0, 3 and 6 days of incubation.

Isolation and characterization of a chloroguaiacol-degrading bacterial strain

 A. lwoffii RB2 was isolated from BKME by its ability to use guaiacol as sole carbon source. The ability to grow and/or dechlorinate typical BKME chloroaromatic pollutants was studied. Growth tests were carried out in minimal medium liquid cultures with 0.2-2 mM carbon source, and incubated for up to 10 days. The strain was only able to grow in simple, non-chlorinated aromatic compounds like benzoate and phenol (Table 2). However, the strain was able to dechlorinate some chloroguaiacols and chlorocatechols (Table 2). These observations suggest that strain RB2 has the ability to produce and partially metabolise chlorocatechols from some representative BKME chloroaromatic compounds. In fact, a gas chromatography- mass spectra analysis, performed as previously described (Céspedes et al. 1996), of supernatant of resting cells pre-grown on guaiacol and exposed to 4- or 5-chloroguaiacol showed the formation of the respective chlorocatechol (data not shown). The ability to produce chlorocatechols and dechlorinate chloroguaiacols was dependent on the growth substrate, because cells grown on LB rich medium were less efficient than cells of strain RB2 grown on guaiacol (Figure 2).

Catabolic properties of bacterial strains engineered with a chlorocatechol-degrading tfd gene cluster

Three miniTn5 derived plasmids containing a differentially regulated tfd_ICDEF gene cluster were constructed: lacI^q/P_trctfd_ICDEF (pITFD), tfdR/P_tfd-Itfd_ICDEF (pDTFD) and P_tfd-Itfd_ICDEF (pTFD). pITFD harbors a tfd gene cluster that is responsive to IPTG through the heterologous lacI regulator. The other two contains the upstream controlling region of the tfd_ICDEF, P_tfd-I, but only one of them (pDTFD) has also the putative master regulatory gene (tfdR) for tfd gene expression. The appropriate expression of tfd genes would allow chlorocatechol produced by initial reactions encoded in the recipient strains to be further metabolised (Figure 1c), and eventually, allow growth of bacterial strains on monochloroaromatic compounds.

Each tfd gene cluster was introduced into the chromosome of the recipient cells by triparental mating which allows miniTn5 mediated integration of each cluster due to the use of a suicide plasmid in recipients lacking the lpir protein (Herrero et al. 1990; de Lorenzo et al. 1990). Transconjugants of strains JMP222 or KT2442 were selected in agar plates with benzoate plus kanamycin, whereas transconjugants of strain RB2 were selected in guaiacol plus kanamycin. All clones tested gave positive PCR amplification for tfd_ICD and tfd_IEF sequences. Confirmation of chromosomal tfd_ICDEF insertions in selected clones was obtained by Southern analysis of a genomic EcoRI digestion probed with biotinylated pITFD DNA (data not shown). Furthermore, the absence of plasmid DNA in cell lysates prepared by a described procedure (Kado and Liu, 1981), the Ap^s phenotype, and the stability of the kanamycin marker after growth on LB medium without antibiotic indicated chromosomal insertion of the internal sequences of each miniTn5 derived vector. Six to ten clones from each strain/gene cluster were checked as indicated above, and no significant differences were found among them. Therefore, two clones of each type were selected for enzyme activities and dechlorination tests. For simplicity, only the results of one clone of each strain/gene cluster are given in Table 3. To make a proper comparison we used cells grown on benzoate in the presence of 3-CB or IPTG as inducers. Induced cells of R. eutropha JMP222 containing the regulated clusters, exhibited 63% to 78% of the chlorocatechol 1,2-dioxygenase specific activity detected in strain JPM134 (0.51 U/mg, Table 3). Non-induced cells gave 0% to 6% of the TfdC activity of the induced strain JMP134, and were as low as in the non-induced strain JMP134 (0.04 U/mg). The chloromuconate cycloisomerase assay indicated that induced cells expressed as high as 38% of the level of the wild type strain (0.08 U/mg). Rates of dechlorination of 3-CB were in agreement with the level of Tfd enzymes (last column, Table 3). Cells of P. putida KT2442 containing the tfd_ICDEF cluster regulated by IPTG or 3-CB, were also analysed (Table 3). TfdC specific activities were also lower than in wild type strain JMP134. Again, rates of dechlorination of 3-CB agreed with Tfd activities. On the other hand, the activity of Tfd enzymes in strains containing P_tfd-Itfd_ICDEF insertions, (therefore lacking tfdR), was five to ten-fold lower than those induced clones containing a regulated tfd_I cluster (Table 3). In the case of A. lwoffii RB2 derivatives containing the tfd_ICDEF cluster, the activity of the Tfd enzymes were almost negligible (Table 3, only results with the lacI^q/P_trctfd_ICDEF gene cluster are shown). Despite of that, 3-CB dechlorination tests gave significant results for the ITFD clone (Table 3, last row). In addition, cells of this derivative grown on guaiacol, but not on LB medium, showed higher dechlorination of 4- and 5-chloroguaiacol in the presence of the inducer IPTG (Figure 2).

Two hundred clones of R. eutropha JMP222, P. putida KT2442 or A. lwoffii RB2 containing one of each regulated tfd_ICDEF cluster, were streaked in agar plates containing 3 mM 3-CB or monochloroguaiacols, respectively, with or without the corresponding inducer. In spite of up to 7 days of incubation only very small colonies were observed. No growth was attained after transfer of these colonies to fresh medium. Liquid cultures were also unsuccessful to allow these clones to proliferate. In contrast, under these conditions, liquid cultures or agar plates containing 1-5 mM 3-CB allowed growth of strain JMP134 after 1-2 days.

Catabolic performance of tfd genes-engineered derivatives in BKME microcosms

The ability to degrade 3-CB in complex systems by the tfd genes-engineered strains R. eutropha JMP222::DTFD4, and A. lwoffii RB2::ITFD3 was assessed in BKME microcosms. The presence of R. eutropha JMP222::DTFD4 provoked significant removal of 3-CB (Table 4). Microcosms non inoculated, or inoculated with the parental strain JMP222, were completely unable to remove this compound. The wild type strain JMP134, containing the full set of tfd genes showed a significantly higher catabolic performance in these microcosms (Table 4). On the other hand, the presence of tfd genes in the A. lwoffii derivative did not have much effect in removal of 3-CB in BKME microcosms (Table 4). No significant reduction of the introduced population was detected with any of the bacterial strains used as inoculant, with the sole exception of R. eutropha JMP222::DTFD4 (Table 4). In sterile BKME microcosms all strains survive, after six days of incubation.

In this work we assessed if the introduction of a specialized gene cluster for chlorocatechol degradation in strains accumulating these intermediates, would improve their catabolic properties. Although expression of Tfd enzymes was obtained, the level of activity with the three catabolic strains used, was always lower than with R. eutropha JMP134, the source of catabolic genes. This lower activity was also evident in dechlorination, which is produced by the presence of Tfd enzymes and, in the catabolic performance of derivatives introduced into BKME microcosms. The presence of one copy of the tfd_ICDEF cluster was also not enough to allow R. eutropha JMP222, P. putida KT2442 or A. lwoffii RB2 to grow on 3-CB. There are several possible explanations for this catabolic behaviour. One possibility is that the regulatory systems used here do not allow adequate gene expression. However, as similar Tfd expression levels were obtained with two different regulatory systems used, this explanation may be disregarded. It is also possible that a particular chromosomal position of the inserted cluster prevented adequate gene expression. However, integration driven by Tn5 does not have strong sequence preference (Ahmed and Podemski, 1995; de Lorenzo et al. 1990), and we analysed two hundred clones of each type, without any success to find growth on 3-CB or monochloroguaiacols. On the other hand, an interesting possibility is that the tfd_ICDEF cluster itself is not enough to support adequate rates of monochlorocatechol transformation in vivo. During growth on 3-CB, part of it is transformed into 3-chlorocatechol, and then to 2-chloromuconate (Pieper et al. 1993). The latter is non-efficiently degraded due to poor activity of chloromuconate cycloisomerases (Pieper et al. 1993; Vollmer et al. 1999; Pérez-Pantoja et al. 2000). This limitation may be avoided in strain JMP134 by the presence of two tfd clusters. Interestingly, a pJP4 rearrangement leading to an enlarged plasmid that includes a duplication of the tfd_ICDEF and tfd_IICDEF sequences has been recently reported to improve growth of R. eutropha JMP134 on 3-CB (Clément et al. 2001). Furthermore, another recent study from our lab showed that the introduction of each tfd gene cluster into R. eutropha JMP222 or P. putida KT2442, cloned in the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene, allows these strains to use 3-CB as sole carbon source (Pérez-Pantoja et al. 2000), suggesting a gene dosage effect.

From a biotechnological and an evolutionary point of view, this work suggests that the sole acquisition of the tfd_ICDEF cluster, which has a similar organization to those clusters from plasmids p51 (Van der Meer et al. 1991) and pAC27 (Ghosal et al. 1985), is not necessarily enough to allow growth on 3-CB. Introduction into the chromosome of P. putida KT2442 of a tcbCDEF gene cluster encoding chlorocatechol degradation neither allow selection of derivatives growing on 3-CB, unless direct selection on 3-CB was applied. In such case, only derivatives containing more than one copy of the tcbCDEF element were selected (Klemba et al. 2000).

The catabolic complementation for monochloroaromatic degradation in A. lwoffii RB2 worked even more poorly than with R. eutropha or P. putida strains. Unfortunately, the introduction in A. lwoffii RB2 of pJP4, or the pBBR1MCS-2 derivatives mentioned above could not be properly assessed because these plasmids are not stably replicated in this strain. Negative effects of heterologous gene expression or the presence of inhibitors may explain such poor performance, and specially the lack of correlation between Tfd enzymes activities and dechlorination rates. The dechlorination substrate profile of A. lwoffii RB2 was narrower than a closer strain A. junii 5ga (González et al. 1993), or the Gram-positive Rhodococcus ruber CA16 (Acevedo et al. 1995). The first strain was only able to dechlorinate monochloroguaiacols whereas the later two also degrade the dichlorinated ones. However, the choice of A. lwoffii as recipient for the tfd genes was based mostly by the fact that good survival may be also required in a bioremediation procedure. In this context, use of endogenous strains like A. lwoffii RB2 may be the first choice with respect to exogenous microorganisms.

Chloroaromatic degrading strains are intended to be used in polluted environments. Therefore, we also tested the catabolic abilities in BKME aerobic treatments microcosms. It has been shown that microbial activity significantly removes several chloroaromatic compounds from BKME, at the low (usually ppb) concentrations found (Céspedes et al. 1996). It has been also shown that BKME microcosms are able to remove higher amounts (usually ppm) of 2,4,6-trichlorophenol, 2,4-dichlorophenol, 3-CB or 2,4-D, if inoculated with R. eutropha JMP134 (Valenzuela et al. 1997). These observations along with that reported here indicate that only very efficient catabolic strains should be tested for removal of pollutants from systems like BKME.

R. Céspedes was a FUNDACION ANDES Ph.D. fellow.

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