Institute of Biology, College of Natural Sciences, Carleton University, Ottawa, Ontario, CanadaK1S 5B61
Faculty of Biology, The University, D-78457, Konstanz, Germany2
Author for correspondence: R. Campbell Wyndham. Tel: +1 613 520 2600 ext. 3651. Fax: +1 613 520 3539. e-mail: cwyndham{at}ccs.carleton.ca
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ABSTRACT |
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Keywords: aromatic, biodegradation, meta ring fission, lig genes
Abbreviations: Ap, ampicillin; Cm, chloramphenicol; HCMS, 2-hydroxy-4-carboxymuconate semialdehyde; HCMSD, HCMS dehydrogenase; Km, kanamycin; MMA, minimal medium A; OCA, 4-oxalocitramalate aldolase; Pca, protocatechuate; PDCH, 2-pyrone-4,6-dicarboxylic acid hydrolase; PMD, Pca 4,5-dioxygenase
The GenBank accession number for the sequence reported in this paper is AF305325.
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INTRODUCTION |
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Comamonas (formerly Pseudomonas) testosteroni, a ß-proteobacterium, was used in pioneering studies on the Pca meta pathway (Dagley et al., 1968 ; Dennis et al., 1973
; Wheelis et al., 1967
) and is the organism from which the ring cleavage enzyme, Pca 4,5-dioxygenase (PMD), was first purified and characterized (Arciero et al., 1990
) and in which the metabolic pathway via the pyrone to oxaloacetate and pyruvate (Fig. 1a
) was elucidated (Kersten et al., 1982
). Four other Pca meta pathway enzymes were also purified and characterized from the non-fluorescent bacterium Pseudomonas ochraceae (Maruyama, 1979
, 1983a
, b
, 1985
, 1990a
, b
; Maruyama et al., 1978
) and five of the corresponding genes have been studied in the
-proteobacterium Sphingomonas paucimobilis SYK-6, an organism used to investigate degradation of model lignin compounds (Hara et al., 2000
; Masai et al., 1999
, 2000
; Noda et al., 1990
). This bacterium also served as the source of PMD in crystal structure studies (Sugimoto et al., 1999
).
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METHODS |
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N-terminal amino acid sequences of blotted proteins (gel filtration step) were determined after Edman degradation (Schläfli et al., 1994 ). Reversed-phase HPLC of the ring cleavage product, 2-hydroxy-4-carboxymuconate semialdehyde (HCMS), was done with the method established by Locher et al. (1989)
with an apparatus described by Laue et al. (1996)
. HCMS was generated in an oxygen-dependent conversion of Pca catalysed by partially purified PMD (DEAE step) from strain BR6020 (see Table 2
).
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Generation of BR6020 with a disrupted pmdA.
A summary of steps for the construction of a recombinational disruption cassette for pmdA is provided in Table 1 and a schematic diagram is provided in Fig. 2
. The cassette contained a site-specific cross-over region and a Cm resistance marker and was cloned into the suicide-delivery vector, pRR1, resulting in pSMpmdACm5. The latter can be transferred by conjugation but possesses an R6K oriV and can thus only be maintained as an independent plasmid in hosts encoding
pir (de Lorenzo et al., 1990
). Recombination at the cross-over region results in duplication of this section, complete integration of the plasmid and insertional inactivation of the gene. The knock-out vector was mobilized from E. coli CC118
pir into C. testosteroni BR6020 via tri-parental filter mating (Nakatsu & Wyndham, 1993
) and transconjugants were recovered on succinate-MMA agar with Cm. Some spontaneous Cm resistance was observed in controls, but a true pmdA mutant, designated BR6020::pmdA, was recognized by its inability to grow after being patched to MMA containing p-hydroxybenzoate and Cm. Proper integration of the knock-out vector was confirmed by Southern blotting. To test whether disruption of pmdA affected complete metabolism of various aromatic growth compounds (see Results), BR6020::pmdA was cultured initially on succinate-MMA agar with Cm, patched to MMA agar containing Cm and an aromatic growth substrate and scored for growth after 2 to 7 d incubation.
Assay for Pca production.
The ability of BR6020 and BR6020::pmdA to generate Pca when grown on succinate in the presence of various aromatic substrates was determined using the method of Parke for detection of vicinal diols (Parke, 1992 ), with minor modifications. In brief, bacteria were cultured for 48 h at 32 °C on MMA agar (
20 ml medium per plate) containing succinate, an aromatic substrate, Cm for BR6020::pmdA and spread onto plates prior to addition of bacteria, 70 µl of a 50 mM aqueous FeCl3 solution (filter-sterilized) and 100 µl of a 0·1 M p-toluidine solution in dimethylformamide. Production of Pca resulted in a dark reddish-brown halo around colonies.
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RESULTS |
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Separation and analysis of PMD from C. testosteroni BR6020
A low level of PMD activity [0·2 mkat (kg protein)-1] was observed in extracts of succinate-grown cells, while high activity was observed in Pca-grown cells [6·9 mkat (kg protein)-1]. The inducible enzyme is unstable and initial purification attempts used the protective buffers described by Arciero et al. (1990) , but they had little effect. The protocol presented here is a modified version of an established procedure used for purification of PMD from C. testosteroni T-2 (Mampel, 2000
). It allowed us to separate sufficiently pure, active enzyme such that we could determine the relative molecular masses (18 and 31 kDa, respectively) and N-terminal amino acid sequences of the
- and ß-subunits (ALEKPYLDVPGTI and ARITASVFTSHVP, respectively). The reaction product from separated enzyme, HCMS, was identified by co-chromatography and identical UV-visible spectra with authentic material generated by whole cells of C. testosteroni BR6020. Analyses were at pH 2·2 (
max 283 nm) and pH 6·7 (
max 411 nm).
Cloning and sequence analysis of the pmd locus in C. testosteroni BR6020
Three clones in the plasmid library, pLIB8H4, pLIB20G12 and pLIB20F2 (Table 1 and Fig. 1b
), were positive for PMD activity, as judged by conversion of Pca to HCMS on plates. Based on restriction mapping, the three clones represented a contiguous 10·8 kb chromosomal region and the complete sequence was determined. Seven ORFs in an area spanning nt 6617740 of this locus were identified based on homologies to entries in the GenBank database (Table 3
) and these were designated pmdKEFDABC (Figs 1b
and 3
). The conceptual translation of the N-termini of pmdA and pmdB (Fig. 3
) and the derived molecular masses of the products (Table 3
) corresponded to data obtained from separated PMD (see above), thus confirming that these genes encode the two subunits of this enzyme. Tentative functions for products of the other ORFs from the pmd locus (Fig. 1a
) were attributed based on sequence identity to entries in the GenBank database and similarity to purified proteins of the Pca meta pathway from S. paucimobilis SYK-6 and P. ochraceae (see Table 3
and Discussion). A 22 nt inverted repeat and a potential stemloop structure were detected between pmdF and pmdE, and another potential stemloop was found following pmdC (Fig. 3
). However, their significance remains to be elucidated. The regions flanking the pmd locus (Fig. 1b
) presumably encode proteins for other aspects of bacterial metabolism and are not discussed here. In all three clones, the pmd genes are read in the same direction as the plasmid promoter (Plac). We attempted to IPTG-induce pmd expression in liquid cultures of E. coli to determine whether Pca was converted to pyruvate by whole cells or cell-free extracts, but negligible metabolism of Pca was measured. The colour change observed on plates by cultures exposed to Pca presumably reflected a low level of initial conversion of the substrate.
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Effects of disrupting pmdA on aromatic metabolism by C. testosteroni BR6020
Strain BR6020::pmdA, containing an interrupted gene for the -subunit of PmdAB (see Methods), was not able to grow with Pca, nor were any residual levels of PMD activity detected in succinate-grown cells. In addition, the mutant could not grow with the aromatic growth substrates shown in Fig. 1(a)
, although each of the compounds could still be converted to Pca. The mutation obviously affected the lower pathway for degradation of Pca and not the upper pathways that generate this compound from a range of aromatic precursors. Strain BR6020::pmdA was able to grow normally with benzoate and o-aminobenzoate, indicating that these are not degraded via Pca. In addition, no vicinal diols were detected when BR6020 or BR6020::pmdA were grown on succinate in the presence of these compounds, indicating that they are not converted to catechol, a known metabolite for these substrates in many other bacteria (see Discussion). Strain BR6020::pmdA also grew with gentisate.
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DISCUSSION |
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Other ORFs physically linked to pmdAB were identified (Figs 1b and 3
) and potential roles were inferred based on high identity to proteins of known function (summarized in Fig. 1a
and Table 3
). PmdK shows similarity to members of the aromatic acid:H+ symporter subclass of the major facilitator superfamily (Pao et al., 1998
). Other examples of this subclass are responsible for transport of Pca, p-hydroxybenzoate, benzoate and 2,4-dichlorophenoxyacetate (Table 3
), and although this remains to be shown, PmdK may mediate uptake of Pca. PmdC, PmdD and PmdE appear to be, respectively, the HCMS dehydrogenase (HCMSD), 2-pyrone-4,6-dicarboxylic acid hydrolase (PDCH) and 4-oxalomesaconate hydratase of BR6020 based on similarity to LigC, LigI and LigJ, respectively, of S. paucimobilis SYK-6 (Table 3
). In addition to high sequence identity as evidence of proposed functions, the derived amino acid compositions (Table 4
) and molecular masses of PmdC and PmdD (35·2 and 34·4 kDa, respectively) are similar to those reported, respectively, for the 35 kDa monomer of the HCMSD and the 33 kDa PDCH from P. ochraceae (Maruyama, 1983b
; Maruyama et al., 1978
). Moreover, with respect to PmdD, we have generated a BR6020 strain with a disrupted pmdD using a method similar to the one described here for pmdA and the growth characteristics of this strain on various aromatic substrates were identical to those obtained with BR6020::pmdA (unpublished data), further evidence showing that the product of pmdD is involved in the Pca meta pathway of BR6020. PmdF appears to be 4-oxalocitramalate aldolase (OCA) based on a similar derived amino acid composition (Table 4
) and molecular mass (24·0 kDa) to the 26 kDa monomer of the homohexameric OCA from P. ochraceae (Maruyama, 1990a
). This aldolase differs from typical Schiffs base-forming (Class I) aldolases (Maruyama, 1990a
) and instead shares biochemical features with an E. coli methyltransferase (Maruyama, 1990b
). PmdF does not possess any of the consensus signature sequences of Class I aldolases but instead shows sequence homology to hypothetical transferases (Table 3
).
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This study provides evidence for a single lower pathway in C. testosteroni for the metabolism of Pca, which is generated by a variety of upper pathways acting on many aromatic substrates. This contrasts with the situation sometimes observed in other bacteria, which can possess alternative lower pathways for metabolism of the same diol. Examples include Pseudomonas putida, which metabolizes catechol generated from benzoate by an ortho pathway (Harwood & Parales, 1996 ), but catechols generated from toluates by a meta pathway (Assinder & Williams, 1990
); or the presence of three dedicated lower meta pathways in Alcaligenes sp. O-1 for metabolism of the catechols generated by distinct upper pathways (Junker et al., 1994
). The upper pathways of BR6020 addressed in this study are all chromosomally encoded (Table 1
), but plasmid-encoded upper pathways in some C. testosteroni strains for conversion of aromatic compounds to Pca have also been reported, such as cba for chlorobenzoates, tsa for p-toluenesulfonate and psb for p-sulfobenzoate (Junker et al., 1997
; Nakatsu & Wyndham, 1993
; Wyndham et al., 1988
). These plasmid-encoded upper pathways, which are widespread in the environment and can be acquired by horizontal gene transfer (Nakatsu et al., 1995a
; Peel & Wyndham, 1999
; Tralau et al., 2001
), require a functional Pca meta pathway for complete metabolism of the respective aromatic substrates. In the case of cba-encoded metabolism of m-chlorobenzoate, a disrupted pmdA also results in growth defects on this compound (unpublished data) and we are currently investigating whether the same occurs with the latter two pathways. As well, we are studying the distribution and degree of conservation of the pmd locus in other C. testosteroni strains and various aromatic-degrading environmental isolates.
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ACKNOWLEDGEMENTS |
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Received 29 January 2001;
revised 18 April 2001;
accepted 26 April 2001.