UFZ Centre for Environmental Research, Department of Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig, Germany
Correspondence
Doreen Hoffmann
hoffmann{at}umb.ufz.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this article is AY078159.
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INTRODUCTION |
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2,4-D can be used as a carbon and energy source by various soil bacteria and therefore has become a model compound to study the distribution and evolution of catabolic genes for chloroaromatic compounds (Ka et al., 1994; Fulthorpe et al., 1995
, 1996
; Top et al., 1995
; Vallaeys et al., 1996
, 1999
; Hogan et al., 1997
; Kamagata et al., 1997
; McGowan et al., 1998
; Itoh et al., 2002
). Although micro-organisms capable of mineralizing 2,4-D have been investigated intensively, knowledge on the microbial degradation of chloroaromatics in extreme environments such as highly alkaline habitats is still very limited (Maltseva et al., 1996
). However, in addition to contaminated soils and (ground)water, pollution problems in the chemical industry often extend to the facilities themselves. One special problem in this context is toxic residues in pesticide factories and the microbial decontamination of building rubble obtained after their demolition, since aqueous eluates from this material are very alkaline. Microbes occupying such an ecological niche have to face both extreme pH values and toxic substrates.
In the case described here, the building rubble of a former herbicide production plant was heavily contaminated with organochlorines, especially chlorinated and methylated phenoxyalkanoates and phenols, and generated pH values of up to 12 in an aqueous environment (Müller et al., 1999a). A microbial consortium was enriched from this material which was able to mineralize the cocktail of contaminants under alkaline conditions. One of the strains isolated from this consortium was Delftia [formerly Comamonas (Wen et al., 1999
)] acidovorans P4a, which can utilize 2,4-D as sole sources of carbon and energy at pH values of up to 10, and has also exhibited degradative activity on concrete material in situ at overall pH values of up to 11·5 (Hoffmann et al., 1996
; Müller et al., 1996
).
Biodegradation of 2,4-D is catalysed sequentially by six degradative enzymes, encoded by tfdA, tfdB, tfdC, tfdD, tfdE and tfdF in the case of the well-characterized strain Ralstonia eutropha JMP134 (Don & Pemberton, 1985; Pieper et al., 1988
, 1993
). Enzymes with regulatory or transport functions encoded by tfdR, tfdS and tfdK, respectively, are also known (Kaphammer et al., 1990
; Kaphammer & Olsen, 1990
; Leveau & van der Meer, 1996
; Leveau et al., 1998
). The presence of tfd genes in D. acidovorans P4a has been revealed by analysis of PCR products generated using tfd-specific primers and genomic DNA of D. acidovorans P4a as a template (Hoffmann et al., 2001
). In the present study, the organization of the genes of the entire 2,4-D degradative pathway in strain P4a was elucidated, and the relationships of the individual genes to those of other strains were probed. In addition, the localization of the respective genes was looked for. Both these aspects were considered essential to understand the function and stability of 2,4-D biodegradation. These aspects are of special interest if one intends to use strains such as D. acidovorans P4a to initiate or improve bioremediation efficiency in such problematic biotopes as building rubble.
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METHODS |
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Screening of recombinant library clones.
To prepare DNA of the recombinant E. coli clones, the biomass of each freshly grown single colony was suspended in 50 µl sterile distilled water, boiled for 10 min, left on ice for 10 min and sedimented by centrifugation at 23 000 g and 4 °C for 10 min. From each supernatant, 4 µl was dripped onto a nylon membrane (Roche Diagnostics) and the DNA was fixed by UV irradiation. Clones were screened for genes encoding the 2,4-D degradation pathway with probes specific for the tfdA, tfdB, tfdC, tfdD and tfdF genes, obtained by PCR using genomic DNA from D. acidovorans P4a as a template and primers derived from conserved amino acid sequence motifs of corresponding homologous enzymes (Hoffmann et al., 2001). Oligonucleotides were synthesized by MWG BIOTECH. PCR and purification of the PCR products were performed as described by Hoffmann et al. (2001)
. The PCR products were digoxigenin (DIG)-labelled using the DIG DNA Labelling Kit (Roche). For hybridization with the DIG-labelled probes and immunodetection, the DIG Easy Hyb Wash and Block Buffer Set and DIG Nucleic Acid Detection Kit (Roche) were used. Hybridizations with tfdA, tfdB and tfdC probes were performed at 50 °C; tfdD and tfdF probes were hybridized at 60 °C. Two library clones, 59 and 1183, which showed hybridization signals with various tfd probes, were selected for further investigation.
Characterization of positive library clones and subcloning.
Cosmid DNA of the tfd-positive E. coli clones was isolated with the NucleoBond PC Kit and NucleoBond AX columns as recommended by the manufacturer (Macherey-Nagel). Restriction analyses were initiated by digesting the cosmid DNA with various restriction endonucleases (Roche; New England Biolabs). DNA fragments were then separated electrophoretically in 0·8 % agarose gels, blotted onto nylon membranes by standard procedures according to Sambrook et al. (1989) and fixed as described above. Southern blotting was performed with an Appligene vacuum blotter. Hybridizations with the tfdA, tfdB, tfdC, tfdD and tfdF probes were carried out as described above. The positive DNA fragments of the two library clones 59 (subclones A34, B108 and C93) and 1183 (subclone N11) were subcloned using pUC18 and pGEM3-Zf(+) as vectors and E. coli DH5
as a host strain. Recombinant subclones were screened by blue/white selection and plasmidal DNA of selected clones was isolated as described above. The presence of the expected insert was verified by restriction analysis.
DNA sequencing and sequence analysis.
The nucleotide sequences of the tfd-positive and subcloned restriction fragments of D. acidovorans P4a (inserts of A34, B108, C93 as well as N11 subclones) were determined according to the method of Sanger et al. (1977) using an ABI PRISM 310 Genetic Analyser (PE Applied Biosystems). To complete the sequence data, sequencing was extended to neighbouring regions in the cosmid DNA of the library clones 59 and 1183 applying the primer-walking technique (Alphey, 1998
; Strauss et al., 1986
). Special primers were derived from respective insert sequences. Each sequence was determined twice. Sequence data were analysed by the SEQUENCE NAVIGATOR software (pe applied biosystems) and contigs were assembled by the AUTO ASSEMBLER software (PE Applied Biosystems).
The comparison of sequences with DNA and protein sequences in sequence databases was performed with BLAST (Altschul et al., 1997; http://www.ncbi.nlm.nih.gov/blast). The sequence determined will appear in the GenBank/EMBL/DDBJ sequence databases under accession number AY078159.
Location of the tfdCDEF and tfdCIIEIIBKA operons.
Chromosomal DNA of D. acidovorans P4a was isolated by using the NucleoSpin C&T Kit (Macherey-Nagel), while plasmidal DNA was isolated by using the NucleoBond PC Kit (Macherey-Nagel) following the standard protocol of the supplier. DNA was digested with EcoRI and BglII (double digestion). DNA fragments were separated electrophoretically, blotted and fixed as described above, and subjected to Southern blot hybridization with the labelled PCR fragments tfdDEF (3·4 kb) and tfdAKB (2·5 kb) as probes at 60 °C. The tfdCDF probe was obtained by PCR carried out with genomic DNA of strain P4a using the specific tfdD forward and tfdF reverse primers described by Hoffmann et al. (2001). To prepare the tfdAKB probe, PCR was carried out with genomic DNA of strain P4a as template using 5'-CAT (A/G)TC (A/G)CA GAA CTC CGT-3' as the tfdA primer and 5'-GA(A/G) ATG AA(C/T) CAG CGC TAT-3' as the tfdB primer (complementary reverse sequences to the tfdA2 and tfdB1 primers described by Vallaeys et al., 1996
).
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RESULTS |
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The 2,4-D degradation pathway of strain P4a
The genome fragments of library clones 59 and 1183 were found to carry different tfd genes and several ORFs; nevertheless, in both library clones orfL, tfdA, tfdK and tfdB occurred. Based on these data, the sequences were assembled, resulting in a 28·4 kb segment of the genome of strain P4a, suggesting the organization of the degradative genes of the entire 2,4-D degradation pathway in P4a (Fig. 1). Accordingly, the degradation pathway apparently consists of the two different gene clusters tfdCDEF and tfdCIIEIIBKA, arranged in opposite transcriptional directions. The tfdR genes evidently associated with each of the two clusters are transcribed in the opposite direction to the respective clusters. Furthermore, the tfdE and tfdC genes occur twice, once in each cluster.
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The tfdCIIEIIBKA cluster.
The second gene cluster comprises five ORFs. An additional tfdC gene was detected which is identical in nucleotide sequence to that within the tfdCDEF cluster except for a short sequence of 19 bp at the 3' terminus. By contrast, a further tfdE gene was found, the sequence of which differs substantially compared to tfdE in the tfdCDEF cluster, corresponding to gene products with only 28 % identity. For differentiation, the tfdC and tfdE genes in this cluster were indexed. The gene cassette is arranged in the order tfdCIIEIIBKA and transcribed in the opposite direction to the tfdCDEF cluster. The tfdR gene, which presumably encodes the positive regulator of the tfdCIIEIIBKA cluster, is located upstream of tfdCII. Downstream of tfdR, another ORF is located which is identical to orfII in the vicinity of the tfdCDEF cluster.
IS sequences.
Sequence data from the 28·4 kb contig show that the genes of the 2,4-D degradation pathway are flanked by insertion elements of the IS1071 and IS1380 families (Mahillon & Chandler, 1998). This indicates that the tfd genes of strain P4a are located within a transposon-like structure.
The insertion elements belonging to the IS1380 families were completely sequenced and found to be 1744 bp long. The transposase gene (1·4 kb) is flanked on either side by inverted and direct repeats. The inverted repeats consist of 17 bp and their nucleotide sequences are nearly identical: 5'-CCCGGATGTTTCATAAA-3' and 5'-CCCGAATTTTTCATAAA-3'. The nucleotide sequence of the direct repeat is CTAG; it is located in front of the inverted repeat.
The insertion element belonging to the IS1071 family was completely sequenced in the right part of the segment (Fig. 1); the one on the left was only partially sequenced. A fragment of a further transposase gene, tnpA, is located in the intergenic region of the insertion elements IS1380 and IS1071 and showed high similarity to the transposase gene of Tn21.
Comparison of the sequence with sequence databases.
The chlorocatechol degradative operon of strain P4a exhibits high similarity in both its nucleotide sequence and in the arrangement of the genes to the homologous operons in other strains such as R. eutropha NH9(pENH91) [99 % identity (Ogawa & Miyashita, 1995, 1999
)], Pseudomonas chlororaphis RW71 [99 % identity (Potrawfke et al., 1998
, 2001
)] and Pseudomonas sp. P51(pP51) [97 % identity (van der Meer et al., 1991a
); Fig. 2
]. The lengths of the homologous ORFs, their overlaps and the lengths of the intervening sequences are identical in these gene clusters. Comparison of the nucleotide sequences of the complete operons indicated that the D. acidovorans P4a sequence differs in only five nucleotides in relation to R. eutropha NH9.
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DISCUSSION |
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Assuming that the tfd genes detected in the present study were adequately expressed, D. acidovorans P4a provides an excellent example of this kind of adaptation. Indeed, implication of the tfd genes in the metabolism of 2,4-D is highly reliable as this strain exhibits growth on this substrate being accompanied, moreover, by expressing respective enzyme activities as measured for TfdA, TfdB, TfdC and TfdD (D. Hoffmann, unpublished data). The genes of the chlorocatechol pathway (i.e. the tfdCDEF genes encoding the enzymes of the modified ortho pathway and the regulatory unit tfdR, controlling their transcriptional activity as shown for R. eutropha JMP134 by Leveau & van der Meer, 1996) are clustered (Fig. 2
), as they are in a range of other strains (van der Meer et al., 1991a
, b
, c
, d
; Leveau et al., 1994
; Potrawfke et al., 1998
, 2001
; Ogawa & Miyashita, 1999
). Sequence similarity even extends to the overlapping regions of some genes as well as to their intergenic regions. Clearly this kind of arrangement has emerged during the course of evolution, enabling the effective use of chlorocatechols, and is distributed as a module for the recruitment of degradative pathways.
A second cluster found in some organisms is distinguished by the presence of tfdA, encoding a 2,4-D/2-oxoglutarate dioxygenase (Streber et al., 1987; Fukumori & Hausinger, 1993
), in addition to tfdK, which was shown to encode a transport protein (Leveau et al., 1998
). The combination of these genes seems logical if active transport is required for exploiting phenoxyalkanoates as carbon and energy sources. Active transport of these compounds has been found to play a significant role in Sphingomonas herbicidovorans MH, in which inducible and substrate-specific uptake systems have been observed (Zipper et al., 1998
). By contrast, the lack of tfdK appears to have only minor negative effects in a mutant strain of R. eutropha JMP134 (Leveau et al., 1998
). Accordingly, uptake may also proceed by simple diffusion, which is a mechanism known for (uncharged) organic acids. In these two studies, the strains were grown under neutral pH conditions. With strain P4a, which is adapted to alkaline conditions, the uptake characteristics remain to be proven. One should take into account, however, that the dissociation equilibrium is shifted strongly towards the anion of the target compounds during growth under alkaline conditions, which means pH values of up to 10 with this strain (Hoffmann et al., 1996
). Hence, uptake by simple diffusion ought to be limited by the concentration of the free acid of 2,4-D, making it likely or even essential to support utilization of this compound by a specific uptake system.
The second tfd cluster, tfdCIIEIIBKA, also includes tfdB, which is known to encode a chlorophenol hydroxylase (Perkins et al., 1990). This apparently completes the 2,4-D degradative pathway in strain P4a. The structure of this gene cassette appears to be identical to that found in other strains (Fig. 2
; Vallaeys et al., 1996
; Vedler et al., 2000
; Poh et al., 2002
) and, again, transcription of the genes is presumably governed by tfdR. Two sets of tfdC and tfdE genes are found in strain P4a. It remains to be elucidated whether the expression of both these genes is essential for the proper function of the 2,4-D pathway in this strain or whether one of each is merely rudimentary. In R. eutropha JMP134, two complete sets of the chlorocatechol genes were also found, the expression of which was shown to speed up growth of this organism on 2,4-D (Leveau et al., 1999
; Laemmli et al., 2000
; Pérez-Pantoja et al., 2000
). According to the differences in their nucleotide sequences, both tfd modules must have been acquired from different evolutionary origins in R. eutropha JMP134. This can also be concluded for strain P4a when the differences between tfdE and tfdEII are considered: their gene products share only 28 % identity with each other but are highly similar to TfdE homologues in different bacterial strains (Table 1
).
This high degree of coincidence between the tfd genes and their arrangement suggests horizontal gene transfer as the likely explanation for the acquisition of the 2,4-D cassettes in strain P4a rather than being a feature evolved in this strain itself. The two different cassettes (tfdCDEF and tfdCIIEIIBKA) hint at two origins, although it cannot be excluded that a complete arrangement was captured by D. acidovorans from any strain. With special reference to building rubble, from which strain P4a was isolated, some questions regarding the generation of the ability to degrade 2,4-D remain obscure. These refer to the fact that the highly alkaline environmental conditions, which exceed pH 11·5 in aqueous surroundings of this material, preclude growth of D. acidovorans on this material. Thus, any conjugative processes are rather unlikely to occur in this very habitat. One can speculate, however, that micro-niches with moderate pH conditions, for example, mixing zones between building rubble and soil, are appropriate breeding areas for this characteristic. Starting from any primitive strain carrying and expressing the genetic information for 2,4-D degradation, strain P4a might have evolved in one of these micro-niches by gene transfer, genetic rearrangements and selection of the fittest. This also applies to the strains D. acidovorans MC1 and Rhodoferax sp. P230, which were isolated from the same habitat and are highly similar to strain P4a with reference to some of their tfd genes (Müller et al., 2001).
The nucleotide sequences found outside the 2,4-D degradation genes, i.e. downstream of the tfdR genes, are indicative of possible maturation processes and complete the picture of the recent strain D. acidovorans P4a. The tfd genes are flanked by transposons of the IS1380 and IS1071 families, each comprising a transposase gene in this DNA segment. Remarkably, the two segments are identical in structure. Moreover, the identity is extended to an ORF with unknown function, named orfII, and to both tfdR and tfdC. A further observation that may help elucidate the evolution of the 2,4-D degradation pathway in strain P4a is that a short sequence of tfdCII (19 bp, located downstream) deviates from that of tfdC in P4a and is identical to homologous sequences of other tfdC genes found in the tfdCEBKA cassettes of some other strains (Fig. 2). This implies that recombination events took place at this position with a gene descending from a different ancestor.
Another finding that should be emphasized is that the complete set of tfd genes is located on the chromosome in strain P4a, while in other strains known to carry chromosomally located tfd genes only parts of the 2,4-D degradation pathway are encoded on the chromosome (Ka et al., 1994; Matheson et al., 1996
; Suwa et al., 1996
). When we started investigating strain P4a we noted plasmids of various sizes ranging from 5 to about 60 kb, and found indications that tfd genes were located on them (Hoffmann et al., 1996
). However, we have had serious problems assigning the tfd genes to the respective plasmids. Recent investigations have shown that strain P4a now carries only one plasmid of about 21 kb, which is not associated with 2,4-D degradation. Therefore, under the selective pressure applied during laboratory cultivation, events occurred in the genetic structure that stabilized the degradative activities, leading to the emergence of the strain we describe as recent. The transposon structure is likely to have promoted these events in a kind of evolutionary optimization process, similar to phenomena observed in various other strains, raising questions about the pros and cons of carrying plasmids (Deutz et al., 1991
; Stouthamer & Kooijman, 1993
).
The chromosomal location of the genes of a pathway should certainly have consequences. Genes localized in the chromosomal matrix of the genome can be considered established, in contrast to plasmid-derived properties which can be classed as temporary or transitional. Taking into account that transposons are elements appropriate to quickly spread genetic information to suitable carriers, and plasmids are carriers capable of distributing genetic information into respective host strains, strains with plasmids harbouring respective information on transposons are fragile for this characteristic as they include 2 d.f. of losing this information. When it becomes localized on the chromosome, by contrast, even as a transposon, the d.f. is accordingly reduced. This prompts the question of whether P4a, as a strain of a species that prefers organic acids as carbon and energy sources (Tamaoka et al., 1987; Wen et al., 1999
), is in the process of adding a further acid, i.e. 2,4-D, to its spectrum of preferred substrates. Only a few mutation events which prevent this genetic element from transposing, for example, by changing target sites of transposase attack, avoiding expression of transposases or leading to inactive transposases, would be required to stabilize this property, making the ability to use the compound an inherent property of the strain. Stability of the degradative performance is by no means under selective pressure but becomes decisive if any strain is intended to be used for bioaugmentation as effective production of biomass will rely more on conventional carbon and energy sources. Even in recent times we have observed the occasional loss of the 2,4-D degradative capability in some clones, accompanied by the lack of all tfd genes.
The metabolic constellation of this alkalitolerant strain (P4a) enables the productive degradation of 2,4-D at pH values up to 10 in laboratory cultures (Hoffmann et al., 1996). Moreover, we have detected activity at up to pH 11·5 when strain P4a has been applied as an inoculum in a bioremediation process with building rubble (Müller et al., 1996
, 2000
). The capacity of the species to metabolize phenoxyalkanoates was also demonstrated with another strain, D. acidovorans MC1, which was isolated from the same alkaline environment and was able to utilize an even wider spectrum of compounds (Müller et al., 1999b
, 2001
). In contrast to these alkalitolerant strains, true alkaliphiles are unlikely to be able to degrade 2,4-D productively. This conclusion is consistent with results obtained with a soda lake isolate, Halomonas sp. EF43, supplied with plasmid pJP4 by conjugative transfer (Kleinsteuber et al., 2001
). Despite the expression of the tfd genes, this strain was unable to use 2,4-D as a sole source of carbon and energy, even though it was able to degrade the compound, at least in the presence of an additional carbon source (Kleinsteuber et al., 2001
). This may well also apply to another alkaliphilic strain belonging to the Halomonadaceae described by Maltseva et al. (1996)
, since the published data do not indicate that this isolate can make productive use of 2,4-D.
The property of catching and getting rid of genetic information is considered a mechanism to effectively settle niches and contributes to the metabolic resilience of biotopes. The present results suggest that the uptake of any genetic information for completing a degradative pathway, even as preformed and optimized cassettes, is only a precondition to enable the degradation of a given compound but may not be sufficient to do this in an effective, ultimately competitive, way. This refers to growth rate first but may include stability too, both of which are an expression of adaptation. The latter also applies for D. acidovorans MC1, a further strain isolated in this laboratory in 1999. In this case, we observed loss of the degradative trait under non-selective conditions in almost all overnight cultures. Today, derivatives of this strain exist which harbour this activity for at least 80 generations during growth on complex medium. Thus, stability is gained at the expense of flexibility.
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Received 27 January 2003;
revised 16 April 2003;
accepted 9 May 2003.
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