German Research Centre for Biotechnology (GBF), Division of Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
Correspondence
Bernd Hofer
bho{at}gbf.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the novel bphA1 core segment sequences reported in this study are AJ544517AJ544525.
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INTRODUCTION |
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Previous studies with class II ARHDO systems have demonstrated that the major determinants of the fundamental catalytic properties such as substrate and product spectra reside within the C-terminal part of the large or -subunit (Erickson & Mondello, 1993
; Kimura et al., 1997
; Mondello et al., 1997
; Beil et al., 1998
; Zielinski et al., 2002
), although the small or
-subunit has occasionally been reported to exert some influence on these properties (Furukawa et al., 1994
; Hurtubise et al., 1998
). Recently, it has been shown that the C-terminal 60 aa of the
-subunit are of minor importance for these properties (Zielinski et al., 2002
). Thus, if consensus oligonucleotide primers could be derived from conserved sequences flanking the part of the gene that encodes the catalytic centre, such segments could be rapidly amplified from, for example, bacterial isolates, microbial consortia or just samples of nucleic acids. These segments could be reconstituted into complete ARHDO gene clusters by fusion with the missing sequences from a helper operon. If the resulting hybrid genes would express catalytically active enzymes, such a system should be a powerful tool for the rapid isolation and characterization of naturally occurring ARHDO activities.
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METHODS |
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Bacterial strains and plasmids.
The following strains were used in this study: Escherichia coli strains DH5 (Grant et al., 1990
), DH10B (Grant et al., 1990
) and BL21(DE3)(pLysS) (Studier, 1991
); Burkholderia sp. strain LB400 (Bopp, 1986
; Fain & Haddock, 2001
); Pseudomonas sp. strains B2A, B3B, B4, B6K and B7A (Bartels et al., 1999
); Ralstonia eutropha H850 (Bedard et al., 1987
; Williams et al., 1997
); Ralstonia sp. strains B11 and B15 (Bartels et al., 1999
); Rhodococcus globerulus P6 (Furukawa et al., 1978
; Asturias et al., 1994
); Rhodococcus opacus BIE-20 (Wagner-Döbler et al., 1998
); Sphingomonas yanoikuyae Q1 (Furukawa et al., 1983
; Wang & Lau, 1996
). pAIA6000 is a pT7-6-based expression plasmid harbouring genes bphA1m2A3A4BC of strain LB400; bphA1m contains silent mutations that generate NdeI and XhoI sites (Zielinski et al., 2002
).
Bacterial cultures and preparation of resting cells.
Soil bacteria were grown at 30 °C in Luria broth (Bopp et al., 1983) or in minimal medium with biphenyl as carbon source as described previously (Bartels et al., 1999
). E. coli DH strains harbouring a pAIA plasmid were grown at 37 °C in LB medium (Sambrook et al., 1989
) containing 100 µg ampicillin ml-1. E. coli BL21(DE3)(pLysS) strains harbouring a pAIA plasmid were grown at 30 °C in LB medium containing 50 µg chloramphenicol ml-1 and 100 µg ampicillin ml-1. For the preparation of resting cells, the latter strains were grown to an OD600 value of about 1·0. IPTG was then added to 0·4 mM (final concentration), and the incubation was continued for another 45 min. Cells were harvested, washed with 50 mM sodium phosphate buffer (pH 7·5) and then resuspended in the same buffer to an OD600 value of 5·0.
DNA techniques.
In vitro DNA modifications, agarose gel electrophoresis (AGE) and transformations were carried out according to standard protocols (Sambrook et al., 1989), unless described in detail.
The introduction of an AflII site into bphA1m-LB400 was done as follows. Plasmid pAIA6000 was used as template to amplify two overlapping parts of its bphA1m gene by PCR (Mullis & Faloona, 1987), using primers BPH2454M and BPH-2651M or BPH2632M and BPH-2711, respectively. Both PCR products (198 and 80 bp, respectively) were purified via AGE and then used as template for overlap extension PCR (Higuchi et al., 1988
) with primers BPH2454M and BPH-2711. The gel-purified product (258 bp) was cut with XhoI and AgeI and then cloned into identically cleaved, dephosphorylated pAIA6000. This yielded plasmid pAIA6100. The integrity of its insert was verified by DNA sequencing.
For PCRs with consensus primers, a small sample of cells was suspended in 20 µl of water and heated to 95 °C for 4 min. After spinning down cell debris with a table top centrifuge, 2 µl of supernatant were used as template in the PCR. Thirty cycles with an annealing temperature of 60 °C, an elongation time of 90 s (with an increment of 3 s per cycle) and otherwise standard conditions were used.
To generate ARHDO -subunit fusion genes, PCR products of the consensus primers were purified by AGE, cut with MluI and AflII and then ligated with identically cleaved, dephosphorylated pAIA6100. The resulting clones were screened for ARHDO activity (see below) and further analysed by restriction and insert sequencing.
For DNA sequence determinations, about 1 µg of plasmid or 0·050·2 µg of PCR product was subjected to Taq DNA polymerase-catalysed cycle sequencing as described previously (Bartels et al., 1999). The sequences of the novel bphA1 core segments have been deposited in the EMBL/GenBank/DDBJ data libraries under accession numbers AJ544517AJ544525.
Protein gel electrophoresis.
Resting cells were lysed with a cracking buffer (Tabor & Richardson, 1985) and proteins were separated by 0·1 % SDS-15 % PAGE as described previously (Hofer et al., 1993
). Gels were stained with Coomassie brilliant blue R250 (Sambrook et al., 1989
).
ARHDO activity measurements.
ARHDO activity was assayed with biphenyl as substrate. Colonies were tested by dispensing some crystals of biphenyl into the lid of the Petri dish. As pAIA6100 and its derivatives also expressed bphB and bphC, ARHDO activity led to the conversion of biphenyl into 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, which was observed by eye (max=434 nm). ARHDO activity of resting cells was determined by incubation of a cell suspension with an OD600 value of 5·0 with 0·1 mM biphenyl at room temperature. At intervals, the A434 values of the supernatants were measured.
Depletion of aromatic compounds.
A mixture of aromatic compounds (see Table 2) and 2,4,6,2',4'-pentachlorobiphenyl (as internal standard) were added to resting cells with an OD600 value of 5·0 to a final concentration of 12 µM each. After vortexing for 10 s, the teflon-sealed tubes were shaken at 30 °C for 24 h. Thereafter, the suspensions were extracted with 1 volume of n-hexane, and 5 µl of the organic phase were analysed in an HP 5890 Series II gas chromatograph (GC) equipped with a flame-ionization detector and an HP Ultra2 column (length, 50 m; inner diameter, 0·2 mm; film thickness, 0·11 µm). The carrier gas was hydrogen. The injector was held at 250 °C. The GC temperature programme used was as follows: 5 min at 40 °C, linear gradient of 4 °C min-1 to 288 °C and 10 min at 288 °C. The flame-ionization detector was operated at 300 °C. GC peak areas were normalized to the internal standard. Background depletion of substrates was determined by using resting cells devoid of ARHDO genes.
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RESULTS AND DISCUSSION |
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The concentrations of wild-type and hybrid -subunits and of wild-type
-subunits were analysed by denaturing PAGE of SDS-lysed cells (Fig. 3
). After staining with Coomassie blue, bands of both subunits were clearly visible in all cases, indicating high level expression of the respective genes.
Substrate specificities of the hybrid ARHDOs were assessed by incubation of a mixture of aromatic compounds with the respective E. coli recombinants and analysis of their depletion. The data show that the substrate spectra of the hybrid enzymes differed from each other and from that of the strain LB400 ARHDO (Table 2). This result confirms that the substrate range is crucially determined by the replaced
core segment. A detailed analysis of substrate and product spectra of selected hybrid ARHDOs will appear elsewhere.
For comparison, the same assays were carried out with the donor strains of the core segments (Table 2). In most cases, similar degrees of substrate depletion were observed as with the respective E. coli recombinants, indicating that the properties of the hybrid ARHDOs often, but not always, reflect the properties of the donor strains. Minor differences were found occasionally that indicated more depletion by the recombinants. This may result from a higher level of gene expression in the E. coli strains that harbour multiple gene copies downstream of a strong phage T7 late promoter. Whenever major deviations in substrate depletion occurred, donors exhibited significantly higher depletions of certain compounds than the recombinants. A straightforward explanation would be the existence of more than one ARHDO in these strains. This has previously been observed in bacteria (Kim & Zylstra, 1999
; Kitagawa et al., 2001
).
The limitations of the approach described here are that it relies upon DNA sequence similarity with the consensus primers and compatibility of protein three-dimensional structures between donor and recipient. An in silico analysis indicated that more than 80 % of the cores of sequenced genes encoding -subunits of ARHDOs that are known or likely to belong to class II or to accept benzene or benzene derivatives as substrates should be amplified with the described primers. The genes of the entire ARHDO family are too diverse to permit amplification by a single pair of oligonucleotides. However, different sets of primers may be designed for different ARHDO subclasses. A model of the three-dimensional structure of the class II biphenyl dioxygenase of strain LB400 (M. Zielinski, S. Kahl, H.-J. Hecht & B. Hofer, unpublished data), based on the known class III naphthalene dioxygenase structure (Carredano et al., 2000
), suggests that the structural similarity between these two classes of ARHDOs is high enough to make it likely that the fusion approach outlined here may also be applied to class III dioxygenases. The formation of a functional ARHDO hybrid will depend upon the structural compatibility of donor and recipient regions. This is not strictly correlated with the amino acid sequence similarity between the fusion components, since rather dissimilar sequences can result in quite similar structures. The class III dioxygenase structure and the class II dioxygenase model show that in the (
)3 hexamer the exchanged core segment has contacts with both terminal parts of its subunit, with the neighbouring
-subunit and with one of the two other
-subunits. It is located at the periphery of the hexamer and forms a fairly compact subdomain in itself. Thus, it is less likely that core replacements will severely disturb the overall structure of the hydroxylase complex.
In summary, our results suggest that the system outlined here will, in many cases, result in catalytically active ARHDOs. These hybrid enzymes possess distinct properties, depending on the donor segment. Therefore, the system could be used to rapidly screen a large number of bacterial isolates for ARHDO activity. It requires no strain selection or any knowledge of the induction of ARDHO activity. Moreover, the system may be applied directly to complex mixtures of organisms or their nucleic acids, respectively, such as present in environmental samples. This application may be particularly useful as only a minority of all microbes can currently be cultivated in the laboratory (Amann et al., 1995). Thus, the approach described here may be helpful in a functional characterization of the natural ARHDO diversity and may also allow access to a so-far-unattainable biocatalytic potential.
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ACKNOWLEDGEMENTS |
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Received 2 September 2002;
revised 10 December 2002;
accepted 18 February 2003.
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