National Research Centre for Biotechnology (GBF), Division of Microbiology, Mascheroder Weg1, D-38124 Braunschweig, Germany1
Author for correspondence: Bernd Hofer. Tel: +49 531 6181467. Fax: +49 531 6181411.
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ABSTRACT |
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Keywords: aerobic bacteria, biphenyl catabolism, bph genes, glutathione S-transferase, 1-chloro-2,4-dinitrobenzene
Abbreviations: BP, biphenyl; BP+/-, phenotype able/unable to grow on biphenyl as sole carbon source; CDNB,1-chloro-2,4-dinitrobenzene; DBDO, 2,3-dihydroxybiphenyl 1,2-dioxygenase; GST, glutathione S-transferase; PAH, polyaromatic hydrocarbon
The EMBL accession numbers for the sequences determined in this work are AJ245981AJ245985.
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INTRODUCTION |
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GSTs form a family of heterogeneous, multifunctional enzymes which are found in animals, plants and micro-organisms (Fahey & Sundquist, 1991 ; Hayes & Pulford, 1995
). The best-studied GSTs are those from mammals, in which typically several isoenzymes are found (Mannervik, 1985
; Pickett & Lu, 1989
). In most instances, their exact physiological functions are not known.
The majority of eukaryotic GSTs are believed to primarily be involved in cellular detoxification of substances of xenobiotic origin (Pickett & Lu, 1989 ). Induction of their synthesis in response to the application of xenobiotics, for example certain polychlorobiphenyls or 2,4-dichlorophenoxyacetic acid, has been observed (Aoki et al., 1992
; Fujita et al., 1994
; Tanno & Aoki, 1996
). GSTs can enhance the aqueous solubility of hydrophobic xenobiotics by mediating coupling of the hydrophilic tripeptide GSH to such compounds. The resulting conjugates are then further metabolized and eventually excreted. Some GSTs appear to be involved in binding and transport of hydrophobic substances (Mannervik, 1985
; Pickett & Lu, 1989
). Others show peroxidase activity with organic peroxides (Prohaska, 1980
). Based on substrate specificity and primary structure, (potentially) cytosolic GSTs have been divided into six classes, alpha, mu, pi, sigma, theta and zeta (Hayes & Pulford, 1995
; Board et al., 1997
); additionally, classes phi (Blocki et al., 1993
) and delta (Toung et al., 1993
) have been proposed.
Prokaryotic GSTs, however, do not fit into this scheme (Di Ilio et al., 1993 ). Some of these enzymes have been shown to possess peroxidase activity (Nishida et al., 1994
), to act as epoxide thiolases (Arca et al., 1988
; van Hylckama Vlieg et al., 1998
), or to catalyse different essential reactions in the metabolism of compounds such as gentisate, dichloromethane, lignin or tetrachlorohydroquinone that can thereby serve as growth substrates for the respective microbes (Crawford & Frick, 1977
; La Roche & Leisinger, 1990
; Masai et al., 1993
; Nohynek et al., 1996
). The in vivo functions of other bacterial GSTs, such as BphK, remain obscure. To obtain more insight into the role of BphK, we examined the expression of its gene in the parental organism, Burkholderia sp. strain LB400, the response of bphK expression to growth of the bacterium on biphenyl (BP) and the requirement of the gene for utilization by the organism of BP as sole carbon source. Furthermore, we investigated the substrate range of the enzyme and the distribution of bphK genes and of BP-inducible 1-chloro-2,4-dinitrobenzene (CDNB)-accepting GST activity in other strains able to use BP as sole carbon source.
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METHODS |
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Enzyme activities.
These were measured in 1 cm quartz cuvettes in a UV2100 Shimadzu spectrophotometer equipped with a temperature-controlled cuvette holder. GST activity was assayed by the method of Habig et al. (1974) at 25 °C, unless otherwise indicated. The standard reaction mixture (600 µl) contained 0·1 M potassium phosphate buffer (pH 6·5), 1·0 mM GSH, 1·0 mM CDNB and 1060 µl cell extract. Other buffers used (Fig. 1
) were also 0·1 M at pH 6·5. The influence of temperature, salt concentration and pH were assayed with potassium phosphate buffer. Enzyme activity was calculated by using a molar absorption coefficient for the CDNBGSH conjugate of 9600 M-1 cm-1 at 340 nm (Habig et al., 1974
). 2,3-Dihydroxybiphenyl 1,2-dioxygenase (DBDO) activity was assayed at 25 °C in a volume of 600 µl containing 50 mM potassium phosphate buffer (pH 7·5), 0·3 mM 2,3-dihydroxybiphenyl (supplied by Wako Chemicals) and 110 µl of cell extract. Absorption was monitored at 434 nm. Enzyme activity was calculated using a molar absorption coefficient for the meta-cleavage product of 13200 M-1 cm-1 at pH 7·5 (Eltis et al., 1993
). For both enzymes, one unit of activity was defined as the amount producing 1 µmol product min-1 at 25 °C.
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Procedures for agarose gel electrophoresis, staining, Southern blotting and restriction enzyme digestions were as described by Sambrook et al. (1989) unless otherwise specified. For dot blots, 0·0050·1 µg of genomic DNA were applied directly to a nylon membrane (Biodyne B; Pall). For Southern blots, digested genomic DNAs were separated in 0·8% (w/v) agarose gels and transferred to nylon membranes with a Posiblot 3030 Pressure Blotter (Stratagene). Hybridizations of probes (performed at 60 °C with non-stringent washings at 45 °C) and their visualization were carried out with a digoxigenin-11-dUTP kit (Boehringer Mannheim) as described in the manufacturers instructions.
PCR amplification of bph genes, including synthesis of probes.
PCR amplifications (Mullis & Faloona, 1987 ) of bph genes were carried out in 50 µl volumes and contained 100 ng genomic DNA, 0·5 µM of each primer, 0·2 mM of each dNTP (Pharmacia), 1·5 U Taq DNA polymerase (Boehringer Mannheim) and 0·1 vol. 10xTaq buffer supplied with the polymerase, under 2 drops of mineral oil. Amplifications were performed in a thermal cycler (Landgraf TC Varius V) programmed for one cycle of 120 s at 95 °C, 40 cycles at 95 °C (30 s), 50 °C (30 s, with an increment of 3 s per cycle), 72 °C (60 s, with an increment of 3 s per cycle) and a final incubation for 10 min at 72 °C. Products were separated electrophoretically in 1% (w/v) agarose gels. The following primers were used: PK1 (5'-TACTACAGCCCTGGTGCC-3'), PK2 (5'-GTATTGCACGATGGCCGG-3'), PK3 (5'-CGAGCACGACGAACAGATAG-3'), PK4 (5'-CTCCTTGATCAAGCCTTCGG-3'), PC1 (5'-TAGAGGTCGAGTATGGCTGG-3'), PH1 (5'-CATCCGTTGCTGAATGTGG-3'), PS4D (5'-ATCGCCGTTCAGCAGGGCGA-3'), PS4U (5'-CTCCAGCCATACTCGACCTC-3'), PS5D (5'-GCATGGCGCAGTTATGCTG-3') and PS5U (5'-TTGCAGTGCATGAAGTAC-3'). The latter four primers were sythesized according to sequences designed previously (Erb & Wagner-Döbler, 1993
). Probes were labelled using a digoxigenin-11-dUTP kit (Boehringer Mannheim). The bphK probe (207 bp) was synthesized using PK1 and PK2. The LB400 bphC probe (643 bp) was obtained with PS4D and PS4U. The Q1 bphC probe (523 bp) was synthesized using PS5D and PS5U. Probes were used for hybridization without further purification. For combinations of primers used in analytical PCRs and for locations of their target sequences in bphCKH of strain LB400 see Results and Fig. 2
.
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Determination of bph gene sequences.
PCR-amplified bph gene segments were sequenced as described in the next section. The primers used were PC1, PK1, PK3 and PK4 (see above).
Determination of 16S rDNA sequences.
Genomic DNA was extracted from 15 colonies picked from agar plates by resuspension in 100 µl 10 mM Tris/HCl, 1 mM EDTA (pH 8·0), heating at 95 °C for 5 min and removal of cell debris by a brief centrifugation. Supernatant (1 µl), containing genomic DNA, was added to a PCR mixture (Mullis & Faloona, 1987 ) for amplification of bp 28519 of 16S rRNA genes using a GeneAmp PCR System 9600 (Perkin-Elmer) and reaction conditions as reported previously (Karlson et al., 1993
). PCR-rDNA was purified using Centricon-100 microconcentrators (Amicon) and sequenced using an Applied Biosystems 373A DNA Sequencer and the protocol of the manufacturer (Perkin Elmer, Applied Biosystems Division) for Taq cycle-sequencing with fluorescent dye-labelled dideoxynucleotides. The sequencing primers have been described by Lane (1991)
. Sequence data were aligned with reference rRNA and rDNA sequences (Maidak et al., 1997
; Stoesser et al., 1997
) using evolutionarily conserved primary and secondary structures as reference (Woese et al., 1983
; Gutell et al., 1985
). Sequence similarities were calculated for sequence pairs using unambiguously determined nucleotide positions.
Computational methods.
Database searches and binary amino acid sequence alignments were carried out at the National Center for Biotechnology Information, Bethesda, MD, USA, using programs BLASTN, TBLASTN, or BLASTP (Altschul et al., 1990 , 1997
). Alignments were performed using the BLOSUM62 comparison matrix (Henikoff & Henikoff, 1992
) with the following parameters: open gap cost, 11; gap extension cost, 1.
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RESULTS |
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BphK had previously been shown to catalyse formation of a GSH conjugate with the so-called universal GST substrate CDNB (Hofer et al., 1994 ). The substrate spectrum of the enzyme was investigated using six other compounds frequently accepted by GSTs, namely 1,2-dichloro-4-nitrobenzene, 4-nitropyridine N-oxide, p-nitrobenzyl chloride, bromosulphophthalein, trans-4-phenyl-3-buten-2-one and 2,3-dichloro-4-(2-methylene-1-oxobutyl)phenoxyacetic acid (ethacrynic acid). None of these compounds was found to be converted into a GSH conjugate, suggesting that the enzyme has a relatively narrow substrate range.
Relationship between CDNB-accepting GST activity of strain LB400 and its bphK gene
Strain LB400 was examined by Southern hybridization of EcoRI-digested DNA with a gene probe derived from the 5'-terminal part of bphK (Fig. 2). A single strong band (see Fig. 5
, lane 8) and two extremely faint bands were observed. The weak bands may not necessarily be due to lower similarity GST genes, but could, for example, result from genes encoding other types of GSH-binding proteins as the probe was directed against the gene segment encoding the GSH-binding domain (Xinhua et al., 1992
). The strong band observed corresponded to a fragment size of 6·6 kbp, which agrees with the sequence data for the bph locus of strain LB400 (Erickson & Mondello, 1992
; Hofer et al., 1994
).
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Requirement for bphK for growth of strain LB400 on biphenyl
The mobilizable plasmid pFY741A containing LB400 genes bphA1A2A3A4BCDHJI, but devoid of bphK, was transferred to the BP- mutant of strain LB400. Transconjugants were obtained that had regained the ability to grow on BP, whilst spontaneous revertants to the BP+ phenotype were not observed. This indicates that bphK is not essential for utilization of this carbon source by strain LB400.
Analysis of strain LB400 and other BP+ bacteria for CDNB-accepting GST activity and its inducibility by growth on BP
The location of bphK in the centre of a gene cluster encoding enzymes for the utilization of BP as carbon source (Fig. 2) suggests that its expression is induced by this compound or its metabolites. GST activity was quantified after growth of strain LB400 on BP or succinate as sole carbon source (Table 2
). Activity was found under both conditions, but the level of specific activity was approximately 20-fold higher after growth on BP.
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To gain insight into the distribution of similar GST activity in other BP-degrading bacteria, a number of randomly chosen strains able to use BP as sole carbon and energy source were investigated. Some of them were well-known laboratory strains, others were recently isolated (Blumenroth, 1997 ) from polluted sediments of two rivers in eastern Germany, the Elbe and the Spittelwasser (Table 1
). A taxonomic characterization of the new isolates was carried out by 16S rDNA sequencing. It revealed that they belong to the genera Pseudomonas and Ralstonia and represent five different species (Table 1
). The taxonomic relationship of all Gram-negative strains used in this work is illustrated in Fig. 3
.
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Induction of GST activity was again compared to that of DBDO activity of the strains (Table 2). However, quantification of the latter proved to be difficult with several of the bacteria. Sometimes activity could not be detected at all in the standard assay. Rapid turnover of the ring fission product could account for this problem. Consistent with this view, a yellow metabolite, presumably the ring fission product, was sometimes transiently observed when a large excess of solid substrate was added to whole cells or cell extracts of strains that had not shown activity under standard conditions. Except for strain LB400, no correlation was found between the induction ratios of GST and DBDO activities. These results could be due to the problems in DBDO activity measurement. The data presented in the following section indicate that in most of the strains bphC genes are indeed located upstream of bphK genes.
Analysis of strain LB400 and other BP+ bacteria for the occurrence and relative location of bphK-like genes
The randomly chosen BP-degrading bacteria were screened in dot blots with the bphK probe described above. Some representative results are illustrated in Fig. 4; an overview is given in Table 3
. Clear signals were obtained with seven strains, all of which had GST activity (above). Weak dots were observed with strains Q1, B11 and B15, although no GST activity had been detected with the latter two. No probe hybridization was detected with strains P6 and RW1, which also were GST-negative. The E. coli K-12 strain DH5
and the E. coli B strain BL21(DE3)[pLysS], assayed as controls, did not give rise to significant signals. An alignment of bphK with the sequenced GST gene from E. coli K-12 (Nishida et al., 1994
) yielded an approximately 60% identity in the region spanned by the probe (not shown). This agrees with the experimental result, as such a degree of homology normally is too low for a probe to stably anneal.
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The Southern blots were rehybridized with bphC probes derived from strains LB400 or Q1 (Table 3). The LB400 probe yielded a single strong signal with all DNAs except for that of strain Q1. It annealed, on the basis of fragment mobility, to the same fragments as the bphK probe, suggesting that both genes belong to the same cluster. The Q1 probe annealed to two DNA fragments of strain Q1, in agreement with the presence of an EcoRI site within its bphC gene (Taira et al., 1988
). However, neither of these two fragments corresponded to the fragment containing the bphK analogue.
PCR was used to locate the bphK analogues more precisely relative to potentially adjacent bph genes. Primers were derived from bphC, bphH and bphK of strain LB400 (Fig. 2). An overview of the PCR data is given in Table 3
. No PCR products were obtained from strain Q1. The other strains that had given signals in Southern blots yielded fragments of the same sizes as LB400. Typical results are presented for strain B6B in comparison with strain LB400 in Fig. 6
. These data indicate a significant similarity between the bph gene clusters of the positive strains and the bph locus of LB400. Strains B11 and B15, which had shown no GST activity but weak signals in dot blot hybridization, gave rise to various bands in PCR. These included very faint bands with the mobility of the corresponding products from strain LB400. Three such PCRs with DNA from B11 (Fig. 6c
, lanes 2, 5 and 8) were used as templates in second, nested or semi-nested PCRs with primers PK1 and PK2. In one of these reactions, a fairly strong band of the mobility of the corresponding product from strain LB400 was obtained in addition to several other bands of comparable intensity (not shown). Sequencing of PCR products derived from strains H850, B3B, B4, B7T and B15 using primers PC1 and PK3 or PK1 and PK4 (Fig. 2
) revealed 100% identity with the LB400 sequence in four cases. The bph sequence of Pseudomonas sp. strain B4, when compared to LB400, showed six silent or nonsilent point mutations and a deletion of 18 base pairs at the 3' terminus of the bphC gene, but only three silent mutations at the 5' end of the bphK gene (not shown).
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DISCUSSION |
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The finding that bphK is not absolutely required for utilization of BP does not exclude a usefulness of the gene. Without being essential, a gene that leads to even only a slight increase in growth rate or viability can give a large competitive advantage to its host. Moreover, bphK could only be helpful or even essential under specific conditions which may, for example, require additional detoxification during BP metabolism. A bphK requirement could well depend on specific features of the organisms BP catabolism and may therefore be strain-specific, thus explaining the absence of the gene in a number of BP degraders. Some experimental results positively support a function of bphK. Firstly, bphK genes are present in several different bph loci. Secondly, these genes possess a high degree of sequence identity. Thus, bphK genes encoding identical enzymes (as far as the sequence has been determined) have been found in bph clusters from six different organisms, including strain LB400. Five of these loci significantly deviate from each other outside the bphK region (our unpublished results). Thirdly, bphK is expressed and its expression is conditionally enhanced in response to the use of BP as carbon source.
Additional evidence against a by-chance occurrence of bphK genes comes from the finding that GST-encoding genes seem to be widely distributed among bacteria able to aerobically degrade different aromatic coumpounds. A closely related but distinct group of GSTs (Table 4) has recently been found to be encoded by several polyaromatic hydrocarbon (PAH)-degrading bacteria, and three of the respective genes, gst of Sphingomonas paucimobilis EPA505 (Lloyd-Jones & Lau, 1997
), phnC of Pseudomonas sp. strain DJ77 (GenBank accession number AF001103) and bphK of plasmid pNL1 of Sphingomonas aromaticivorans F199 (GenBank accession number AF079317) have been entirely sequenced. One of the gene products has been shown to accept CDNB as substrate (Lloyd-Jones & Lau, 1997
), and, on the basis of the high similarities in the sequenced regions, this is very likely to also hold for the rest of these GSTs. Their genes, like the LB400-type bphK genes, typically appear to be located at a similar, unique position within a certain type of gene cluster. Their downstream neighbours have invariably been identified as encoding a hydrolase (e.g. phnD, an analogue of bphD) and an extradiol dioxygenase (e.g. phnE, an analogue of bphC) that are possibly involved in PAH catabolism (Lloyd-Jones & Lau, 1997
; Shin et al., 1997
). Upstream adjacent genes have thus far only been desribed for bphK of strain F199. Based on sequence similarity, they encode the large and small subunits of an aromatic-ring hydroxylating mono- or dioxygenase. We note that, similar to the situation with BP degraders, gene clusters encoding PAH degradative pathways exist that are structured differently from the phn-type cluster and probably do not contain a GST-encoding gene (e.g. the nah cluster of the prototype plasmid NAH7; Eaton, 1994
). This suggests that such a gene is not generally required for PAH utilization. The first member of another closely related, but distinct, group of GSTs appears to be XylK of Cycloclasticus oligotrophus RB1 (Table 4
), a marine
-proteobacterium that grows on several aromatic hydrocarbons including toluene, BP, naphthalene and phenanthrene (Wang et al., 1996
). XylK accepts CDNB and is encoded by a gene which is also located in the vicinity of genes that, based on their sequences, are very likely to be involved in aromatic hydrocarbon metabolism, although their substrates are presently not known (Wang et al., 1996
). The genetic environment of xylK is clearly different from those of bphK- and of phnC-type genes. Recently, the gene gst of a CDNB-accepting GST with a high degree of sequence similarity to BphK of strain LB400 (Table 4
) has been detected in the soil bacterium Ochrobactrum anthropi (Favaloro et al., 1998b
), which is able to use the heteroaromatic herbicide atrazine as sole carbon source (Favaloro et al., 1998a
). However, nothing is yet known about its genetic context. The soil bacteria Burkholderia cepacia AC1100, Sphingomonas sp. strain RW1 and Sphingomonas sp. strain RW5 are able to aerobically degrade 2,4,5-trichlorophenoxyacetate, dibenzodioxin and BP, or gentisate, respectively (Daubaras et al., 1996
; Armengaud & Timmis, 1997
; Werwath et al., 1998
). Genes encoding members of the GST superfamily have also been discovered in gene clusters of these organisms that encode enzymes for the breakdown of these or other aromatic compounds. However, the sequences of these GSTs are rather dissimilar to one another and to the above-mentioned GSTs. In agreement with this result, the enzyme of strain RW1 appears not to accept CDNB (Armengaud & Timmis, 1997
).
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Finally, we have shown that BphK of strain LB400 is a physically fairly stable protein that is active under a variety of conditions ranging, for example, up to at least 1 M in salt concentration and up to 60 °C in temperature. Moreover, the product of the reaction catalysed with the artificial substrate CDNB appeared not to be further metabolized. These results, together with the finding that bphK is co-expressed with other bph genes, make bphK and its analogues interesting candidates for the use as reporters for the monitoring of expression of gene clusters encoding such degradative pathways. This is of particular interest in cases when the activity of the commonly used extradiol-cleaving dioxygenases cannot be measured accurately or at all due to, for example, rapid further conversion of the cleavage product or to instability of the enzyme. We encountered such problems with several strains used in this study (cf. Table 2). Whilst the dioxygenases will remain extremely helpful for analyses such as the screening of gene banks, due to the chromogenic nature of the catalysed reaction, the BphK assay has, in our hands, largely replaced the BphC assay in the quantification of bph gene expression.
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ACKNOWLEDGEMENTS |
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Received 6 April 1999;
revised 15 June 1999;
accepted 22 June 1999.