Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z31
Author for correspondence: Julian Davies. Tel: +1 604 221 8896. Fax: +1 604 221 8881. e-mail: jed{at}unixg.ubc.ca
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ABSTRACT |
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Keywords: actinomycetes, Streptomyces, vanillic acid, biotransformation, enzyme
The GenBank accession number for the sequence reported in this paper is AF134589.
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INTRODUCTION |
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Vanillic acid is an abundant component of solubilized lignin biomass, and degradative mechanisms by which this compound is catabolized have been elucidated for several prokaryotic organisms. The vanA and vanB genes responsible for vanillic acid demethylation have been cloned and sequenced from Pseudomonas sp. strain ATCC 19151 (Brunel & Davison, 1988 ), Acinetobacter sp. ADP1 (A. Segura & N. L. Ornston, accession no. AF009672) and, most recently, Sphingomonas paucimobilis (Nishikawa et al., 1998
). In these organisms, vanillic acid is converted to protocatechuate by the enzyme vanillate demethylase; subsequently the protocatechuate is mineralized by enzymes of the ß-ketoadipate pathway. However, in some strains of Streptomyces and Bacillus (Crawford & Olson, 1978
), vanillic acid is catabolized via an alternative pathway involving non-oxidative decarboxylation to guaiacol, with further catabolism via demethylation and mineralization through the intermediate catechol. In fact, Sutherland et al. (1981
) demonstrated that Streptomyces isolates degraded vanillic acid by both routes, i.e. via both catechol and protocatechuate.
Non-oxidative decarboxylation of aromatic acids involves the removal of the carboxyl moiety from the benzene nucleus via an enzymic reaction that requires neither oxygen nor cofactors typical of the oxidative process. The non-oxidative process results in the complete removal of the carboxyl group, in contrast to the oxidative reaction, which substitutes a hydroxyl group at the relevant carbon atom. For biotransformation and metabolic engineering applications, both oxidative and non-oxidative decarboxylation processes are valuable as components of hybrid pathways for the production of various industrially useful chemicals. For example, a Klebsiella pneumoniae (formerly Klebsiella aerogenes) protocatechuate non-oxidative decarboxylase was engineered into a hybrid pathway to produce catechol, a useful building block for pharmaceuticals, using glucose as a renewable starting material (Frost & Draths, 1995 ).
In this study, we demonstrate that soil isolate Streptomyces sp. D7 decarboxylates vanillic acid to guaiacol non-oxidatively (Fig. 1), and performs this reaction via the combined activity of the products of vdcB, vdcC and vdcD. While there are a number of reports of microbial aromatic acid non-oxidative decarboxylases in the literature (Grant & Patel, 1969
; Yoshida & Yamada, 1982
; Nakajima et al., 1992
; Huang et al., 1993
; Santha et al., 1995
; He & Wiegel, 1995
, 1996
; Zeida et al., 1998
), this is, to our knowledge, the first report of the gene sequences associated with such processes. Further study of this system should allow these genes to be incorporated into metabolically engineered micro-organisms for the production of industrially useful chemical products such as guaiacol, catechol and adipic acid.
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METHODS |
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Isolate catabolic-screening procedures.
Screening for the catabolism of aromatic acids was carried out on agar minimal medium containing aromatic acid (0·53 g l-1), trace elements, phosphate buffer and the pH indicator bromothymol blue, pH 7·2 (Grund et al., 1990 ). At pH values 7·2 and below, the indicator appears green to yellow, while at pH values above 7·2 the indicator is blue. Catabolism of the aromatic acid being tested results in decreased acidity in the medium; this rise in pH can be scored visually by a change of the medium colour from green (at pH 7·2) to blue (at pH >7·2). Isolates that caused the medium to turn blue were also observed to grow well in the presence of the aromatic acid being tested. Organisms that expressed agarase also grew on assay plates, but did not cause a colour change in the medium. Isolates which were positive in the bromothymol blue plate assay were grown in liquid minimal medium in the presence of the appropriate aromatic acid, and culture supernatants sampled during time-course studies were analysed by UV spectrophotometry to monitor substrate disappearance. Decrease in absorbance at the wavelength for the particular aromatic acid being studied (e.g. 250 nm
max for vanillic acid) as compared to abiotic controls was evidence that the aromatic compound tested was being transformed or specifically degraded.
Bacterial strains and plasmids.
Bacterial strains and plasmids used in this study are shown in Table 1. Streptomyces sp. D7 was isolated from forest soil on the University of British Columbia campus, as described above. Subcloning of Streptomyces DNA was performed in Escherichia coli DH5
with pUC19. Expression studies of the vdc genes in Streptomyces lividans 1326 were performed using pIJ680 (Hopwood et al., 1985
), a vector that places target genes under the control of the aminoglycoside phosphotransferase (aph) constitutive promoter, and pIJ702 (Katz et al., 1983
), which provides the weaker constitutive tyrosinase (mel) promoter. S. lividans 1326 was converted to protoplasts and transformed according to published methods (Bibb et al., 1978
; Thompson et al., 1982
). Protoplasts were plated on R5 solid medium (Thompson et al., 1980
) and allowed to regenerate for 14 h before transformants were selected by an overlay of soft nutrient agar containing thiostrepton to achieve a final concentration of 50 µg thiostrepton ml-1 per plate. Chromosomal DNA was extracted from Streptomyces strains by the method of Fisher (Hopwood et al., 1985
). Streptomyces plasmids were isolated by the alkaline lysis method (Kieser, 1984
) and E. coli plasmid DNA was routinely isolated using the Qiaprep Spin miniprep kit (Qiagen) or the NucleoSpin miniprep kit (Clontech) for sequencing and routine manipulations.
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Time-course analysis of gene expression.
To analyse gene expression during growth in the presence of aromatic acids, the following strategy was employed. Ten millilitres of MSMYE supplemented with 6 mM vanillic acid in a 50 ml baffled flask (Bellco) was inoculated with one colony of sporulating Streptomyces sp. D7 grown on ISP4 agar. The seed culture was incubated at 30 °C, 260 r.p.m., for approximately 21 h, at which time the cells were centrifuged, washed twice and then resuspended in 10 ml sterile distilled water. One millilitre of the seed suspension was used to inoculate 50 ml MSMYE in separate 250 ml baffled flasks. One flask was treated as a control, uninduced culture; the second flask was supplemented with 3·6 mM vanillic acid after early exponential growth was reached. Immediately prior to induction of the test culture, 1 ml of the control culture was removed and radiolabelled with 100 µCi (3·7 MBq) [35S]methionine/cysteine (DuPont-NEN) at 30 °C for 30 min in a 15 ml polypropylene tube with shaking. After incubation, the labelled sample was pelleted, quick frozen in a dry ice ethanol bath and stored at -70 °C. One millilitre aliquots of both induced and uninduced cultures were labelled as above at 1, 2, 5, 12·5 and 18 h after addition of vanillic acid to the experimental culture. These frozen pellets were lysed (as described in the next section) to obtain protein extracts for proteomic analysis by 2D-PAGE.
Protein extraction and sample preparation.
Cell pellets were thawed on ice and washed twice in 2 ml lysis buffer [10 mM Tris/HCl pH 7·5, 5 mM EDTA, 100 µg PMSF ml-1 (Sigma), 1 µg pepstatin A ml-1], then resuspended in 0·5 ml of the same buffer. Samples were sonicated on ice, with a microtip set at medium power setting, in three bursts, 15 s each, with 15 s delays to prevent samples from overheating. Immediately after sonication, samples were centrifuged at 14000 g for 15 min at 4 °C. The pellet was discarded, and DNase/RNase solution (24 mM Tris base, 476 mM Tris/HCl, 50 mM MgCl2, 1 mg DNase I ml-1, 0·25 mg RNase A ml-1) was added to 10-1 the total volume of each supernatant, followed by incubation for 10 min on ice. Total cell extracts (approx. 1 ml per time-point sample) were concentrated by ultrafiltration at a molecular mass cut-off of 10000 Da, then resuspended in 20 µl IEF sample buffer. In addition to concentrating the sample, the ultrafiltration step removed unincorporated 35S-labelled amino acids.
Protein 2D-PAGE.
Protein two-dimensional (2D) gel electrophoresis was performed according to Garrels (1979 ) using the protocols, chemicals and equipment of the Investigator system (Genomic Solutions). First dimension IEF tube gels incorporated ampholytes in the pH range 310, optimized for analysis of total cell extracts. Second dimension high-tensile-strength slab gels contained 12·5% acrylamide and an acrylamide to N',N'-methylene-bisacrylamide ratio of 30:0·65 (Duracryl, Genomic Solutions). Ten microlitres of each sample containing 1x106 c.p.m. was applied to each IEF tube and focused for 18000 V h-1 (17·5 h total run time). After second dimension electrophoresis, slab gels were agitated in fix solution (40% methanol, 10% glacial acetic acid) for 1 h, followed by treatment with a fluor solution (Enhance; DuPont-NEN), and then dried. Proteins were visualized by exposure of dried gels to Kodak Bio-Max MR film for 710 d at -70 °C.
Computer-aided analysis of 2D-PAGE gels.
Autoradiograms were scanned and analysed using PDQuest 2D analysis software version 5.0 (PDI) for 2D gels. Automated spot detection was performed using the PDQuest standard algorithms for 35S-labelled gels. Spot quantification was performed by computer-generated 2D Gaussian modelling. Gels were compared by a process of landmarking and matching spots among all gels in the experiment. A correction factor for each 2D gel protein spot was calculated as the total optical density units detected in the standard gel (a master reference gel created by merging all gels in a time-course matchset) divided by the total optical density units detected in the particular gel to which each protein spot belonged. The PDQuest gel analysis software was run on a SparcStation 5 (Sun Microsystems).
Protein amino-terminal sequencing.
Soluble cell protein from a non-radiolabelled, vanillic-acid-induced sample was run in 10 replicate gels, each containing 150 µg protein, and stained post-electrophoretically by the zinc-imidazole method (Oritz et al., 1992 ). The zinc-imidazole stain results in a white background with clear spots that correlate with protein locations. These protein spots are not fixed in the gel matrix. Protein spots of interest were cut from each gel and pooled for loading in a single 1·5-mm-thick well of a 12·5% acrylamide slab gel. SDS-PAGE sample buffer (Laemmli, 1970
) was added on top of the gel pieces in the well, and the protein was electrophoresed into the slab gel. The slab gel was blotted to Immobilon-PSQ PVDF membrane (Millipore) using the semi-dry graphite blotter supplied with the Investigator 2D system. A three-buffer protocol was used according to the manufacturers instructions, in which
-amino-n-caproic acid was substituted for glycine. The membrane was stained with a Coomassie blue R-250 solution for several seconds, destained in 40% methanol, then washed with 18 M
cm-1 distilled water several times. The purified, blotted protein band was excised from the membrane and air-dried prior to amino-terminal sequencing by the Nucleic Acid Protein Sequencing (NAPS) Unit at the University of British Columbia.
DNA sequencing and analysis.
Automated DNA sequencing was performed using the AmpliTaq PRISM kit (Applied Biosystems) with a standard thermocycling program provided by the manufacturer, with variations in the annealing temperature to match the melting temperature of the sequencing primer being used. Sequence reaction products were sent to the NAPS Unit at the University of British Columbia and electrophoresed on an ABI model 377 DNA sequencing apparatus (Applied Biosystems). Nucleic acid sequence was analysed by the Wisconsin Package Version 10 (Genetics Computer Group) on a Sun Microsystems SparcStation 5.
Library construction and gene cloning.
A Lambda DASH II (Stratagene) genomic DNA phage library of chromosomal Streptomyces sp. D7 fragments was constructed by ligating 922 kb Sau3AI partially digested chromosomal DNA fragments into the BamHI site of the phage arms. This library was screened by high-stringency hybridization at 60 °C with [-32P]ATP-labelled oligonucleotide probes derived from protein amino-terminal sequencing data. The vdcB, vdcC and vdcD genes were subcloned as a 4·4 kb BamHI fragment into pUC19 in E. coli DH5
MCR (Gibco-BRL).
Chemical analytical methods.
Culture supernatants were filtered through 0·45 µm syringe filters and processed through a C-18 hydrophobic interaction column attached to a HPLC system (Hewlett Packard, model 1050). Conditions for separation were 30% phosphoric acid/water, 70% methanol, with a flow rate of 1·0 ml min-1. Retention times for vanillic acid and guaiacol under these conditions are 5 min and 4 min, respectively. Integrated peak areas corresponding to compounds in supernatant samples were calibrated against known concentrations of vanillic acid and guaiacol standards. Additional analysis of supernatant samples was performed using a Cary 1 Bio ultraviolet/visible spectrophotometer (Varian). For UV analysis, vanillic acid characteristically displays a primary absorbance at 250 nm and a secondary absorbance at 285 nm, while guaiacol absorbs at 275 nm.
RNA isolation and transcript detection.
Total RNA was isolated from cells grown under two different conditions. Primary cultures were grown in 25 ml YEME for 48 h before cells were pelleted by centrifugation, washed twice with sterile water and resuspended in minimal media (MSMYE). For induced cultures, the media were supplemented with 3·6 mM vanillic acid, while no additional substrates were added to the uninduced control. These cultures were then allowed to grow an additional 3 h after which cells were pelleted and washed as before. Total RNA was isolated using standard RNA isolation techniques (Hopwood et al., 1985 ; Kirby et al., 1967
). For transcript detection, Northern gel electrophoresis and transfer were performed according to the manufacturers recommendations for the NorthernMax kit (Ambion). Fifteen micrograms of total RNA was loaded on the gel and 1 µg of an RNA standard ladder (NEB) was included for size comparison. After electrophoresis was complete, the RNA ladder lane was excised and stained with ethidium bromide to allow for visualization and to confirm RNA integrity. Transcript was detected using a probe specific for the vdcC gene. To generate the probe, traditional double-stranded PCR was performed on pKCE1 using oligonucleotides vdcC.F and vdcC.R (Table 2
). Following amplification, excess dNTPs and oligonucleotides were removed using a QIAquick PCR Purification kit (Qiagen). This product was then used as template for asymmetric PCR with only the vdcC.R primer. This resulted in a single-stranded PCR product that was complementary to the predicted RNA transcript. During chain elongation, [32P]dCTP was provided in place of dCTP in the dNTP mix to allow for direct incorporation of radiolabel. The extension product was purified and allowed to hybridize with the immobilized RNA at 60 °C for 24 h. Excess probe was removed by washing as directed by the NorthernMax protocol, and the hybridizing transcript was visualized by exposure to film for autoradiography. The membrane was then stripped and rehybridized using a vdcB-specific single-stranded PCR product generated in a similar manner, using the padx.F and padx.R primers (Table 2
).
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Reagents and enzymes.
All reagents used were of the highest quality, and purchased from Sigma unless noted otherwise. Restriction endonucleases and other modification enzymes were obtained from Gibco-BRL, New England Biolabs, Boehringer Mannheim or Pharmacia.
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RESULTS |
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Streptomyces sp. D7 conversion of vanillic acid to guaiacol
When mycelia of Streptomyces sp. D7 were grown in the presence of vanillic acid, a strong odour characteristic of guaiacol was apparent several hours after inoculation. This biotransformation was confirmed by spectroscopic and chromatographic analysis of culture supernatants as described in Methods. By HPLC analysis of culture supernatants, it was observed that vanillic acid was decarboxylated to guaiacol in equimolar amounts (Fig. 2).
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Sequence analysis revealed that the gene encoding protein 3717 was contained on a 4·4 kb BamHI fragment, and was determined to be the second gene in a cluster of at least three genes, designated vdcB (602 bp), vdcC (1424 bp) and vdcD (239 bp), as depicted in Fig. 4. blast-x sequence analyses (Altschul et al., 1990
) revealed that the gene cluster, in whole or in part, is present in a variety of micro-organisms. The vdcB translation product is highly similar to phenylacrylate decarboxylase (PAD) from Saccharomyces cerevisiae. The yeast PAD contains a putative membrane-binding domain close to the amino-terminus, which is highly conserved among other hypothetical PAD homologues from various micro-organisms, as revealed by genome projects. The vdcC translation product is also highly similar to hypothetical proteins from various microbial genome projects, in addition to the amino-terminal similarity to 4-hydroxybenzoate carboxy-lyase as revealed from the Edman degradation sequencing of protein 3717. Dendrograms of the vdcB and vdcC translation products in comparison to other microbial homologues are shown in Fig. 5
. Unlike the first two genes in the cluster, the vdcD translation product shows similarity only to a hypothetical protein from Bacillus subtilis. Although these genes have homologues in a number of microbial genomes, they are not always clustered and only B. subtilis contains all three genes in the same order as Streptomyces sp. D7. Other micro-organisms contain vdcB and vdcC homologues, but at different chromosomal locations. Interestingly, Sphingomonas aromaticivorans strain F199 possesses homologues to both vdcB and vdcC on its 184 kb catabolic plasmid pNL1 (Romine et al., 1999
), although they are not clustered. Plasmid pNL1 contains a variety of genes encoding catabolic enzymes for the degradation of a number of organic chemicals.
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Expression of the gene cluster in S. lividans 1326
The 4·4 kb BamHI DNA fragment containing the vdcBCD gene cluster was inserted into the Streptomyces cloning vector pIJ702 at the unique BglII site. Insertion at this site places the cluster downstream of the mel promoter, thereby disrupting transcription of the tyrosinase gene that serves as a colour selection marker for transformants. pIJ702 carrying the insert in the same orientation as the mel promoter was designated pKCS1; conversely, a vector construct with the insert in the opposite orientation to the promoter was designated pKCS2. S. lividans 1326 carrying pKCS1 acquired the ability to efficiently decarboxylate vanillic acid to guaiacol, while S. lividans 1326 carrying pKCS2 produced extremely low amounts of guaiacol (Fig. 6). S. lividans 1326 wild-type cells did not decarboxylate vanillic acid. These results suggest that transcription of the genes required for vanillic acid decarboxylation by S. lividans 1326 carrying pKCS1 is being driven by the constitutive mel promoter. There is possibly a low level of transcription from a natural promoter, however, as observed for S. lividans 1326 carrying pKCS2, which contains the gene cluster in the opposite orientation from the mel promoter (Fig. 6b
).
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Substrate specificity
Sonicated cell extracts of S. lividans 1326(pKCS3) were used to test the specificity of the vdc system towards aromatic acids similar to vanillic acid. The following compounds were tested: 4-methoxy-3-hydroxybenzoate (isovanillic acid), 3,4-dimethoxybenzoate (veratrate), 3,4-dihydroxybenzoate (protocatechuate), 4-hydroxy-3,5-dimethoxybenzoate (syringate), 3,4,5-trihydroxybenzoate (gallate), 3-phenylpropenoate (trans-cinnamate). No decarboxylation of any of the substrates tested was observed, as assayed by UV spectrophotometry.
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DISCUSSION |
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Microbial non-oxidative decarboxylase systems characterized in the literature (Grant & Patel, 1969 ; Yoshida & Yamada, 1982
; Nakajima et al., 1992
; Huang et al., 1993
; Santha et al., 1995
; He & Wiegel, 1995
, 1996
; Zeida et al., 1998
) have minimal or no cofactor requirements for activity. The vanillate decarboxylase system of Streptomyces sp. D7 is active in the absence of oxygen, and amino acid sequence analysis of all three vdc gene products failed to reveal any cofactor binding motifs, e.g. for NAD and FAD, characteristic of oxidative enzymes.
The distribution of homologues of the vdc genes throughout the microbial world, mostly on chromosomes, but also plasmid-borne as in the case of pNL1 in Sphingomonas aromaticivorans, suggests that these gene products provide useful metabolic abilities for their hosts. However, non-oxidative decarboxylation of most aromatic acids yields toxic phenolic compounds and, in many cases cited in the literature (e.g. He & Wiegel, 1996 ), the micro-organisms do not possess appropriate mechanisms to further degrade the compounds produced by these dead-end pathways. In this study, Streptomyces sp. D7 was able to rapidly convert vanillic acid to guaiacol, but was unable to further degrade the guaiacol, which is a toxic phenol. In another example, C. hydroxybenzoicum decarboxylated p-hydroxybenzoate to phenol, and protocatechuate to catechol, without further metabolism. The functions of these seemingly toxic metabolic reactions of micro-organisms are not readily apparent; however, in natural ecosystems, it can be imagined that other organisms in a consortium would mineralize and remove these toxins from the environment. For example, Streptomyces setonii 75Vi2 (Pometto et al., 1981
; Sutherland, 1986
) and a Moraxella sp. (Sterjiades et al., 1982
) demethylate methoxylated aromatic compounds such as guaiacol to catechol, leading to subsequent mineralization. Evidence implicating cytochrome P-450 systems was provided in both cases. S. setonii 75Vi2 was also observed to degrade phenol (Antai & Crawford, 1983
). Indeed, there is a growing focus on biodegradation, not from the standpoint of an individual micro-organism, but rather as a coordinated function of the entire gene pool (Wackett et al., 1999
). Efficient biodegradation is therefore likely the result of natural consortiums of micro-organisms. The observation that Streptomyces sp. D7 converts vanillic acid to the toxic guaiacol but is unable to remove the guaiacol from its environment appears to support this view.
The proteomics analysis presented in this work demonstrates that genes encoding proteins linked to vanillic acid decarboxylation are induced by vanillic acid itself. This is supported by the Northern blot data, indicating that the vdc gene cluster is transcribed in one polycistronic mRNA product upon induction by vanillic acid. These results suggest that transcription of the gene cluster is under tight regulatory control. In fact, preliminary nucleotide sequence analysis upstream of the vdc gene cluster is highly indicative of a divergent regulatory gene. blast-x analysis of the region matched the translation product strongly to a putative positive transcriptional activator from the Streptomyces coelicolor A3(2) genome.
Cloning the genes for vanillic acid non-oxidative decarboxylation expands our knowledge of this reaction. Non-oxidative decarboxylases represent crosstalk between the two major branches of aromatic acid catabolism, characterized by either catechol or protocatechuate central intermediates. By joining these two pathways, this class of enzymes supports the concept of aromatic catabolism as a web of interconnecting biodegradative processes (Crawford & Olson, 1978 ). It will be interesting to determine if the numerous sequence homologues of vanillic acid decarboxylase from Streptomyces sp. D7 from microbial genome databases are indeed other non-oxidative decarboxylases. Knowledge gained from such endeavours should ultimately allow this class of enzyme to be used for a variety of metabolic engineering applications.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Anderson, J. J. & Dagley, S. (1981). Catabolism of tryptophan, anthranilate, and 2,3-dihydroxybenzoate in Trichosporon cutaneum. J Bacteriol 146, 291-297.[Medline]
Antai, S. P. & Crawford, D. L. (1983). Degradation of phenol by Streptomyces setonii. Can J Microbiol 29, 142-143.
Bibb, M. J., Ward, J. M. & Hopwood, D. A. (1978). Transformation of plasmid DNA into Streptomyces protoplasts at high frequency. Nature 274, 398-400.[Medline]
Brunel, F. & Davison, J. (1988). Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase. J Bacteriol 170, 4924-4930.[Medline]
Crawford, R. L. & Olson, P. R. (1978). Microbial catabolism of vanillate: decarboxylation to guaiacol. Appl Environ Microbiol 36, 539-543.[Medline]
Frost, J. W. & Draths, K. M. (1995). Environmentally compatible synthesis of catechol from d-glucose. J Am Chem Soc 117, 2395-2400.
Garrels, J. I. (1979). Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J Biol Chem 254, 7961-7977.[Abstract]
Grant, D. J. W. & Patel, J. C. (1969). The non-oxidative decarboxylation of p-hydroxybenzoic acid, gentisic acid, protocatechuic acid and gallic acid by Klebsiella aerogenes (Aerobacter aerogenes). Antonie Leeuwenhoek 35, 325-343.
Grund, E., Knorr, C. & Eichenlaub, R. (1990). Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp. Appl Environ Microbiol 56, 1459-1464.[Medline]
He, Z. & Wiegel, J. (1995). Purification and characterization of an oxygen-sensitive reversible 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum. Eur J Biochem 229, 77-82.[Abstract]
He, Z. & Wiegel, J. (1996). Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum. J Bacteriol 178, 3539-3543.[Abstract]
Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
Huang, Z., Dostal, L. & Rosazza, J. P. N. (1993). Mechanisms of ferulic acid conversions to vanillic acid and guaiacol by Rhodotorula rubra. J Biol Chem 268, 23954-23958.
Kamath, A. V., Dasgupta, D. & Vaidyanathan, C. S. (1987). Enzyme-catalyzed non-oxidative decarboxylation of aromatic acids. I. Purification and spectroscopic properties of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger. Biochem Biophys Res Commun 145, 586-595.[Medline]
Katz, E., Thompson, C. J. & Hopwood, D. A. (1983). Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol 129, 2703-2714.[Medline]
Kieser, T. (1984). Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid 12, 19-36.[Medline]
Kirby, K. S., Fox-Carter, E. & Guest, M. (1967). Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria. Biochem J 104, 258-262.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Nakajima, H., Otani, C. & Niimura, T. (1992). Decarboxylation of gallate by cell-free extracts of Streptococcus faecalis and Klebsiella pneumoniae isolated from rat feces. J Food Hyg Soc Jpn 33, 371-376.
Nakazawa, T. & Hayashi, E. (1978). Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase. Appl Environ Microbiol 36, 264-269.[Medline]
Nishikawa, S., Sonoki, T., Kasahara, T., Obi, T., Kubota, S., Kawai, S., Morohoshi, N. & Katayama, Y. (1998). Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O-demethylation of vanillate and syringate. Appl Environ Microbiol 64, 836-842.
Oritz, M. L., Calero, M., Patron, C. F., Castellanos, L. & Mendez, E. (1992). Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins. FEBS Lett 296, 300-304.[Medline]
Pometto, A. L.III, Sutherland, J. B. & Crawford, D. L. (1981). Streptomyces setonii: catabolism of vanillic acid via guaiacol and catechol. Can J Microbiol 27, 636-638.[Medline]
Pujar, B. G. & Gibson, D. W. (1985). Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase. Appl Environ Microbiol 49, 374-376.[Medline]
Romine, M. F., Stillwell, L. C., Wong, K.-K., Thurston, S. J., Sisk, E. C., Sensen, C. W., Gaasterland, T., Fredrickson, J. K. & Saffer, J. D. (1999). Complete sequence of a 184 kb catabolic plasmid from Sphingomonas aromaticivorans strain F199. J Bacteriol 181, 1585-1602.
Santha, R., Savithri, H. S., Rao, A. & Vaidyanathan, C. S. (1995). 2,3-Dihydroxybenzoic acid decarboxylase from Aspergillus niger. A novel decarboxylase. Eur J Biochem 230, 104-110.[Abstract]
Sterjiades, R., Sauret-Ignazi, G., Dardas, A. & Pelmont, J. (1982). Properties of a bacterial strain able to grow on guaiacol. FEMS Microbiol Lett 14, 57-60.
Sutherland, J. B. (1986). Demethylation of veratrole by cytochrome P-450 in Streptomyces setonii. Appl Environ Microbiol 52, 98-100.
Sutherland, J. B., Crawford, D. & Pometto, A. L.III (1981). Catabolism of substituted benzoic acids by Streptomyces species. Appl Environ Microbiol 41, 442-448.
Thompson, C. J., Ward, J. M. & Hopwood, D. A. (1980). DNA cloning in Streptomyces: resistance genes from antibiotic-producing species. Nature 286, 525-527.[Medline]
Thompson, C. J., Ward, J. M. & Hopwood, D. A. (1982). Cloning of antibiotic resistance and nutritional genes in streptomycetes. J Bacteriol 151, 668-677.[Medline]
Wackett, L. P., Ellis, L. B. M., Speedie, S. M. & 9 other authors (1999). Predicting microbial biodegradation pathways. ASM News 65, 8793.
Yoshida, H. & Yamada, H. (1982). Microbial production of pyrogallol through decarboxylation of gallate. Agric Biol Chem 49, 659-663.
Zeida, M., Wieser, M., Yoshida, T., Sugio, T. & Nagasawa, T. (1998). Purification and characterization of gallic acid decarboxylase from Pantoea agglomerans T71. Appl Environ Microbiol 64, 4743-4747.
Received 15 March 1999;
accepted 10 May 1999.