1 Department of Microbiology, University College Cork, National University of Ireland Cork, Cork, Ireland
2 School of Biological Sciences, University of Liverpool, Liverpool, UK
Correspondence
A. D. W. Dobson
a.dobson{at}ucc.ie
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ABSTRACT |
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The GenBank accession number for the nucleotide sequence of the portion of the pks gene cloned as part of this study is AY272043.
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INTRODUCTION |
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OTA is produced by Aspergillus ochraceus and Penicillium verrucosum, with the latter being the most common OTA producer in temperate regions such as northern Europe and Canada and the former is more common in warmer climates (Pitt, 2000). Penicillium spp. producing OTA are now classified as either P. verrucosum or Penicillium nordicum (Castella et al., 2002
). Other Aspergillus spp. known to produce OTA include Aspergillus auricomus, Aspergillus melleus, Aspergillus ostianus, Aspergillus petrakii, Aspergillus sclerotiorum and Aspergillus sulfureus (in the A. ochraceus group); Aspergillus alliaceus and Aspergillus albertensis (in section Flavi); Aspergillus carbonarius and Aspergillus niger (in section Nigri); and Aspergillus glaucus (in section Aspergillus) (Abarca et al., 2001
; Bayman et al., 2002
; Dalcero et al., 2002
). Few of these species are thought to contaminate foods or animal feeds with OTA, but the toxin has been detected in animal feed (Dalcero et al., 2002
). The screening of A. niger strains currently used in biotechnological applications for their ability to produce OTA has also been recommended (Schuster et al., 2002
).
OTA has been detected in food products such as wine, beer, grape juice, dried fruit, meat, figs, coffee and cereals (Abarca et al., 1994; Bayman et al., 2002
; Cabanes et al., 2002
; Creppy, 2002
; Gareis & Scheurer, 2000
; Hussein & Brasel, 2001
; Stefanaki et al., 2003
; Taniwaki et al., 2003
; Visconti et al., 2000
). Cereals, or cereal-based products, normally account for 5080 % of average consumer intake of OTA (Jorgensen & Jacobsen, 2002
); consequently, prevention of OTA formation by fungi in cereals would significantly reduce the overall levels of human exposure.
The biosynthetic pathway for OTA has not yet been completely established; however, the isocoumarin group is a pentaketide skeleton formed from acetate and malonate via a polyketide synthesis pathway with the L-phenylalanine being derived from the shikimic acid pathway (Moss, 1996, 1998
). There is no information currently available on either the enzymes or the genes responsible for any of these biosynthetic steps.
We report here on the cloning and molecular characterization of a polyketide synthase gene (pks) involved in OTA biosynthesis that was isolated using a suppression subtractive hybridization PCR (SSH-PCR)-based technique (Diatchenko et al., 1996). We also clearly demonstrate that this pks gene is involved in OTA biosynthesis since mutant strains of A. ochraceus in which the pks gene has been interrupted lose their ability to produce the mycotoxin.
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METHODS |
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RNA preparation and cDNA synthesis.
Mycelia were stored frozen at -70 °C prior to extraction of total RNA. RNA was extracted by using the RNeasy plant mini-kit (Qiagen) and frozen mycelia that had been ground to a fine powder in liquid nitrogen, with a mortar and pestle. The extracted RNA was treated with DNase I (Roche) to remove contaminating DNA and stored at -70 °C until used. cDNA was synthesized from each sample with a SMART cDNA synthesis kit (Clontech Laboratories).
SSH-PCR.
This was performed with a PCR-Select kit (Clontech) as specified by the manufacturer. The permissive cDNA served as the tester DNA and the restrictive cDNA as the driver DNA. All PCR amplifications during the cDNA synthesis and SSH-PCR were performed with an Advantage-2 PCR kit (Clontech). The resulting cDNAs were cloned into the pGEM-T easy vector (Promega) and the ligation mixtures were transformed into Escherichia coli TOP 10 cells (Invitrogen). The transformants obtained were replica plated onto LuriaBertani agar (96 per 155 mm Petri dish).
Screening for up-regulated clones.
Nested primers from the SSH-PCR kit were used to amplify the cloned insert and the PCR products were transferred to duplicate membrane filters and cross-linked following exposure to UV light (Stratalinker; Stratagene). Up-regulated clones were identified in Southern hybridizations to cDNA probes prepared from both permissive and restrictive cells by labelling 25 ng of cDNA from each fungal culture with [-32P]ATP (New England Nuclear) by random prime labelling (Prime-a-Gene; Promega). Hybridization was detected by autoradiography with KODAK biomax film and an exposure time of 5 h at -70 °C.
DNA sequencing.
Clones to be sequenced were grown in 96-well microtitre plates at 37 °C for 24 h. Glycerol was added to a concentration of 40 % (v/v) and the plates were frozen at -70 °C. All DNA preparation and sequencing reactions were performed by Lark Technologies (Essex, UK). Nucleotide and protein sequences of each clone were compared to the NCBI protein databases by using the BLAST-X algorithm. A 408 bp clone with a deduced amino acid sequence that shared a high degree of similarity to a number of polyketide synthase (PKS) proteins was selected for further study.
Cloning of DNA sequences flanking the cloned cDNA.
Genomic DNA flanking the 408 bp SSH-PCR clone was cloned by using a single-specific primer-PCR (SSP-PCR)-based approach as described previously (Shyamala & Ames, 1989). A. ochraceus genomic DNA was digested with various restriction enzymes and the fragments were ligated into the appropriate site in the pUC18 vector. The ligation mixture served as a template for PCRs in which a primer specific to the end regions of the pks gene sequence was used with a primer specific to the pUC18 vector to amplify the inserted sequences. A pair of PCR primers (PKS4-KOF and PKS4-KOR; Table 1
) was designed to amplify a 1·4 kb fragment of the completed sequence. These primers were used to amplify this sequence from A. ochraceus genomic DNA and the PCR product was cloned into the pGEM-T vector (Promega), generating pAOPKS-J4.
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OTA mutant construction.
The pks gene sequence was disrupted by inserting the E. coli hygromycin B phosphotransferase gene (hph) flanked by the Aspergillus nidulans trpC promoter and terminator sequences (Cullen et al., 1987) from plasmid pID21 (Tang et al., 1992
). The ends of the excised fragment were converted to blunt ends by treatment with the Klenow fragment of DNA polymerase (Promega) and the fragment was ligated into a SmaI site in the pks portion of pAOPKS-J4 to produce plasmid pAOPKS-J5 (Fig. 3a
). A. ochraceus protoplasts were prepared and transformed with pAOPKS-J5, as described previously (Tilburn et al., 1995
) for A. nidulans. Hygromycin-resistant transformants were selected on A. nidulans regeneration medium supplemented with 200 U hygromycin B ml-1 (Calbiochem). For the preparation of protoplasts and selection of transformants A. ochraceus was incubated at 30 °C, except that immediately after transformation the regeneration plates were held at 20 °C for 24 h. Approximately 120 transformants were obtained per microgram of plasmid DNA. Putative transformants were transferred to hygromycin-supplemented PDA for purification prior to screening for OTA production.
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Confirmation of disruption of the pks gene by Southern blot.
Genomic DNA from both the wild-type and the mutant strain was digested with SmaI, electrophoresed on a 0·8 % agarose gel and transferred to duplicate nitrocellulose membranes (Southern, 1992). One membrane was hybridized to a pks gene probe amplified with primers PKS4-KOF and PKS4-KOR by PCR from the genome of the wild-type strain, the other was hybridized to a hygromycin gene probe amplified with primers HPH-1F and HPH-1R from pID21 (Table 1
). Both probes were labelled with [
-32P]dATP by priming with random hexanucleotides (Prime-a-Gene; Promega). Hybridizations were performed at 55 °C for 12 h in hybridization buffer (0·5 M sodium phosphate/5 % SDS, pH 7·0), washed once in 2x SSC/0·1 % SDS and twice in 0·2x SSC/0·1 % SDS before autoradiography on KODAK MR film.
HPLC analysis of OTA production.
The extraction procedure for the HPLC assay was identical to that outlined for the TLC assays except that the dried extract was dissolved in 100 µl of methanol rather than in 20 µl. The HPLC method was as described by Sibanda et al. (2002) using a Beckman System Gold HPLC apparatus and a Beckman Ultrasphere C18 (250x4·6 mm, 5 µM) reversed-phase column. An aliquot (50 µl) of sample was injected using a Beckman 508 autosampler, the mobile phase was acetonitrile/water/acetic acid (99 : 99 : 2) at a flow rate of 1·0 ml min-1 and OTA was detected by UV absorbance at 330 nm on a Beckman 166 UV-VIS detector. Under these conditions OTA eluted from the column at 9·15 min.
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RESULTS |
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DISCUSSION |
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The gene we sequenced includes a highly conserved acyl transferase region typical of PKS proteins, with this region displaying a high level of similarity (5560 % at the amino acid level) to other fungal PKS acyl transferases. PKSs and fatty acid synthases (FASs) contain an acyl transferase domain. The primary reaction in both cases is condensation of short-chain fatty acids to form -ketoacyl thioesters. It is in the subsequent reactions that the PKSs and FASs diverge. The deduced amino acid sequence of our gene is most similar to PKS proteins involved in the biosynthesis of mycotoxins and other bioactive compounds. For example, the PKS proteins with the highest degree of similarity to the A. ochraceus PKS were from C. heterostrophus (T-toxin) (Yang et al., 1996
), A. terreus (lovastatin) (Hendrickson et al., 1999
), G. moniliformis (fumonisin biosynthesis) (Proctor et al., 2003
) and Penicillium citrinum (compactin) (Abe et al., 2002
).
The pks gene was not expressed when A. ochraceus was grown on the OTA restrictive medium, but was expressed in permissive medium. Thus, the expression of this pks gene in A. ochraceus is similar to the expression of other PKS-encoding genes such as the pksP1 gene from A. parasiticus and the stcA gene from A. nidulans, which are also differentially regulated under different physiological conditions (Feng & Leonard, 1995). Expression of the pks gene appeared strongest during days 4 and 5 of growth in the permissive medium and occurred to a much lesser degree at later time points. This expression appeared to correlate with OTA production as assessed by TLC analyses of fungal cultures.
We knocked out the pks gene in A. ochraceus through insertional inactivation with the E. coli hygromycin B phosphotransferase gene (hph). The A. ochraceus NUIC118 mutant no longer produces OTA (Fig. 6), indicating a functional role for the pks gene in OTA biosynthesis.
The cloning of the pks gene should allow us to identify key physiological parameters affecting OTA production in A. ochraceus and in other aspergilli, and allow us to potentially clone OTA biosynthetic gene homologues in P. verrucosum. As genes for secondary metabolite production are often arranged in clusters it will also allow the cloning of other genes involved in OTA biosynthesis from these fungi, and the elucidation of the OTA biosynthetic pathway in both A. ochraceus and P. verrucosum. Continued molecular studies on these fungi should provide information that will help in the development of new strategies for controlling OTA contamination of foods.
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ACKNOWLEDGEMENTS |
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Received 2 July 2003;
revised 22 September 2003;
accepted 29 September 2003.
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