From the Microbial Toxicology Laboratory, the ¶ Animal and
Cellular Systems Laboratory, and the
Division of Molecular
Characterization, The Institute of Physical and Chemical Research
(RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Trichothecene mycotoxins such as deoxynivalenol, 4,15-diacetoxyscirpenol, and T-2 toxin, are potent protein synthesis inhibitors for eukaryotic organisms. The 3-O-acetyl derivatives of these toxins were shown to reduce their in vitro activity significantly as assessed by assays using a rabbit reticulocyte translation system. The results suggested that the introduction of an O-acetyl group at the C-3 position in the biosynthetic pathway works as a resistance mechanism for Fusarium species that produce t-type trichothecenes (trichothecenes synthesized via the precursor trichotriol).
A gene responsible for the 3-O-acetylation reaction, Tri101, has been successfully cloned from a Fusarium graminearum cDNA library that was designed to be expressed in Schizosaccharomyces pombe. Fission yeast transformants were selected for their ability to grow in the presence of T-2 toxin, and this strategy allowed isolation of 25 resistant clones, all of which contained a cDNA for Tri101. This is the first drug-inactivating O-acetyltransferase gene derived from antibiotic-producing organisms. The open reading frame of Tri101 codes for a polypeptide of 451 amino acid residues, which shows no similarity to any other proteins reported so far. TRI101 from recombinant Escherichia coli catalyzes O-acetylation of the trichothecene ring specifically at the C-3 position in an acetyl-CoA-dependent manner. By using the Tri101 cDNA as a probe, two least overlapping cosmid clones that cover a region of 70 kilobase pairs have been isolated from the genome of F. graminearum. Other trichothecene biosynthetic genes, Tri4, Tri5, and Tri6, were not clustered in the region covered by these cosmid clones. These new cosmid clones are considered to be located in other parts of the large biosynthetic gene cluster and might be useful for the study of trichothecene biosynthesis.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Trichothecenes belong to a family of sesquiterpenoid secondary metabolites produced by Fusarium species and other molds (1). Considerable variation exists in the oxygenation pattern of individual trichothecenes, but all are characterized by a 9,10 double bond and a 12,13 epoxide group. These compounds are potent inhibitors of protein synthesis in eukaryotes (2) and prevent polypeptide chain initiation or elongation by binding to 60 S ribosomal subunits (3). From pharmacological and toxicological points of view, trichothecenes are an important group of mycotoxins that cause serious problems of food pollution. They have been implicated in incidents of mycotoxicosis such as vomiting, dermatitis, and hemorrhagic septicemia in humans and livestock (4).
The trichothecene biosynthetic pathway has been studied in detail by
the use of blocked mutants and by precursor feeding experiments. The
proposed pathway in Fusarium species (see Fig. 1) proceeds from mevalonate via farnesyl pyrophosphate, trichodiene (5), isotrichodiol (tricho-9-ene-2,11
-diol) (6), isotrichotriol (tricho-9-ene-2
,3
,11
-triol) (7, 8), trichotriol
(tricho-10-ene-2
,3
,9
-triol) (8, 9), isotrichodermol
(3
-hydroxytrichothecene) (8, 9), and isotrichodermin (3
-acetyl
trichothecene) (8, 10) to produce a group of t-type trichothecenes
(trichothecenes synthesized via the precursor trichotriol; see
"Discussion"). Although the pathway intermediates and the sequence
of the reactions were well characterized, recent investigation has
revealed an unexpected result; i.e. the radiolabeled
3-O-acetyl group of isotrichodermin was mostly lost in its
metabolism to 3-acetyldeoxynivalenol (11). Thus, the fate of the
3-O-acetyl group in the pathway still remains an open
question for further study.
The structure-activity relationships of trichothecenes were investigated, and the stereospecific nature of their action has been demonstrated in a previous study (12). However, the significance of substituents at the C-3 position seems to have been underestimated. Our results on in vitro toxicity, standing on the elucidated biosynthetic pathway (see Fig. 1), led to the assumption that specific O-acetylation at the C-3 position following the second cyclization reaction to give isotrichodermol must be related to a self-defense mechanism for the fungus to produce t-type trichothecenes. This assumption afforded an efficient strategy to isolate a new trichothecene biosynthetic gene, Tri101, that is responsible for the 3-O-acetylation. The present report describes cloning and analysis of Tri101 and its DNA-flanking regions, and the results are discussed in relation to the mechanism of trichothecene biosynthesis.
|
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microorganisms and Chemicals--
The deoxynivalenol
(DON)1 producing strain of
Fusarium graminearum F15 was cultured in liquid YPD medium
(1.0% w/v yeast extract, 2.0% w/v polypeptone, and 2.0% w/v
glucose). Schizosaccharomyces pombe
ura4-294 h (ATCC 38436) was
grown on YEA agar medium (0.5% w/v yeast extract, 3.0% w/v glucose,
and 2.0% agar). Highly competent cells of Escherichia coli
DH5 (Toyobo; Osaka, Japan) were used for all E. coli
transformations. Trichothecenes were purchased from Sigma and Wako Pure
Chemical Co. (Osaka, Japan). Transformed products of trichothecenes
were detected on precoated TLC plates of Kieselgel 60 F254
(Merck). 4-(4-Nitrobenzyl)pyridine and tetraethylenepentamine were
purchased from Wako.
Effects of Trichothecenes on Protein Synthesis-- Effects of trichothecenes on protein synthesis in cultured mammalian cells were determined with virus-infected cells. Monolayer cultures of BHK-21 cells in 96-well titer plates were infected with Newcastle disease virus, and trichothecenes at various concentrations were added at the time of infection. Virus glycoprotein synthesis was quantified by determining hemagglutination units as described previously (13). Effects of trichothecenes on protein synthesis in a cell-free system were investigated by using the rabbit reticulocyte in vitro translation system (Amersham Inc.). The translation reaction was initiated by adding components of the kit and luciferase mRNA (Promega Inc.) to the tubes containing trichothecenes at designated concentrations. Proteins were synthesized at 30 °C for 20 min, and the products were quantitated by measuring luciferase activity using a Luciferase assay system kit (PicaGene PGK-L100, Toyo Ink Co., Tokyo).
Trichothecene Analysis-- Trichothecenes in the cultured broth were extracted three times with an equal volume of ethyl acetate and concentrated in vacuo. The samples were analyzed on TLC by using ethyl acetate/toluene (3:1) as the solvent. The developed TLC plate was sprayed with 1% 4-(4-nitrobenzyl)pyridine and then heated in an oven for 30 min at 150 °C. Trichothecenes on silica gel layers were made visible by treatment with 10% tetraethylenepentamine (14). Structures of trichothecenes acetylated by the crude recombinant enzyme were determined by 1H NMR spectra obtained with a Joel JNM A400 spectrometer in CDCl3 using CHCl3 (7.26 ppm) as an internal standard.
Construction of a F. graminearum cDNA Library-- Poly(A)-rich mRNA was isolated from young mycelia germinated in the presence of T-2 toxin by using a Fast Track mRNA isolation kit (Invitrogen, San Diego, CA). The Superscript cDNA synthesis system (Life Technologies, Inc.) was used for construction of a directional library (cDNA with a NotI site at the poly(A) proximal end and a SalI site at the opposite end). After double digestion with NotI and SalI, the synthesized cDNA was ligated into the expression cloning vector pcDSP21 (15) and transformed to E. coli DH5.
Construction of a F. graminearum Cosmid Library-- A cosmid library was constructed by using the SuperCos1 cosmid vector (Stratagene, La Jolla, USA). Genomic DNA was partially digested with Sau3AI and checked by electrophoresis on a 0.3% agarose gel. The partial digest that showed the most appropriate size distribution (30-45 kb) was ligated between the arms of the SuperCos1 vector after dephosphorylation. Packaging of DNA was performed by using Gigapack III gold packaging extract (Stratagene) as described in the manufacturer instructions.
Cloning of the cDNA for Tri101 and Screening of the Tri5- and Tri101-containing Cosmid Clones-- Screening of the cDNA library was carried out either directly or indirectly by selecting an aliquot of S. pombe-transforming mixture with 20 µg/ml T-2 toxin. The molecular cloning techniques of S. pombe were the same as described previously (16). For screening of the cosmid library, the digoxigenin system (Boehringer Mannheim) was used. The digoxigenin-labeled probes were prepared from the 1.2-kb PCR product for Tri5 or the 1.4-kb PCR product for Tri101 (see the last section of "Experimental Procedures"). Colony hybridization and purification of positive colonies were carried out as described in the manufacturer instructions.
Expression of Tri101 in E. coli and Cell-free Acetyltransferase
Assays--
The coding region of Tri101 was amplified by
PCR with primers EU101 (5-CACCACCCATATGGCTTTC-3
) and ED101
(5
-GTCTAGGATCCTAATCTAAC-3
). The amplification consisted of 25 cycles
under the following conditions: template denaturation for 30 s at
94 °C, primer annealing for 1 min at 55 °C, and primer extension
for 2 min at 72 °C (5 min at last cycle). The amplified product was
cloned into the expression vector pET-19b (Novagen) after double
digestion with BamHI and NdeI. The resulting
plasmid, pET-19bTri101, was transformed into E. coli strain HMS174 (DE3) (Novagen) and used for overproduction of
the enzyme. The E. coli cells were harvested after a 2-h
induction by 1 mM
isopropyl-1-thio-
-D-galactopyranoside and suspended in 0.01 volume of 10 mM Tris-HCl (pH 7.5). The cells were then
disrupted with a sonicator and centrifuged at 12,000 × g for 20 min to obtain the supernatant (crude enzyme
fraction). Acetyltransferase assays were initiated by the addition of
100 µl of the crude enzyme fraction to a reaction mixture containing
3.66 ml of 10 mM Tris-HCl (pH 7.5), 200 µl of
trichothecenes in 50% ethanol (5 mg/ml), and 40 µl of acetyl-CoA in
water (100 mM). The reaction mixture was incubated at
37 °C for 1 h. After the ethyl acetate extraction and
concentration,
of the products was analyzed by TLC, and the
rest was used to confirm their structure by 1H NMR.
Mapping of the Cosmid Clones Containing Tri101-- Cosmid mapping was performed by using FLASH nonradioactive mapping kit (Stratagene). Each cosmid clone containing Tri101 was completely digested with NotI, an enzyme that cut off the insert DNA from the vector, and then partially digested with EcoRI, ClaI, and KpnI. The partial digests were separated by electrophoresis on a 0.4% agarose gel and transferred to a Nytran N membrane (Schleicher & Schuell). The membrane was first hybridized with the T3 probe, washed, and exposed to a film as described in the supplier instructions and then re-hybridized with the T7 probe after stripping off the T3 probe. Based on the size of hybridization signals that appeared on the film, a restriction map was constructed for the large genomic DNA region that extends for 70 kb around Tri101.
Analysis of the Trichothecene Biosynthetic Gene Cluster by
PCR--
PCR was performed to check if a portion of Tri4
(17), Tri5 (18), and Tri6 (19) could be amplified
from the cosmid clones containing Tri101. The conditions for
PCR were as described above, and the following primers were used for
amplification: U4 (5-TCGCAGCATCGACCGTTATCTACTTC-3
) and D4
(5
-GTTGGTAATGTACTT CTTGAGGGGG-3
) for Tri4, U5
(5
-GCATGGAGAACTTTCCCACCGAGT-3
) and D5 (5
-GCTCATT
CCACTAGCTCAATTGAC-3
) for Tri5, U6
(5
-CAGTGCCAAGTCAGCTCATCGCTCTC-3
) and D6
(5
-TGCTGCCAAGGGCACCGGACTGTTG-3
) for Tri6, and U101
(5
-ATGGCTTTCAAGATACAGCT CGAC-3
) and D101
(5
-CTAACCAACGTACTGCGCATACTTG-3
) for Tri101. PCR products
were cloned directly into TA cloning vector pCRII (Invitrogen), which
were confirmed to contain the corresponding genes by partial nucleotide
sequence analyses.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Difference between in Vivo and in Vitro Protein Synthesis Inhibition by 3-O-Acetyl Trichothecenes-- The effect of the 3-O-acetyl group attached to the trichothecene ring on protein synthesis was examined by quantitating hemagglutination units in Newcastle disease virus-infected BHK cells. Acetylation at the C-3 position reduced the inhibitory actions of trichothecenes to about one-fourth of their 3-hydroxy derivatives, i.e. the concentration required to reduce virus hemagglutinin biosynthesis by 50% was estimated to be 300 ng/ml for DON, 2000 ng/ml for 3-acetyldeoxynivalenol, 12 ng/ml for 4,15-diacetoxyscirpenol (DAS), 35 ng/ml for 3,4,15-triacetoxyscirpenol (3-ADAS), 3 ng/ml for T-2 toxin, and 15 ng/ml for 3-acetylT-2 toxin (3-AT-2 toxin) (Fig. 2A). This unexpected slight decrease in the activity of 3-O-acetyl trichothecene was predicted to be caused by deacetylation. To evaluate this possibility, trichothecenes were extracted after incubation with BHK cells and analyzed by TLC. Intense blue spots corresponding to 3-hydroxytrichothecenes were detected with cultures treated with 3-O-acetyl trichothecenes. The acetyl substituents of trichothecenes were quite unstable under alkaline conditions, and even in buffer at pH 7.5, a considerable amount of deacetylated products were generated during an overnight incubation period. Therefore, the apparently potent inhibitory action of 3-O-acetyl trichothecenes might be attributed to the deacetylation of C-3.
|
Cloning Strategy for Tri101, the Trichothecene 3-O-Acetyltransferase Gene-- The new finding raised the possibility that the biosynthetic gene involved in 3-O-acetylation may be responsible for the resistance mechanism in producing strains. The biosynthetic pathway involves 3-O-acetylation of isotrichodermol, the first proposed tricyclic intermediate that inhibits growth of the fungi, to produce a nontoxic derivative isotrichodermin (11). After isotrichodermin, the route proceeds along a series of 3-O-acetylated intermediates that are devoid of biological activity, followed by the conversion to 3-deacetylated products at the last step (Fig. 1). Such metabolic shielding is reminiscent of the self-defense mechanism for the antibiotic-producing bacteria (20) from which the corresponding genes were isolated by using drug-sensitive organisms as a heterologous host.
Based on this consideration, we searched for an appropriate host organism to establish an efficient cloning strategy for the 3-O-acetyltransferase gene, which we designated Tri101. Several different trichothecenes were tested for their ability to inhibit growth of yeasts, and as a consequence, S. pombe was found to be very sensitive to trichothecenes. Above all, T-2 toxin inhibited its growth quite effectively. We thus examined whether the DON-producing strain of F. graminearum F15 has an ability to convert T-2 toxin to an 3-O-acetyl derivative. Spore germination was not profoundly affected by 100 µg/ml T-2 toxin in YPD medium, and analysis of the biotransformed products revealed production of HT-2 toxin (a 4-hydroxy derivative of T-2 toxin), 3-acetylHT-2 toxin, and 3-AT-2 toxin. The inhibitory effects of these toxins on growth of S. pombe were then investigated as shown in Table I. In sharp contrast with the severe toxicity to cultured mammalian cells, complete loss of biological activity was observed by 3-O-acetylation of the trichothecene ring. These results suggested that Tri101 would be isolated by T-2 toxin selection of fission yeast transformants carrying an expression library of F. graminearum.
|
Isolation and Analysis of the T-2 Toxin-resistant Plasmid Cloned in Fission Yeast-- A cDNA expression library consisting of 2.0 × 104 independent E. coli colonies was constructed and recovered as a pool of plasmids to transform S. pombe. Although as many as 21 positive clones were obtained by replica-plating the 4.2 × 104 Ura+ transformants to the T-2 toxin-containing medium, only 4 positive clones were identified by direct selection with the drug. All these colonies showed a stable resistance phenotype after transfer to YEA medium containing 100 µg/ml T-2 toxin. Southern analysis revealed that sequences homologous to the 1.6-kb cDNA insert for Tri101 (see below) were contained in DNA from all the 25 resistant colonies (data not shown).
A resistant plasmid was recovered in E. coli from one of the positive clones and subjected to further analysis. As shown in Fig. 3, the original structure of the library was identified on the 6.9-kb NotI fragment of pTR19-1 in which the 1.6-kb cDNA insert was correctly positioned between the SalI and NotI cloning sites of pcDSP21 (pTR19-1-4 in Fig. 3). Both plasmids pTR19-1 and pTR19-1-4 conferred resistance to T-2 toxin when transformed in S. pombe. Since pcDSP21 was constructed so that the initiator AUG codon was located between the 16 S splicing (21) donor and acceptor sequences upstream of the cloning site (15), we could not rule out the possibility that an incomplete cDNA lacking an initiation codon might have been rescued by producing a functionally active fusion protein. To examine whether the positive clone contained a full-length cDNA, plasmid pUCSVTri101 was constructed. It placed the cDNA insert under the direct control of the SV40 early promoter and was transduced into S. pombe by cotransformation with pAU9 (16). The transformed cells showed enough resistance to T-2 toxin, which strongly suggests that the cloned cDNA insert contained a complete open reading frame of the resistance gene.
|
T-2 Toxin Resistance Is Conferred by O-Acetylation at the C-3
Position--
S. pombe cells transformed by this resistant
plasmid were incubated with T-2 toxin to confirm the specific
O-acetylation of the trichothecene ring at the C-3 position.
A bright blue spot having Rf 0.83, a
value of 3-AT-2 toxin, was detected. However, HT-2 toxin
(Rf 0.20) was not detected in
extracts of the fission yeast transformant. The transformed product
showed the following signals on 1H NMR analysis: 5.92 (d, J = 2.9 Hz, H-4), 5.75 (br d, J = 5.9 Hz,
H-10), 5.29 (br d, J = 5.9 Hz, H-8), 5.18 (d, J = 2.9 Hz, H-3), 4.35 (d, J = 12.2 Hz, H-15a), 4.22 (d, J = 5.9 Hz, H-11), 4.10 (d, J = 12.2 Hz, H-15b), 3.86 (d, J = 4.9 Hz, H-2), 3.07 (d, J = 3.9 Hz, H-13a), 2.82 (d, J = 3.9 Hz, H-13b), 2.37 (dd, J = 5.9 Hz and 15.1 Hz,
H-7a), 2.16, 2.10, 2.07 (each s, AcO-3, 4, and 15), 2.17-2.05 (m, H-2
and 3
), 0.96 (d, J = 6.3 Hz, H-4
), and 0.96 (d,
J = 6.3 Hz, H-5
). The 1H NMR result was consistent
with the structure of 3-AT-2 toxin. This implies that the resistance
phenotype should be attributed to the 3-O-acetyl conjugation
of T-2 toxin, not to the deacetylation at the C-4 position, which also
reduced the toxicity to less than one-tenth (Table I). Therefore, the
cloned cDNA insert proved to encode Tri101.
Sequencing of the cDNA for Tri101--
The primary structure
of the 1.6-kb cDNA insert in pUC18 (i. e. pUCTR19-1-4 in
Fig. 3) was determined from both directions by sequencing a series of
deletion clones. The cDNA contained a single potential open reading
frame that encoded a polypeptide of 451 amino acid residues (Fig.
4). Around the putative initiation codon,
a consensus sequence for filamentous fungi (22) was observed;
consistencies at 5 (C or T),
2 (C or A), and
1 (C or A) were
noticed in addition to the highly conserved C, A, and C at positions
4,
3, and +5, respectively. No sequence similarities were found
when the nucleotide and amino acid sequences were compared with
DDBJ/EMBL/GenbankTM and SWISS-PROT data base sequences,
including those of Tri3 (23), the trichothecene
15-O-acetyltransferase gene.
|
Use of Tri101 as a Selectable Marker in S. pombe-- There are no selectable marker genes of eukaryotic origin, except blasticidin S (BS) resistance BSD (15), which confer resistance to drugs by means of inactivation. To compare the frequency of transformation with URA3 and BSD, pBRURA3 and pBRARS were constructed from pAU9 (16) by eliminating the EcoRI fragment of ARS or the PvuII-HindIII fragment of URA3, respectively. S. pombe cells were cotransformed by either pBRURA3, pUCSVBSD (Fig. 3), or pUCSVTri101 (Fig. 3) with the transducing vector pBRARS and plated on agar media that were selective for the desired type of transformants. As shown in Table II, Tri101 was not as good as URA3 or BSD for use as a selectable marker in S. pombe. Although the efficiency was low, the result suggests that Tri101 may be used as a marker gene for eukaryotic organisms for which only a few drugs that effectively inhibit the cell growth are available.
|
In Vitro-specific 3-O-Acetylation of Trichothecene Ring by Recombinant TRI101-- Using the T7 transcription/expression system, Tri101 was overproduced in E. coli (Fig. 5A), and the crude recombinant enzyme was used for in vitro acetylation assays. Each trichothecene was converted to a product showing the same Rf value as that of the 3-O-acetyl derivative in the presence of acetyl-CoA (Fig. 5B). Since there are three potential hydroxyl groups to be acetylated in DON, the structure of each product was confirmed by 1H NMR analysis. Each product showed a signaling pattern consistent with that of the 3-O-acetyl derivative (data not shown). These results indicate that TRI101 could transfer the acetyl group from acetyl-CoA to the hydroxyl group at the C-3 position of the 12,13-epoxytrichothec-9-ene structure but not at the C-7 and C-15 positions.
|
Isolation of the Tri5- and Tri101-containing Cosmid Clones and Mapping of the Regions around Tri101-- We have screened a cosmid library of F. graminearum (containing 8 × 103 unamplified bacterial colonies) and obtained one clone pCosTr032 containing Tri5 and nine clones containing Tri101. Detailed restriction mapping analysis of these Tri101-containing cosmid clones (Fig. 6A) revealed that two least overlapping clones, pCosTr135 and pCosTr137, cover the adjacent regions that extend to 35 kb on both sides of the gene. The genomic DNA region for Tri101 was mapped to a 3-kb XhoI-XbaI fragment (data not shown).
|
Tri101 Is Not Located Near Other Trichothecene Biosynthetic Genes That Are Closely Linked to Tri5-- The biosynthetic genes contained in the Tri5- or Tri101-containing cosmid clones were identified by PCR amplification of regions within the coding sequences of the genes. The primers described under "Experimental Procedures" proved to work well for specific amplification of Tri4, Tri5, and Tri6 as well as for specific amplification of Tri101. No products were generated from the Tri101-containing cosmid clones (pCosTr135 and pCosTr137) when primers for Tri4, Tri5, and Tri6 were used in PCR reactions (Fig. 6B). On the other hand, fragments of Tri5 and Tri6 were successfully amplified from the Tri5-containing cosmid clone (pCosTr032), although Tri101 was not included in this region. A similar conclusion was obtained by Southern analysis (data not shown). The above results indicate that clustering of several trichothecene biosynthetic genes also occurs in F. graminearum and that Tri101 is located at least 35 kb apart from Tri4, Tri5, and Tri6, which were shown to be clustered within 7.1 kb in the genome of Fusarium sporotrichioides (17).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies classified trichothecenes into two groups according to the types of substituents at the C-8 position (2) or the types of protein synthesis inhibition at 60 S ribosomal subunits (3). Here we propose that trichothecenes could also reasonably be divided into two types according to the structure of the pathway intermediates immediately before the second cyclization reaction, i.e. trichothecenes that are derived from di-oxygenated trichodiol and tri-oxygenated trichotriol could be referred to as d-type and t-type trichothecenes, respectively. Several pieces of circumstantial evidence suggest that 3-O-acetylation works as a self-defense mechanism for the t-type-producing fungi.
Is Tri101 a Self-defense Gene for the t-type Trichothecene Producer?-- Although T-2 toxin was inactive on spore germination of F. graminearum F15, trichothecin (Fig. 1), a d-type trichothecene similarly toxic as T-2 toxin to S. pombe (Table I), exerted a potent inhibitory action on the fungal germination at 100 µg/ml in YPD medium. This inhibitory action would be attributed to the lack of the 3-hydroxyl group to be acetylated in d-type trichothecenes. Other t-type trichothecenes did not inhibit spore germination of the fungus due to the presence of the 3-O-acetyltransferase activity. The result indicates that resistance in t-type-producing strains does not involve modification or replacement of the target ribosomes, as opposed to the d-type resistance mechanisms of the Saccharomyces cerevisiae mutant (24) and the trichothecin-producing strain of Trichothecium roseum (25).
Due to the commercial unavailability of the intermediates, we could not check whether S. pombe cells expressing Tri101 have an ability to convert isotrichodermol to isotrichodermin. However, it seems very likely that Tri101 is the only gene responsible for the reaction, considering that all of the 25 T-2 toxin-resistant S. pombe transformants carried Tri101 and that no homologous genes were contained in the genome of the t-type-producing fungus. Furthermore, recombinant TRI101 from E. coli was specific to the C-3 hydroxyl group of the trichothecene ring irrespective of the substituents at other positions. These results suggest that all the compounds with 3-hydroxy-12,13-epoxytrichothec-9-ene structure served as substrates for the enzyme. Thus, the primary defensive strategy of F. graminearum would be modification of t-type trichothecenes by TRI101 through O-acetylation at the C-3 position. N-Acetylation and O-phosphorylation are representatives of self-defense mechanisms against various kinds of antibiotics. Inactivation of a drug by O-acetylation is known to occur in the case of chloramphenicol, but it was not found in organisms that produce chloramphenicol. To our knowledge, Tri101 is the first antibiotic-inactivating O-acetyltransferase gene derived from a producing organism. The existence of an apparent 3-O-acetyl exchange mechanism in the biosynthetic pathway (11) may possibly be explained by the relative instability of the 3-O-acetyl group attached to the trichothecene ring and the importance of the substituent that has to be re-acetylated for the well being of the producer.Utility of Tri101--
The idea that the tricyclic intermediates
of t-type trichothecenes are modified by Tri101 would have
important implications for understanding the mechanism of trichothecene
production. If the gene is essential for the self-defense mechanism,
the Tri101 mutant generated through a genetic
transformation technique would be lethal when the toxic intermediates
accumulated within the cell. An alternative way to rescue the lethal
Tri101
mutation is to introduce a second
mutation on genes that catalyze a trichothecene biosynthetic step
before the formation of isotrichodermol. Such oxygenation genes can be
inactivated by integration of a selectable marker using the restriction
enzyme-mediated integrationtechnique (26, 27). This implies that
use of a Tri101
mutant provides an opportunity
to clone other biosynthetic genes before the second cyclization
reaction by simply selecting for the ability of the restriction
enzyme-mediated integration transformants to grow under conditions that
permit trichothecene biosynthesis.
Location of Tri101 in the Genome of the Producer Fungus--
The
first successful cloning of a trichothecene biosynthetic gene yielded
Tri5 (formerly Tox5), encoding trichodiene
synthase (34). Tri5 was cloned by screening a gt11
library of F. sporotrichioides with antiserum raised against
the purified enzymes (35). Recently, other trichothecene biosynthetic
genes, Tri3 (23) and Tri4 (17), and a regulatory
gene, Tri6 (19), have been identified in cosmid clones that
cover a region of 60 kb around Tri5. Thus, it was of
interest whether Tri101 was also clustered in the same
region in the genome of the producer fungus. PCR analysis indicated
that Tri101 was not located near these biosynthetic genes,
but this does not necessarily rule out the possibility of its existence
in the trichothecene biosynthetic gene cluster. Rather, the
Tri101-containing cosmid clones might be useful in isolating
other genes, not linked to Tri5 in the biosynthetic gene
cluster. In fact, transformations with the Tri5-containing
cosmid clones did not result in restoration of T-2 toxin production in
a strain carrying mutations at the Tri1 locus (36),
suggesting that the gene cluster is too large to be covered by several
cosmid clones. Further investigation may shed light on the structure of
the trichothecene biosynthetic gene cluster and the mechanism of
trichothecene production.
![]() |
ACKNOWLEDGEMENT |
---|
We express our thanks to Dr. S. Maeda for critical reading of the manuscript and valuable suggestions.
![]() |
Note Added in Proof |
---|
Tri101 shows a high similarity to open
reading frame YLL063c of Saccharomyces cerevisiae, which
appeared in the GenBankTM after submission of this manuscript
(accession number Z73168). The BLASTX 2.0.3 program at NCBI (Nov. 14, 1997) generated a statistically significant alignment of its translated
sequence to this yeast hypothetical protein at an E value of
e103 (similarities = 63%, identities = 44%). Open
reading frame YLL063c is located on chromosome XII and codes for a
probable membrane protein of unknown function. The yeast homolog would
not be a functional trichothecene 3-O-acetyltransferase, but
its existence raises a question as to the evolutional origin of this
biosynthetic gene.
![]() |
FOOTNOTES |
---|
* This research was supported in part by a grant for a Biodesign Research Group from the Institute of Physical and Chemical Research (RIKEN).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000874.
§ To whom correspondence should be addressed: Tel.: 81-048-467-9518; Fax: 81-048-462-4676.
** Present address: Plant Pathology Laboratory, Faculty of Agriculture, Meiji University, Higashimita 1-1-1, Tama-ku, Kawasaki-shi, Kanagawa 214, Japan.
1 The abbreviations used are: DON, deoxynivalenol; BHK, baby hamster kidney; kb, kilobase pair(s); PCR, polymerase chain reaction; DAS, 4,15-diacetoxyscirpenol; 3-ADAS, 3,4,15-triacetoxyscirpenol; 3-AT-2, 3-acetylT-2; BS, blasticidin S.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|