Aflatoxins and Anthraquinones from Diploids of Aspergillus parasiticus

Bennett, Joan W.

doi:10.1099/00221287-113-1-127

Cited by 43 publications

(35 citation statements)

References 42 publications

(35 reference statements)

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“…The aflatoxin biosynthesis gene homologs from A. oryzae RIB 40 and their deduced amino acid sequences were compared with those from A. flavus or A. parasiticus available in the NCBI nucleotide database. The accession numbers for the nucleotides used in the analysis are AY 510453 (complete sequence of the aflatoxin biosynthesis gene cluster from A. flavus AF70) (12,15) and AY371490 (complete sequence of the aflatoxin biosynthesis gene cluster from A. parasiticus SU-1 [ATCC 56775]) (2,54). Both strains are aflatoxin-producing fungi.…”

Section: Methodsmentioning

confidence: 99%

Molecular Analysis of an Inactive Aflatoxin Biosynthesis Gene Cluster in Aspergillus oryzae RIB Strains

Tominaga

Lee

Hayashi

et al. 2006

Appl Environ Microbiol

122

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To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40. Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.Koji molds, Aspergillus oryzae and Aspergillus sojae, have traditionally been used in the brewing industry for the production of sake, miso, and soy sauce. A history of safety (1) and nonproductivity of aflatoxin is well established for industrial strains, and A. oryzae is considered "generally recognized as safe" by the U.S. Food and Drug Administration (41). These fungi belong to the Aspergillus section Flavi, which includes Aspergillus flavus and Aspergillus parasiticus, some of which produce the procarcinogen aflatoxin. It is thought that A. oryzae and A. sojae are taxonomically differentiated from A. flavus and A. parasiticus, respectively (20, 21, 23, 35, 37). It has been demonstrated that most of the 25 identified genes clustered within a specific 70-kb region of the fungal genome are involved in aflatoxin biosynthesis (reviewed in references 3, 13, 22, 40, 45, 49, and 54). Among them, the aflR gene is known to encode a major transcriptional regulator of aflatoxin biosynthesis genes (6,9,19,28,47,55). AflR binds to the consensus sequence 5Ј-TCGN 5 CGR-3Ј (16) found in the promoters of most of the aflatoxin biosynthesis genes (54), including aflR (14, 17). Putative binding sites (14, 17) for the transcription factors AreA (10, 34, 36), PacC (18), and FacB (43) have been identified in the aflR promoter.For A. sojae, several studies (30, 31, 42) have suggested an inability to produce aflatoxin, because mutations have been found in the aflR homolog (46). On the other hand, Kusumoto et al. (25) reported that 39 strains of A. oryzae could be classified into three groups based on fragment analysis by a lo...

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Section: Methodsmentioning

confidence: 99%

Molecular Analysis of an Inactive Aflatoxin Biosynthesis Gene Cluster in Aspergillus oryzae RIB Strains

Tominaga

Lee

Hayashi

et al. 2006

Appl Environ Microbiol

122

View full text Add to dashboard Cite

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“…parasiticus which accumulated norsolorinic acid and less aflatoxin than the wild-type. This mutant was recessive in diploids (Bennett, 1979). Several aflatoxin mutants having no detectable pigmented intermediates have been recovered in A.Javus (Papa, 1979).…”

Section: Introductionmentioning

confidence: 99%

Norsolorinic Acid Mutant of Aspergillus flavus

Papa

1982

Microbiology

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A mutant of Aspergillus Javus accumulating norsolorinic acid and approximately 50 % less aflatoxin than the parent strain was recovered after treatment of conidia with Nmethyl-N'-nitro-N-nitrosoguanidine. The gene nor controlling this mutant phenotype was recessive and linked to aJl-1 and leu on linkage group VII. Diploids homozygous for nor were similar to haploids in norsolorinic acid accumulation. The analysis of recombinant diploids and haploids showed that aJ-1 and nor were both distal to leu on the same chromosome arm. I N T R O D U C T I O NAflatoxins, secondary metabolites produced by Aspergillus Javus and Aspergillus parasiticus, have attracted considerable attention, and much has been reported about their biosynthesis and biological effects. Several intermediates in the aflatoxin biosynthetic pathway have been established through studies of induced mutants (Hsieh et al., 1976). Lee et al. (197 1 ) recovered a mutant of A . parasiticus which accumulated norsolorinic acid and less aflatoxin than the wild-type. This mutant was recessive in diploids (Bennett, 1979). Several aflatoxin mutants having no detectable pigmented intermediates have been recovered in A.Javus (Papa, 1979). This paper describes the genetic analysis of a norsolorinic acid mutant of A . Jlavus, including linkage detection and mapping. M E T H O D SStrains. Five auxotrophic strains of A.Javus having either white (w) or tan ( t ) spore colour were used in this study. Their individual genotypes were as follows: 1. w lys (requiring lysine); 2. tpdx leu (pyridoxine, leucine); 3. I leu; 4. afl-1 t leu; and 5 . t arg (arginine). Strain 4 was also impaired in aflatoxin production (aJ-1 ). The induction of auxotrophy, spore colour variation, and aflatoxin mutants has been described previously (Leaich & Papa, 1974; Papa, 1976 Papa, , 1979.Following the exposure of conidia from strain 1 to N-methyl-N'-nitro-N-nitrosoguanidine (NG), a mutant, designated l-N and having an orange-red mycelium, was recovered. The gene controlling the production of this orange-red pigment (norsolorinic acid) has been designated nor (Bennett, 1979).Media. The complete medium (CM) consisted of Czapek-Dox broth, 0.75 % (w/v) malt extract, 0.25 % (w/v) yeast extract and 1.5% (w/v) agar. The minimal medium (MM) was CM without the malt and yeast extracts. Diploids were plated on CM containing 0.007 % (w/v) p-fluorophenylalanine to induce haploidization. Appropriately supplemented MM was used for the identification of nutritional requirements of haploid segregants. The medium for production of aflatoxin consisted of 2 % (w/v) yeast extract and 20% (w/v) sucrose.Aflatoxin and norsolorinic acid determinations. Aflatoxin and norsolorinic acid were extracted according to procedures described by Lillard et al. (1970) and Papa (1976). Extracts were suspended in chloroform, appropriately diluted, and spotted on thin-layer chromatographic plates (pre-coated silica gel) from EM Laboratories, Elmsford, N.Y., U.S.A. Chromatograms for aflatoxin and norsolorinic acid separations were...

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“…Norsolorinic acid is bright orange, therefore, mutants blocked in the conversion of NA are readily identifiable. NA-accumulating mutants have been identified in both A. flavus (Papa, 1979(Papa, , 1982Payne et al, 1993) and in A. parasiticus (Detroy et al, 1973;Bennett, 1979). These mutants are not completely blocked in aflatoxin biosynthesis, but in all cases produce less aflatoxin than wild-type strains.…”

Section: Fig 3 Organization Of Fungal Fatty Acid Synthase (Fas) Andmentioning

confidence: 96%