Emericella astellata, a new producer of aflatoxin B, B and sterigmatocystin

AIMS: To report on aflatoxin B(1) and B(2) production from a species of Emericella. METHODS AND RESULTS: Aflatoxins and sterigmatocystin were determined by high-pressure liquid chromatography (HPLC) with diode array detection and confirmed by HPLC with mass spectrometry detection. Among 30 known species of Emericella only one species produced aflatoxin. Strains originating from the same geographical source material had different patterns of aflatoxin and sterigmatocystin production on different media, indicating that epigenetic factors may be involved in the regulation of aflatoxin production. However, two cultures from the same original genet were very similar. CONCLUSIONS: Emericella astellata can produce small amounts of sterigmatocystin and aflatoxin B(1) and B(2). SIGNIFICANCE AND IMPACT OF THE STUDY: Emericella has been used extensively in genetic studies and therefore the isolates producing aflatoxin can be used to elucidate the genetic, evolutionary and maybe ecological role of aflatoxins using molecular genetic methods.     

Genetic modification of an echinocandin B-producing strain ofAspergillus nidulans to produce mutants blocked in sterigmatocystin biosynthesis

Summary The production of echinocandin B (ECB), a lipopolypeptide used for chemical manufacture of the anti-Candida agent Cilofungin^TM, was accomplished by fermentation using a strain ofAspergillus nidulans. In addition to ECB, this fermentation also produces a significant amount of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Mutants blocked in the ST biosynthetic pathway were created by genetic modification of the polyploid production strain C747. The following steps were involved: (i) reduction of the genotype to haploid by treatment with the spindle fiber poison methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate (MBC), using colony morphology, conidia size, and the ability to obtain 5-fluoro-orotic acid (5-FOA)-resistant mutants as criteria for ploidy; (ii) mutagenesis of a haploid isolate using UV irradiation; and (iii) screening of mutants for inability to produce ST by thin layer chromatography. Six mutants blocked in ST production were isolated. All six remained capable of producing ECB equivalent in quantity to the haploid strain C747-GR14. One of the mutants was shown to be the result of a chromosomal translocation.     

Phylogenetic analysis of homologous fatty acid synthase and polyketide synthase involved in aflatoxin biosynthesis

The first two steps of aflatoxin biosynthesis are catalyzed by the HexA/B and by the Pks protein. The phylogenetic analysis clearly distinguished fungal HexA/B from FAS subunits and from other homologous proteins. The phylogenetic trees of the HexA and HexB set of proteins share the same clustering. Proteins involved in the synthesis of fatty acids or in the aflatoxin or sterigmatocystin biosynthesis cluster separately. The Pks phylogenetic tree also differentiates the aflatoxin-related polypeptide sequences from those of other kinds of secondary metabolism. The function of some of the A. flavus Pks homologues may be deduced from the phylogenetic analysis. The conserved sequence motifs of protein domains shared by HexA/B and Pks - namely, β-polyketide synthase (KS), acetyl transferase (AT) and acyl carrier protein (ACP) - have been identified, and the HexA/B and Pks involved in aflatoxin biosynthesis have been distinguished from those involved in primary metabolism or other kinds of secondary metabolism.     

A strain of Aspergillus flavus from China shows potential as a biocontrol agent for aflatoxin contamination

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Strains of A. flavus that are non-aflatoxigenic (i.e., incapable of secreting aflatoxins) have proven effective in controlling contamination by these aflatoxin producing species in the field. In the present study, a non-aflatoxigenic A. flavus strain, GD-3, was isolated from a peanut field in Guangdong Province, China. Polymerase chain reaction (PCR) analysis showed that 12 aflatoxin biosynthesis genes (aflT, pksA, nor-1, fas-2, fas-1, aflR, aflJ, adhA, estA, norA, ver-1 and verA) were deleted in GD-3. Co-inoculation with a toxigenic strain, GD-15, at the ratio of 1:10, 1:1 or 10:1 (GD-3:GD-15), showed that GD-3 was capable of reducing detectable aflatoxin levels on three different substrates. This reduction ranged from 33% to 99% and correlated with competitor ratio. These results demonstrated that GD-3 was successful at reducing aflatoxin contamination and showed promise as a potential agent of biocontrol for local farmers.     

Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5 kb from 20 kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A. flavus not captured with other methods.

Significance and Impact of the Study

Aflatoxins, potent naturally‐occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A. flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A. flavus populations.     

假单胞菌胞外酶降解黄曲霉毒素B1的酶学性质

[背景]黄曲霉毒素B1(AflatoxinB1,AFB1)毒性强、污染普遍,目前尚无有效的防治办法.[目的]为了发掘高效的AFB1降解菌并探索其降解特性,对红树林污泥样品中一株AFB1降解菌株(HAI2)的酶学性质进行分析.[方法]以AFB1结构类似物为唯一碳源,筛选出一株高效的AFB1降解菌,利用16SrRNA基因测...     

Differential expression of the chsE gene encoding a chitin synthase of Aspergillus nidulans in response to developmental status and growth conditions

Expression of chsE encoding one of the five chitin synthases of Aspergillus nidulans was analyzed. Expression of chsE was moderate in conidiophores, but somewhat weaker in vegetative mycelia. During sexual development, chsE was expressed strongly in young cleistothecia and hülle cells, but little in mature sexual structures. Deletion of chsE caused a significant decrease in the chitin content of the cell wall during early sexual development. Expression of chsE was increased by substituting glucose with lactose or by addition of 0.6M KCl or NaCl, but affected little by substituting glucose with sodium acetate. Consequently, chsE was shown to have a mode of expression distinct from those of the other chitin synthase genes, chsA, chsB and chsC.     

Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAP

In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1–299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The β-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC₁-receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors.     

A novel nuclear factor, SREB, binds to a cis-acting element, SRE, required for inducible expression of the Aspergillus oryzae Taka-amylase A gene in A. nidulans

The Taka-amylase A gene (taaG2) of Aspergillus oryzae is inducibly expressed in A. nidulans upon exposure to inducing carbon sources, such as starch and maltose. In order to identify nuclear factor(s) possibly involved in the induction of the taaG2 gene, gel mobility shift assays and DNase I footprinting analyses were carried out, and revealed a novel nuclear factor in A. nidulans extracts, which specifically bound to two sites in the taaG2 promoter region, −204 to −189 and −182 to −168, which share the common sequence GGAAATT. The nuclear factor was detected in nuclei from both induced and uninduced mycelia. Mutational analysis within and around the binding sequences demonstrated that only the upstream binding sequence, designated SRE (starch responsive element), was required for the inducible expression of the taaG2 gene, and thus we designated the nuclear factor SREB (SRE binding factor). The downstream binding site contained an inverted SRE (ISRE) and played no role in the induction of taaG2 expression. SREB was shown by gel retardation assays to have higher affinity for SRE than for ISRE. Received: 26 January 1999 / Accepted: 10 November 1999     

Expression in a wine yeast strain of the Aspergillus niger abfB gene

Abstract A recombinant wine yeast strain has been constructed expressing the gene coding for a-L-arabinofuranosidase B from Aspergillus niger under the control of the yeast actin gene promoter. The protein is efficiently secreted by the recombinant yeast, allowing its purification and characterisation. The heterologous α-l-arabinofuranosidase B shows similar physico-chemical properties to the native enzyme. The wine produced in microvinification experiments using the recombinant yeast presents the same oenological characteristics as obtained with the untransformed strain. The a-L-arabinofuranosidase B protein is detected throughout the fermentation.     

Evidence for de novo synthesis of an aflatoxin pathway methyltransferase near the cessation of active growth and the onset of aflatoxin biosynthesis in Aspergillus parasiticus mycelia

The accumulation of both activity and protein of a methyltransferase (MTase) from Aspergillus parasiticus, which catalyzes conversion of sterigmatocystin to O-methylsterigmatocystin in the aflatoxin pathway, was detected in fungal mycelia slightly before the onset of aflatoxin biosynthesis in the same cultures. MTase protein was identified in mycelial postmicrosomal (soluble protein) fractions by electrophoresis and subsequent immunoblotting using antiserum raised against purified MTase protein; MTase activity was determined by measuring the rate of conversion of sterigmatocystin to O-methylsterigmatocystin in the presence of soluble protein fractions. Using the above technique, it was determined that MTase protein as well as MTase activity increased sharply in mycelia 30 to 45 h after inoculation, shortly after which, mycelial growth rate began to decline. During the subsequent time interval (45 to 70 h after inoculation), a sharp increase in aflatoxin levels was detected in the culture medium. Results obtained from an experiment in which cycloheximide was added to cultures at various times to inhibit protein synthesis and from an experiment in which mycelial proteins were radiolabelled to identify newly synthesized proteins indicated that accumulation of MTase activity and protein in late growth phase mycelia is due to de novo protein synthesis.