Expression Of aflR Research Articles

Streptomyces–Aspergillus flavus interactions: impact on aflatoxin B accumulation

The aim of this work was to investigate the potential of Streptomyces sp. as biocontrol agents against aflatoxins in maize. As such, we assumed that Streptomyces sp. could provide a complementary approach to current biocontrol systems such as Afla-guard® and we focused on biocontrol that was able to have an antagonistic contact with A. flavus. A previous study showed that 27 (out of 38) Streptomyces sp. had mutual antagonism in contact with A. flavus. Among these, 16 Streptomyces sp. were able to reduce aflatoxin content to below 17% of the residual concentration. We selected six strains to understand the mechanisms involved in the prevention of aflatoxin accumulation. Thus, in interaction with A. flavus, we monitored by RT-qPCR the gene expression of aflD, aflM, aflP, aflR and aflS. All the Streptomyces sp. were able to reduce aflatoxin concentration (24.0–0.2% residual aflatoxin B1). They all impacted on gene expression, but only S35 and S38 were able to repress expression significantly. Indeed, S35 significantly repressed aflM expression and S38 significantly repressed aflR, aflM and aflP. S6 reduced aflatoxin concentrations (2.3% residual aflatoxin B1) and repressed aflS, aflM and enhanced aflR expression. In addition, the S6 strain (previously identified as the most reducing pure aflatoxin B1) was further tested to determine a potential adsorption mechanism. We did not observe any adsorption phenomenon. In conclusion, this study showed that Streptomyces sp. prevent the production of (aflatoxin gene expression) and decontamination of (aflatoxin B1 reduction) aflatoxins in vitro.

Prevention of aflatoxin contamination by a soil bacterium of Stenotrophomonas sp. that produces aflatoxin production inhibitors

A soil bacterium, designated strain no. 27, was found to produce aflatoxin-production inhibitors. The strain was identified as a species of the genus Stenotrophomonas, and was found to be closely related to Stenotrophomonas rhizophila. Two diketopiperazines, cyclo(L-Ala-L-Pro) and cyclo(L-Val-L-Pro), were isolated from the bacterial culture filtrate as main active components. These compounds inhibited aflatoxin production of Aspergillus parasiticus and Aspergillus flavus in liquid medium at concentrations of several hundred µM without affecting fungal growth. Both inhibitors inhibited production of norsorolinic acid, a biosynthetic intermediate involved in an early step of the aflatoxin biosynthetic pathway, and reduced the mRNA level of aflR, which is a gene encoding a key regulatory protein necessary for the expression of aflatoxin-biosynthetic enzymes. These results indicated that the inhibitors targets are present in early regulatory steps leading to AflR expression. Co-culture of strain no. 27 with aflatoxigenic fungi in liquid medium effectively suppressed aflatoxin production of the fungus without affecting fungal growth. Furthermore, application of the bacterial cells to peanuts in laboratory experiments and at a farmer's warehouse in Thailand by dipping peanuts in the bacterial cell suspension strongly inhibited aflatoxin accumulation. The inhibitory effect was dependent on bacterial cell numbers. These results indicated that strain no. 27 may be a practically effective biocontrol agent for aflatoxin control.

Characterization of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B1, while 47.2% aflatoxins B1 and B2. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson’s correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB1 and AFB2, but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.

The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression

The influence of varying combinations of water activity (aw) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17-42°C and 0.90-0.99 aw. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 aw. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B1 or G1 biosynthesis. The temperature optima of aflatoxin B1 and G1 were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G1, 20-30°C) or above (aflatoxin B1, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B1 (aflR) or G1 (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G1 biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B1 biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B1, whereas it was water activity for G1 biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.

Analysis of aflatoxin regulatory factors in serial transfer-induced non-aflatoxigenic Aspergillus parasiticus

Aflatoxins (AFs) are carcinogenic secondary metabolites of Aspergillus parasiticus. In previous studies, non-toxigenic A. parasiticus sec− (for secondary metabolism negative) variants were generated through serial transfer of mycelia from their toxigenic sec+ (for secondary metabolism positive) parents for genetic and physiological analysis for understanding regulation of AF biosynthesis. Previous studies have shown no difference in the DNA sequence of aflR, a positive regulator of AF production, in the sec+ and sec− strains. In this study, AflJ, another positive regulator of AF production, laeA, a global regulator of secondary metabolism, and the intergenic region between aflR and aflJ, were analysed to determine if they play a role in establishment of the sec− phenotype. The study showed that while this sequence identity extended to the aflJ as well as the aflJ–aflR intergenic region, expression of aflR in the sec− strain was several fold lower than that observed in the sec+ strain, while aflJ expression was barely detectable in the sec− strain. Western blot analysis indicated that despite AflR protein being present in the sec− strain, no toxin production resulted. Introduction of a second copy of aflR into the sec− strain increased aflR expression, but did not restore AF production. Lastly, reverse transcription-PCR analysis revealed that laeA was expressed in both sec+ and sec− strains. These results suggest that although aflR, aflJ and laeA are necessary for AF production, they are not sufficient. We propose that the aflR and aflJ expression may be regulated by element(s) downstream from laeA or from pathways not influenced by laeA.

Evidence that a wortmannin-sensitive signal transduction pathway regulates aflatoxin biosynthesis

A signal transduction pathway involving cAMP and protein kinase A (PKA) regulates aflatoxin accumulation and nor-1 and ver-1 (aflatoxin structural genes) promoter function in Aspergillus parasiticus by modulating expression of a key transcriptional activator, AflR. To understand the function of this pathway in greater detail we treated A. parasiticus in culture with wortmannin, a frequently used probe of phosphatidyl inositol (PI)-3 kinase activity. A. parasiticus D8D3 (nor-1::GUS reporter) and I4 (ver-1::GUS reporter) were grown on a defined solid growth medium (GMS agar) under aflatoxin-inducing conditions. GMS containing wortmannin (1 microM) reduced aflatoxin B1 accumulation up to 15-fold accompanied by a similarly large decrease in ver-1 and nor-1 promoter activity. Wortmannin inhibited growth (colony diameter) and asexual sporulation but to a much smaller extent. Wortmannin treatment increased intracellular cAMP levels up to 25-fold; total PKA activity also increased within 10 min of wortmannin exposure. These data support a regulatory model in which PI-3 kinase activity modulates intracellular cAMP accumulation and PKA activity. This in turn regulates AflR expression and activity, aflatoxin gene expression and aflatoxin accumulation.

Evidence that a wortmannin-sensitive signal transduction pathway regulates aflatoxin biosynthesis

A signal transduction pathway involving cAMP and protein kinase A (PKA) regulates aflatoxin accumulation and nor-1 and ver-1 (aflatoxin structural genes) promoter function in Aspergillus parasiticus by modulating expression of a key transcriptional activator, AflR. To understand the function of this pathway in greater detail we treated A. parasiticus in culture with wortmannin, a frequently used probe of phosphatidyl inositol (PI)-3 kinase activity. A. parasiticus D8D3 (nor-1::GUS reporter) and I4 (ver- ::GUS reporter) were grown on a defined solid growth medium (GMS agar) under aflatoxin-inducing conditions. GMS containing wortmannin (1 μM) reduced aflatoxin B1 accumulation up to 15-fold accompanied by a similarly large decrease in ver-1 and nor-1 promoter activity. Wortmannin inhibited growth (colony diameter) and asexual sporulation but to a much smaller extent. Wortmannin treatment increased intracellular cAMP levels up to 25-fold; total PKA activity also increased within 10 min of wortmannin exposure. These data support a regulatory model in which PI-3 kinase activity modulates intracellular cAMP accumulation and PKA activity. This in turn regulates AflR expression and activity, aflatoxin gene expression and aflatoxin accumulation.

Putative Calmodulin-Binding Domains in Aflatoxin Biosynthesis–Regulatory Proteins

The inhibition of aflatoxin production by trifluoperazine, an anticalmodulin (CaM) agent and the relevance of Ca(2+)/CaM-dependent phosphorylation and dephosphorylation during aflatoxin biosynthesis was previously reported. To identify proteins that may be regulated by CaM, an in silico analysis for putative CaM-binding domains (CaMBDs) in the aflatoxin-related proteins of Aspergillus parasiticus was performed using the CaM target database. Interestingly, the key regulators of aflatoxin biosynthesis such as AflR and AflJ contained predicted CaMBDs at their C-termini. Furthermore, potential phosphorylation sites for CaM-kinase II were present within these CaMBDs. In addition to other aflatoxin biosynthesis enzymes--such as Vbs, DmtA and OmtA, and the VeA protein (known to regulate the expression of AflJ and AflR)--also showed the presence of putative CaMBDs. Although the present report reaffirms earlier observations on CaM-mediated regulation of aflatoxin biosynthesis, it also opens new avenues for identifying the specific targets of CaM and elucidating the exact mechanism of initiation and regulation of aflatoxin biosynthesis.

The Phosducin-Like Protein PhnA Is Required for Gβγ-Mediated Signaling for Vegetative Growth, Developmental Control, and Toxin Biosynthesis in Aspergillus nidulans

Phosducin or phosducin-like protein (PhLP) is a positive regulator of Gbetagamma activity. The Gbeta (SfaD) and Ggamma (GpgA) subunits function in vegetative growth and developmental control in the model filamentous fungus Aspergillus nidulans. To better understand the nature of Gbetagamma-mediated signaling, phnA, encoding an A. nidulans PhLP, has been studied. Deletion of phnA resulted in phenotypes almost identical to those caused by deletion of sfaD, i.e., reduced biomass, asexual sporulation in liquid submerged culture, and defective fruiting body formation, suggesting that PhnA is necessary for Gbeta function. The requirement for the RGS protein FlbA in asexual sporulation could be bypassed by the DeltaphnA mutation, indicating that PhnA functions in FlbA-controlled vegetative growth signaling, primarily mediated by the heterotrimeric G protein composed of FadA (Galpha), SfaD, and GpgA. However, whereas deletion of fadA restored both asexual sporulation and the production of sterigmatocystin (ST), deletion of sfaD, gpgA, or phnA failed to restore ST production in the DeltaflbA mutant. Further studies revealed that SfaD, GpgA, and PhnA are necessary for the expression of aflR, encoding the transcriptional activator for the ST biosynthetic genes, and subsequent ST biosynthesis. Overexpression of aflR bypassed the need for SfaD in ST production, indicating that the results of SfaD-mediated signaling may include transcriptional activation of aflR. Potential differential roles of FadA, Gbetagamma, and FlbA in controlling ST biosynthesis are further discussed.

The aflatoxin pathway regulator AflR induces gene transcription inside and outside of the aflatoxin biosynthetic cluster

Aflatoxin contamination of food and feed is a major concern due to the carcinogenic properties of this mycotoxin. Previous studies using classical approaches have identified a cluster of genes responsible for aflatoxin production under the control of the pathway-specific transcriptional regulator aflR, but it is unknown whether aflR controls expression of other genes within the genome. Transcription profiling comparing wild type and DeltaaflR strains of Aspergillus parasiticus grown under conditions conducive for aflatoxin production identified only 23 upregulated genes in the wild type. These included 20 genes in the aflatoxin biosynthetic cluster, and three additional genes outside the aflatoxin biosynthetic cluster (nadA, hlyC, and niiA), all with AflR binding sites. This report is the first to demonstrate genes outside the biosynthetic cluster as being associated with aflR expression.

Identification of a Maize Kernel Stress-Related Protein and Its Effect on Aflatoxin Accumulation

ABSTRACT Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize. Through proteomic comparisons of maize kernel embryo proteins of resistant and susceptible genotypes, several protein spots previously were found to be unique or upregulated in resistant embryos. In the present study, one of these protein spots was sequenced and identified as glyoxalase I (GLX-I; EC 4.4.1.5). The full-length cDNA of the glyoxalase I gene (glx-I) was cloned. GLX-I constitutive activity was found to be significantly higher in the resistant maize lines compared with susceptible ones. After kernel infection by A. flavus, GLX-I activity remained lower in susceptible genotypes than in resistant genotypes. However, fungal infection significantly increased methylglyoxal (MG) levels in two of three susceptible genotypes. Further, MG was found to induce aflatoxin production in A. flavus culture at a concentration as low as 5.0 muM. The mode of action of MG may be to stimulate the expression of aflR, an aflatoxin biosynthesis regulatory gene, which was found to be significantly upregulated in the presence of 5 to 20 muM MG. These data suggest that GLX-I may play an important role in controlling MG levels inside kernels, thereby contributing to the lower levels of aflatoxins found in resistant maize genotypes.

Genetic analysis of morphological variants of Aspergillus parasiticus deficient in secondary metabolite production

Aflatoxins (AFs) are secondary metabolites produced mainly by Aspergillus parasiticus and A. flavus. To study AF regulation, previously isolated non-toxigenic A. parasiticus sec- (for secondary metabolism minus) variants were genetically analysed. In parasexual crossing, the sec- strains failed to form heterokaryons and diploids with other sec- strains. Heterokaryon test results suggested that involvement of cytoplasmic elements in the formation of sec- phenotype was unlikely. At the molecular level, the coding sequence of the sec- aflR (the only known positive regulator of AF pathway) was identical to that of their toxigenic sec+ (for secondary metabolism plus) parents. However, the sec- aflR expression was 5- to 10-fold lower compared to that in the sec+ forms. RT-PCR analysis demonstrated that the AF pathway genes were expressed in the sec- forms but in trace amounts and in their unprocessed forms. Combined, these results suggest that aflR is necessary but not sufficient for AF production and that elements involved in fungal development directly or indirectly influence its proper function.

Variability in nitrogen regulation of aflatoxin production by Aspergillus flavus strains.

Aflatoxins are toxic and carcinogenic metabolites of several Aspergillus species. The effect of nitrate on aflatoxin production and expression of the key regulatory genes involved in aflatoxin biosynthesis, aflR and aflJ, were compared among isolates of the S(B) and S(BG) strains of A. flavus. Aflatoxin production by two of the three strain S(B) isolates did not differ significantly between the two media tested, whereas for S(BG) A. flavus isolates, the level of aflatoxins in buffered nitrate medium was as much as 20-fold lower than in ammonium salts medium. Expression of aflR was not significantly affected by growth of cultures in nitrate medium for most of the isolates. However, on nitrate medium, expression of aflJ was 2.6-fold higher for the S(B) isolates than it was on ammonium salts medium, whereas for the S(BG) isolates aflJ expression was 2-fold lower on nitrate than on ammonium salts medium. This difference may result from the presence in the aflJ/aflR intergenic region of S(BG) isolates of fewer putative binding sites (HGATAR sites) for AreA, the positive-acting, wide domain transcription factor involved in regulation of nitrogen metabolism.

Absence of aflatoxin biosynthesis in koji mold (Aspergillus sojae).

Ten strains isolated from industrial soy sauce producing koji mold were identified as Aspergillus sojae and distinguished from Aspergillus parasiticus morphologically and physiologically. There was no detectable aflatoxin in any culture extracts of A. sojae strains. Strain 477 was chosen as a representative strain of industrial A. sojae for further molecular analysis. All enzymatic activities associated with the aflatoxin biosynthesis were not detected or negligible in strain 477 compared with that of the A. parasiticus strain. Southern analysis suggested that the genomic DNA of strain 477 contained aflatoxin biosynthetic pathway genes. In contrast, all industrial strains lacked detectable transcripts of aflR, the main regulatory gene for aflatoxin biosynthesis, under the aflatoxin-inducing condition. Our data suggest that defects in aflR expression cause the lack of expression of aflatoxin-related genes which results in the absence of aflatoxin biosynthesis in A. sojae strains.

RcoA has pleiotropic effects on Aspergillus nidulans cellular development.

Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins. The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1. Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression. RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression. In A. nidulans, deletion of rcoA (DeltarcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis. Expression of the asexual and ST pathway-specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e. flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a DeltarcoA strain. Overexpression of aflR in a DeltarcoA strain could not rescue normal expression of downstream targets of AflR. CreA-dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a DeltarcoA strain. The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N. crassa.

Generation of aflR disruption mutants of Aspergillus parasiticus.

The aflR gene of Aspergillus parasiticus and A. flavus encodes a binuclear zinc-finger, DNA-binding protein, AflR, responsible for activating the transcription of all known aflatoxin biosynthetic genes including itself. Studies to determine how environmental and nutritional factors affect aflR expression and hence aflatoxin production in A. parasiticus have been difficult to perform due to the lack of aflR "knockout" mutants. Transformation of an O-methylsterigmatocystin (OMST)-accumulating strain of A. parasiticus with an aflR-niaD gene disruption vector resulted in clones harboring a recombinationally inactivated aflR gene which no longer produced OMST or aflR transcript. By transformation of this aflR disruptant strain with constructs containing mutated versions of the aflR promoter, we identified three cis-acting sites that were necessary for aflR function: an AflR-binding site, a PacC-binding site, and a G + A-rich site near the transcription start site of aflR.

Blasticidin A derivatives with highly specific inhibitory activity toward aflatoxin production in Aspergillus parasiticus.

Blasticidin A (1), an antibiotic, has strong inhibitory activity toward aflatoxin production by Aspergillus parasiticus. We prepared some derivatives of 1 and examined their biological activities. Among them, derivatives 3 and 4 without the tetramic acid moiety of 1 maintained inhibitory activity toward aflatoxin production, but did not show antifungal activity or toxicity. RT-PCR experiments indicated that derivatives 3 and 4 as well as 1 significantly reduced the expression of genes encoding aflatoxin biosynthetic enzymes (pksA, ver-1 and omtA) and a regulatory gene (aflR) in A. parasiticus. These results suggested that derivatives 3 and 4 can inhibit aflatoxin production more specifically than 1 by inhibiting an early step prior to the expression of aflR in the pathway of aflatoxin biosynthesis.

Aspergillus nidulans mutants defective in stc gene cluster regulation.

The genes involved in the biosynthesis of sterigmatocystin (ST), a toxic secondary metabolite produced by Aspergillus nidulans and an aflatoxin (AF) precursor in other Aspergillus spp., are clustered on chromosome IV of A. nidulans. The sterigmatocystin gene cluster (stc gene cluster) is regulated by the pathway-specific transcription factor aflR. The function of aflR appears to be conserved between ST- and AF-producing aspergilli, as are most of the other genes in the cluster. We describe a novel screen for detecting mutants defective in stc gene cluster activity by use of a genetic block early in the ST biosynthetic pathway that results in the accumulation of the first stable intermediate, norsolorinic acid (NOR), an orange-colored compound visible with the unaided eye. We have mutagenized this NOR-accumulating strain and have isolated 176 Nor(-) mutants, 83 of which appear to be wild type in growth and development. Sixty of these 83 mutations are linked to the stc gene cluster and are likely defects in aflR or known stc biosynthetic genes. Of the 23 mutations not linked to the stc gene cluster, 3 prevent accumulation of NOR due to the loss of aflR expression.

Repressor-AFLR interaction modulates aflatoxin biosynthesis in Aspergillus parasiticus.

Regulation of aflatoxin (AF) biosynthesis likely involves a complex interplay of positive- and negative-acting factors that are affected by physiological cues responsive to internal and external stimuli. These factors, presumably, modulate the expression of the AF pathway-specific regulatory gene, aflR, whose product, AFLR, a zinc cluster transcription factor, then turns on or off the transcription of other AF genes. To determine if the AFLR carboxyl region (AFLRC) interacts with positive- or negative-acting proteins, we fused the Aspergillus parasiticus aflR carboxyl coding region (aflRC) to the promoter of A. parasiticus nitrite reductase gene (niiA(p)::aflRC), and transformed it into A. parasiticus SRRC 2043. Transformants that contained two copies of niiA(p)::aflRC, one at the niaD locus and another at the aflR locus, overproduced AF precursors independent of the nitrogen source. The higher copy number of the integrated niiA(p)::aflRC correlated with increased production of AF precursors by the transformants as well as increased expression of both aflRC and native aflR in potato dextrose broth and A&M medium. Since aflRC does not encode a DNA-binding domain, the expressed AFLRC should not bind to the promoters of AF pathway genes and affect transcription directly. The results are consistent with AFLRC titrating out a putative repressor that interacts with AFLR under different growth conditions and modulates AF biosynthesis. This interaction also indirectly affects sclerotial development.

Regulation of aflR and its product, AflR, associated with aflatoxin biosynthesis.

We studied the role of the regulatory gene aflR and its product, AflR, in the biosynthesis of aflatoxin in Aspergillus. Western blot and enzyme-linked immunosorbent assay analyses revealed that aflatoxin B1 accumulation was directly related to AflR expression and was regulated by various environmental and nutritional conditions, including temperature, air supply, carbon source, nitrogen source, and zinc availability. Expression of an aflatoxin biosynthetic pathway structural gene, omtA, was regulated by the presence of AflR. Induction patterns for aflR mRNA and AflR were correlated with that for omtA mRNA in an aflatoxin-producing strain of Aspergillus parasiticus. Analysis of non-aflatoxin-producing strains of A. flavus, A. sojae, and A. oryzae grown in medium suitable for aflatoxin B1 production showed that both aflR mRNA and AflR production were present; however, omtA mRNA production was not detected in any of these examined strains. AflR in the A. oryzae strain was regulated by carbon source and temperature in a manner similar to that seen with A. parasiticus.