Vibrio gazogenes inhibits aflatoxin production through downregulation of aflatoxin biosynthetic genes in Aspergillus flavus

Aspergillus flavus is an opportunistic pathogen of oilseed crops such as maize, peanut, cottonseed, and tree nuts and produces carcinogenic secondary metabolites known as aflatoxins during seed colonization. Aflatoxin contamination not only reduces the value of the produce but also is a health hazard to humans and animals. Previously, we observed inhibition of A. flavus aflatoxin biosynthesis upon exposure to the marine bacterium, Vibrio gazogenes (Vg). In this study, we used RNA sequencing to examine the transcriptional profiles of A. flavus treated with both live and heat-inactivated dead Vg and control samples. Fungal biomass, total accumulated aflatoxins, and expression profiles of genes constituting secondary metabolite biosynthetic gene clusters were determined at 24, 30, and 40 h after treatment. Statistically significant reductions in total aflatoxins were detected in Vg-treated samples as compared to control samples at 40 h. But no statistical difference in fungal biomass was observed upon these treatments. The Vg treatments were most effective on aflatoxin biosynthesis as was reflected in significant downregulation of majority of the genes in the aflatoxin gene cluster including the aflatoxin pathway regulator gene, aflR. Along with aflatoxin genes, we also observed significant downregulation in some other secondary metabolite gene clusters including cyclopiazonic acid and aflavarin, suggesting that the treatment may inhibit other secondary metabolites as well. Finally, a weighted gene correlation network analysis identified an upregulation of ten genes that were most strongly associated with Vg-dependent aflatoxin inhibition and provide a novel start-point in understanding the mechanisms that result in this phenomenon.

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Differential regulation and production of secondary metabolites among isolates of the fungal wheat pathogen Zymoseptoria tritici

10.1101/2021.08.12.456184 ◽

2021 ◽

Author(s):

M. Amine Hassani ◽

Ernest Oppong-Danquah ◽

Alice Feurty ◽

Deniz Tasdemir ◽

Eva H Stukenbrock

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Expression Profiles ◽

Pathogenic Fungus ◽

Gene Clusters ◽

Secondary Metabolite Production ◽

Zymoseptoria Tritici ◽

Host Colonization ◽

Metabolite Production

The genome of the wheat pathogenic fungus, Zymoseptoria tritici, represents extensive presence-absence variation in gene content. Here, we addressed variation in biosynthetic gene clusters (BGCs) content and biochemical profiles among three isolates. We analysed secondary metabolite properties based on genome, transcriptome and metabolome data. The isolates represent highly distinct genome architecture, but harbor similar repertoire of BGCs. Expression profiles for most BGCs show comparable patterns of regulation among the isolates, suggesting a conserved 'biochemical infection program'. For all three isolates, we observed a strong up-regulation of an abscisic acid (ABA) gene cluster during biotrophic host colonization, indicating that Z. tritici potentially interfere with host defenses by the biosynthesis of this phytohormone. Further, during in vitro growth the isolates show similar metabolomes congruent with the predicted BGC content. We assessed if secondary metabolite production is regulated by histone methylation using a mutant impaired in formation of facultative heterochromatin (H3K27me3). In contrast to other ascomycete fungi, chromatin modifications play a less prominent role in regulation of secondary metabolites. In summary, we show that Z. tritici has a conserved program of secondary metabolite production contrasting the immense variation in effector expression, some of these metabolites might play a key role during host colonization.

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Genome sequence of Novosphingobium malaysiense strain MUSC 273T, novel alpha-proteobacterium isolated from intertidal soil

Progress In Microbes & Molecular Biology ◽

10.36877/pmmb.a0000003 ◽

2018 ◽

Vol 1 (1) ◽

Author(s):

Hooi-Leng Ser ◽

Wai-Fong Yin ◽

Kok-Gan Chan ◽

Nurul-Syakima Ab Mutalib ◽

Learn-Han Lee

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Genome Sequence ◽

Catalase Activity ◽

Gene Clusters ◽

Rrna Genes ◽

Gram Negative ◽

Genomic Features

Novosphingobium malaysiense strain MUSC 273T is a recently identified Gram-negative, aerobic alpha-proteobacterium. The strain was isolated from intertidal soil with strong catalase activity. The genome sequence comprises 5,027,021 bp, with 50 tRNA and 3 rRNA genes. Further analysis identified presence of secondary metabolite gene clusters within genome of MUSC 273T. Knowledge of the genomic features of the strain may allow further biotechnological exploitation, particularly for production of secondary metabolites as well as production of industrially important enzymes

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Phylogenetic Distribution of Secondary Metabolites in the Bacillus subtilis Species Complex

mSystems ◽

10.1128/msystems.00057-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Kat Steinke ◽

Omkar S. Mohite ◽

Tilmann Weber ◽

Ákos T. Kovács

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Species Complex ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Content Type ◽

Frameshift Mutations ◽

Metabolite Production

ABSTRACT Microbes produce a plethora of secondary (or specialized) metabolites that, although not essential for primary metabolism, benefit them to survive in the environment, communicate, and influence cell differentiation. Biosynthetic gene clusters (BGCs), responsible for the production of these secondary metabolites, are readily identifiable on bacterial genome sequences. Understanding the phylogeny and distribution of BGCs helps us to predict the natural product synthesis ability of new isolates. Here, we examined 310 genomes from the Bacillus subtilis group, determined the inter- and intraspecies patterns of absence/presence for all BGCs, and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in the distribution of both known and unknown products. Further, we analyzed variations in the BGC structures of particular families encoding natural products, such as plipastatin, fengycin, iturin, mycosubtilin, and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and a few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Surprisingly, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed genomic analysis of BGCs and the remarkable phylogenetically conserved erosion of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCE Members of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those, the production of antifungals, which makes them promising biocontrol strains. While there are studies examining the distribution of well-known secondary metabolites in Bacilli, intraspecies clade-specific distribution has not been systematically reported for the B. subtilis group. Here, we report the complete biosynthetic potential within the B. subtilis group to explore the distribution of the biosynthetic gene clusters and to reveal an exhaustive phylogenetic conservation of secondary metabolite production within Bacillus that supports the chemodiversity within this species complex. We identify that certain gene clusters acquired deletions of genes and particular frameshift mutations, rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview guides the assignment of the secondary metabolite production potential of newly isolated Bacillus strains based on genome sequence and phylogenetic relatedness.

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Genome mining and UHPLC-MS/MS illuminate the specificity of secondary metabolite synthetic gene clusters in Bacillus subtilis NCD-2

10.21203/rs.3.rs-33091/v1 ◽

2020 ◽

Author(s):

Zhenhe Su ◽

Xiuye Chen ◽

Xiaomeng Liu ◽

Qinggang Guo ◽

Shezeng Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Biological Activities ◽

Genome Mining ◽

Gene Clusters ◽

Synthetic Gene ◽

Integrative Approach ◽

Amino Acid Sequence Similarity ◽

Genetic Characteristics

Abstract Background Bacillus subtilisstrain NCD-2 is anexcellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore all the secondary metabolite synthetic gene clusters and related bioactive compounds in NCD-2. An integrative approach, which coupled genome mining with structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was conducted to interpret the chemical origins of the significant biological activities in NCD-2. Results Genome mining revealed that NCD-2 contained nine gene clustershaving predicted functionsinvolving secondary metabolites with bioactive abilities. They encoded six known products-fengycin, surfactin, bacillaene, subtilosin, bacillibactin, and bacilysin-as well as three unknown products.Interestingly, the synthetic gene clusters for surfactin and fengycin showed 83% and 92% amino acid sequence similarity levels with the corresponding productsin Bacillus velezensisstrain FZB42. A further comparison of gene clusters encoding fengycin and surfactinrevealed that strain NCD-2 had lost thefenC and fenDgenes in the fengycinbiosynthetic operon, and that the surfactin synthetic enzyme-related gene srfAB was divided into two parts.Abioinformatics analysis showed that fenEAmay function as fenCD in synthesizing fengycinand that the structure of thisfengycin synthetic gene clusteris likely unique to NCD-2.Five kinds of fengycin,with 26 homologs, and surfactin,with 4 homologs,were detectedfrom strain NCD-2, which indicated the non-typical and unique nature of this fengycin biosynthetic gene cluster.To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis. Conclusions The data provide the genetic characteristics of secondary metabolite synthetic gene clusters for fengycinand surfactin in B. subtilis NCD-2, including the unique synthetic mechanism for fengycin, and suggest that bioactive secondary metabolites explain the biological activities of NCD-2.

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A detailed in silico analysis of secondary metabolite biosynthesis clusters in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum

10.21203/rs.2.11845/v2 ◽

2019 ◽

Author(s):

Carolyn Graham-Taylor ◽

Lars G Kamphuis ◽

Mark Derbyshire

Keyword(s):

Secondary Metabolites ◽

Host Range ◽

Secondary Metabolite ◽

Sclerotinia Sclerotiorum ◽

Plant Pathogens ◽

Pathogenic Fungus ◽

Gene Clusters ◽

Sequence Diversity ◽

Broad Host Range ◽

Data Set

Abstract Background The broad host range pathogen Sclerotinia sclerotiorum infects over 400 plant species and causes substantial yield losses in crops worldwide. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but little is known about the secondary metabolite repertoire of S. sclerotiorum. In this study, we predicted secondary metabolite biosynthetic gene clusters in the genome of S. sclerotiorum and analysed their expression during infection of Brassica napus using an existing transcriptome data set. We also investigated their sequence diversity among a panel of 25 previously published S. sclerotiorum isolate genomes.Results We identified 80 putative secondary metabolite clusters. Over half of the clusters contained at least three transcriptionally coregulated genes. Comparative genomics revealed clusters homologous to clusters in the closely related plant pathogen Botrytis cinerea for production of carotenoids, hydroxamate siderophores, DHN melanin and botcinic acid. We also identified putative phytotoxin clusters that can potentially produce the polyketide sclerin and an epipolythiodioxopiperazine. Secondary metabolite clusters were enriched in subtelomeric genomic regions, and those containing paralogues showed a particularly strong association with repeats. The positional bias we identified was borne out by intraspecific comparisons that revealed putative secondary metabolite genes suffered more presence / absence polymorphisms and exhibited a significantly higher sequence diversity than other genes.Conclusions These data suggest that S. sclerotiorum produces numerous secondary metabolites during plant infection and that their gene clusters undergo enhanced rates of mutation, duplication and recombination in subtelomeric regions. The microevolutionary regimes leading to S. sclerotiorum secondary metabolite diversity have yet to be elucidated. Several potential phytotoxins documented in this study provide the basis for future functional analyses.

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Variation Among Biosynthetic Gene Clusters, Secondary Metabolite Profiles, and Cards of Virulence Across Aspergillus Species

Genetics ◽

10.1534/genetics.120.303549 ◽

2020 ◽

Vol 216 (2) ◽

pp. 481-497 ◽

Cited By ~ 1

Author(s):

Jacob L. Steenwyk ◽

Matthew E. Mead ◽

Sonja L. Knowles ◽

Huzefa A. Raja ◽

Christopher D. Roberts ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Secondary Metabolite ◽

Immune Cell ◽

Sequence Divergence ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Host Immune System ◽

Biosynthetic Gene ◽

Genetic Determinants

Aspergillus fumigatus is a major human pathogen. In contrast, Aspergillus fischeri and the recently described Aspergillus oerlinghausenensis, the two species most closely related to A. fumigatus, are not known to be pathogenic. Some of the genetic determinants of virulence (or “cards of virulence”) that A. fumigatus possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. To examine whether secondary metabolism-associated cards of virulence vary between these species, we conducted extensive genomic and secondary metabolite profiling analyses of multiple A. fumigatus, one A. oerlinghausenensis, and multiple A. fischeri strains. We identified two cards of virulence (gliotoxin and fumitremorgin) shared by all three species and three cards of virulence (trypacidin, pseurotin, and fumagillin) that are variable. For example, we found that all species and strains examined biosynthesized gliotoxin, which is known to contribute to virulence, consistent with the conservation of the gliotoxin biosynthetic gene cluster (BGC) across genomes. For other secondary metabolites, such as fumitremorgin, a modulator of host biology, we found that all species produced the metabolite but that there was strain heterogeneity in its production within species. Finally, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis. A. fumigatus biosynthesized fumagillin and pseurotin, while A. oerlinghausenensis biosynthesized fumagillin and A. fischeri biosynthesized neither. These biochemical differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light onto the genetic and phenotypic changes associated with the evolution of fungal pathogenicity.

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Comparative Analyses of Cytochrome P450s and Those Associated with Secondary Metabolism in Bacillus Species

International Journal of Molecular Sciences ◽

10.3390/ijms19113623 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3623 ◽

Cited By ~ 9

Author(s):

Bongumusa Mthethwa ◽

Wanping Chen ◽

Mathula Ngwenya ◽

Abidemi Kappo ◽

Puleng Syed ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

In Silico Analysis ◽

Gene Clusters ◽

Bacillus Species ◽

Cytochrome P450 Monooxygenases ◽

Enzymatic Reactions ◽

P450 Monooxygenases ◽

Stereo Selectivity

Cytochrome P450 monooxygenases (CYPs/P450s) are among the most catalytically-diverse enzymes, capable of performing enzymatic reactions with chemo-, regio-, and stereo-selectivity. Our understanding of P450s’ role in secondary metabolite biosynthesis is becoming broader. Among bacteria, Bacillus species are known to produce secondary metabolites, and recent studies have revealed the presence of secondary metabolite biosynthetic gene clusters (BGCs) in these species. However, a comprehensive comparative analysis of P450s and P450s involved in the synthesis of secondary metabolites in Bacillus species has not been reported. This study intends to address these two research gaps. In silico analysis of P450s in 128 Bacillus species revealed the presence of 507 P450s that can be grouped into 13 P450 families and 28 subfamilies. No P450 family was found to be conserved in Bacillus species. Bacillus species were found to have lower numbers of P450s, P450 families and subfamilies, and a lower P450 diversity percentage compared to mycobacterial species. This study revealed that a large number of P450s (112 P450s) are part of different secondary metabolite BGCs, and also identified an association between a specific P450 family and secondary metabolite BGCs in Bacillus species. This study opened new vistas for further characterization of secondary metabolite BGCs, especially P450s in Bacillus species.

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Resistance to Aflatoxin Contamination in Corn as Influenced by Relative Humidity and Kernel Germination

Journal of Food Protection ◽

10.4315/0362-028x-59.3.276 ◽

1996 ◽

Vol 59 (3) ◽

pp. 276-281 ◽

Cited By ~ 35

Author(s):

B. Z. GUO ◽

J. S. RUSSIN ◽

R. L. BROWN ◽

T. E. CLEVELAND ◽

N. W. WIDSTROM

Keyword(s):

Relative Humidity ◽

Aspergillus Flavus ◽

Aflatoxin B1 ◽

Resistance Mechanisms ◽

Aflatoxin Contamination ◽

Aflatoxin Biosynthesis ◽

Aflatoxin Accumulation ◽

Moisture Conditions ◽

Very High ◽

Fungus Growth

Kernels of corn population GT-MAS:gk, resistant to aflatoxin B1 production by Aspergillus flavus, and susceptible Pioneer hybrid 3154 were tested for aflatoxin when incubated under different relative humidities (RH). High aflatoxin levels were not detected in either genotype at RH < 91%. Resistance in GT-MAS:gk was consistent across all RH levels (91 to 100%) at which significant aflatoxin accumulation was detected. Aflatoxin levels in GT-MAS:gk averaged about 98% less than those in susceptible Pioneer 3154, which suggests that storage of this or other genotypes with similar resistance mechanisms may be possible under moisture conditions less exacting than are required with susceptible hybrids. Results for fungus growth and sporulation ratings on kernel surfaces were similar to those for aflatoxin levels. When kernels of both genotypes were preincubated 3 days at 100% RH prior to inoculation with A. flavus, germination percentages increased to very high levels compared to those of kernels that were not preincubated. In preincubated kernels aflatoxin levels remained consistently low in GT-MAS:gk but decreased markedly (61%) in Pioneer 3154. When eight susceptible hybrids were evaluated for aflatoxin accumulation in preincubated kernels, seven of these supported significantly lower toxin levels than kernels not subjected to preincubation. Average reduction across hybrids was 83%, and reductions within hybrids ranged from 68 to 96%. Preincubated kernels of one susceptible hybrid (Deltapine G-4666) supported aflatoxin levels comparable to those in resistant GT-MAS:gk. Data suggest that an inhibitor of aflatoxin biosynthesis may be induced during kernel germination. Possible mechanisms for embryo effects on resistance to aflatoxin accumulation are discussed.

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Chromatin-dependent regulation of secondary metabolite biosynthesis in fungi: is the picture complete?

FEMS Microbiology Reviews ◽

10.1093/femsre/fuz018 ◽

2019 ◽

Cited By ~ 4

Author(s):

Jérôme Collemare ◽

Michael F Seidl

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Biological Activities ◽

Gene Clusters ◽

Research Field ◽

Chromatin Modifications ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Post Translational Modifications ◽

Fungal Secondary Metabolites

ABSTRACTFungal secondary metabolites are small molecules that exhibit diverse biological activities exploited in medicine, industry and agriculture. Their biosynthesis is governed by co-expressed genes that often co-localize in gene clusters. Most of these secondary metabolite gene clusters are inactive under laboratory conditions, which is due to a tight transcriptional regulation. Modifications of chromatin, the complex of DNA and histone proteins influencing DNA accessibility, play an important role in this regulation. However, tinkering with well-characterised chemical and genetic modifications that affect chromatin alters the expression of only few biosynthetic gene clusters, and thus the regulation of the vast majority of biosynthetic pathways remains enigmatic. In the past, attempts to activate silent gene clusters in fungi mainly focused on histone acetylation and methylation, while in other eukaryotes many other post-translational modifications are involved in transcription regulation. Thus, how chromatin regulates the expression of gene clusters remains a largely unexplored research field. In this review, we argue that focusing on only few well-characterised chromatin modifications is significantly hampering our understanding of the chromatin-based regulation of biosynthetic gene clusters. Research on underexplored chromatin modifications and on the interplay between different modifications is timely to fully explore the largely untapped reservoir of fungal secondary metabolites.

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Comprehensive Analyses of Cytochrome P450 Monooxygenases and Secondary Metabolite Biosynthetic Gene Clusters in Cyanobacteria

International Journal of Molecular Sciences ◽

10.3390/ijms21020656 ◽

2020 ◽

Vol 21 (2) ◽

pp. 656 ◽

Cited By ~ 4

Author(s):

Makhosazana Jabulile Khumalo ◽

Nomfundo Nzuza ◽

Tiara Padayachee ◽

Wanping Chen ◽

Jae-Hyuk Yu ◽

...

Keyword(s):

Cytochrome P450 ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Gene Clusters ◽

Cytochrome P450 Monooxygenases ◽

Biosynthetic Gene ◽

Mycobacterial Species ◽

Biosynthetic Gene Clusters ◽

P450 Monooxygenases ◽

Cyanobacterial Species

The prokaryotic phylum Cyanobacteria are some of the oldest known photosynthetic organisms responsible for the oxygenation of the earth. Cyanobacterial species have been recognised as a prosperous source of bioactive secondary metabolites with antibacterial, antiviral, antifungal and/or anticancer activities. Cytochrome P450 monooxygenases (CYPs/P450s) contribute to the production and diversity of various secondary metabolites. To better understand the metabolic potential of cyanobacterial species, we have carried out comprehensive analyses of P450s, predicted secondary metabolite biosynthetic gene clusters (BGCs), and P450s located in secondary metabolite BGCs. Analysis of the genomes of 114 cyanobacterial species identified 341 P450s in 88 species, belonging to 36 families and 79 subfamilies. In total, 770 secondary metabolite BGCs were found in 103 cyanobacterial species. Only 8% of P450s were found to be part of BGCs. Comparative analyses with other bacteria Bacillus, Streptomyces and mycobacterial species have revealed a lower number of P450s and BGCs and a percentage of P450s forming part of BGCs in cyanobacterial species. A mathematical formula presented in this study revealed that cyanobacterial species have the highest gene-cluster diversity percentage compared to Bacillus and mycobacterial species, indicating that these diverse gene clusters are destined to produce different types of secondary metabolites. The study provides fundamental knowledge of P450s and those associated with secondary metabolism in cyanobacterial species, which may illuminate their value for the pharmaceutical and cosmetics industries.

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