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

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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Secondary metabolism in the gill microbiota of shipworms (Teredinidae) as revealed by comparison of metagenomes and nearly complete symbiont genomes

10.1101/826933 ◽

2019 ◽

Author(s):

Marvin A. Altamia ◽

Zhenjian Lin ◽

Amaro E. Trindade-Silva ◽

Iris Diana Uy ◽

J. Reuben Shipway ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Gene Clusters ◽

The United States ◽

Biosynthetic Gene Cluster ◽

The Philippines ◽

Symbiotic Bacteria ◽

Biosynthetic Pathways ◽

Biosynthetic Gene ◽

Bioactive Secondary Metabolites

AbstractShipworms play critical roles in recycling wood in the sea. Symbiotic bacteria supply enzymes that the organisms need for nutrition and wood degradation. Some of these bacteria have been grown in pure culture and have the capacity to make many secondary metabolites. However, little is known about whether such secondary metabolite pathways are represented in the symbiont communities within their hosts. In addition, little has been reported about the patterns of host-symbiont co-occurrence. Here, we collected shipworms from the United States, the Philippines, and Brazil, and cultivated symbiotic bacteria from their gills. We analyzed sequences from 22 shipworm gill metagenomes from seven shipworm species and from 23 cultivated symbiont isolates. Using (meta)genome sequencing, we demonstrate that the cultivated isolates represent all the major bacterial symbiont species and strains in shipworm gills. We show that the bacterial symbionts are distributed among shipworm hosts in consistent, predictable patterns. The symbiotic bacteria encode many biosynthetic gene cluster families (GCFs) for bioactive secondary metabolites, only <5% of which match previously described biosynthetic pathways. Because we were able to cultivate the symbionts, and sequence their genomes, we can definitively enumerate the biosynthetic pathways in these symbiont communities, showing that ∼150 out of ∼200 total biosynthetic gene clusters (BGCs) present in the animal gill metagenomes are represented in our culture collection. Shipworm symbionts occur in suites that differ predictably across a wide taxonomic and geographic range of host species, and collectively constitute an immense resource for the discovery of new biosynthetic pathways to bioactive secondary metabolites.ImportanceWe define a system in which the major symbionts that are important to host biology and to the production of secondary metabolites can be cultivated. We show that symbiotic bacteria that are critical to host nutrition and lifestyle also have an immense capacity to produce a multitude of diverse and likely novel bioactive secondary metabolites that could lead to the discovery of drugs, and that these pathways are found within shipworm gills. We propose that, by shaping associated microbial communities within the host, the compounds support the ability of shipworms to degrade wood in marine environments. Because these symbionts can be cultivated and genetically manipulated, they provide a powerful model for understanding how secondary metabolism impacts microbial symbiosis.

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Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa082 ◽

2021 ◽

Vol 85 (3) ◽

pp. 714-721

Author(s):

Risa Takao ◽

Katsuyuki Sakai ◽

Hiroyuki Koshino ◽

Hiroyuki Osada ◽

Shunji Takahashi

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Secondary Metabolite ◽

Response Regulator ◽

Bac Library ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Gene Organization ◽

Biosynthetic Gene ◽

Streptomyces Sp

ABSTRACT Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

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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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IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites

mBio ◽

10.1128/mbio.00932-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 66

Author(s):

Michalis Hadjithomas ◽

I-Min Amy Chen ◽

Ken Chu ◽

Anna Ratner ◽

Krishna Palaniappan ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Genomic Data ◽

Gene Clusters ◽

Metagenomic Data ◽

Integrated Analysis ◽

Analysis Tool ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Analysis Tools

ABSTRACTIn the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time inAlphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules.IMPORTANCEIMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world.

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Screening of tenuazonic acid production-inducing compounds and identification of NPD938 as a regulator of fungal secondary metabolism

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab143 ◽

2021 ◽

Author(s):

Takayuki Motoyama ◽

Tomoaki Ishii ◽

Takashi Kamakura ◽

Hiroyuki Osada

Keyword(s):

Secondary Metabolism ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Tenuazonic Acid ◽

Cellular Functions ◽

Biosynthetic Gene Clusters ◽

Chemical Library ◽

Tea Production ◽

Fungal Secondary Metabolism

Abstract The control of secondary metabolism in fungi is essential for the regulation of various cellular functions. In this study, we searched the RIKEN Natural Products Depository (NPDepo) chemical library for inducers of tenuazonic acid (TeA) production in the rice blast fungus Pyricularia oryzae and identified NPD938. NPD938 transcriptionally induced TeA production. We explored the mode of action of NPD938 and observed that this compound enhanced TeA production via LAE1, a global regulator of fungal secondary metabolism. NPD938 could also induce production of terpendoles and pyridoxatins in Tolypocladium album RK99-F33. Terpendole production was induced transcriptionally. We identified the pyridoxatin biosynthetic gene cluster among transcriptionally induced secondary metabolite biosynthetic gene clusters. Therefore, NPD938 is useful for the control of fungal secondary metabolism.

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IMG-ABC v.5.0: an update to the IMG/Atlas of Biosynthetic Gene Clusters Knowledgebase

Nucleic Acids Research ◽

10.1093/nar/gkz932 ◽

2019 ◽

Cited By ~ 5

Author(s):

Krishnaveni Palaniappan ◽

I-Min A Chen ◽

Ken Chu ◽

Anna Ratner ◽

Rekha Seshadri ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Microbial Genomes ◽

Initial Release ◽

Systematic Identification ◽

Biomedical Potential ◽

Biosynthetic Machinery

Abstract Microbial secondary metabolism is a reservoir of bioactive compounds of immense biotechnological and biomedical potential. The biosynthetic machinery responsible for the production of these secondary metabolites (SMs) (also called natural products) is often encoded by collocated groups of genes called biosynthetic gene clusters (BGCs). High-throughput genome sequencing of both isolates and metagenomic samples combined with the development of specialized computational workflows is enabling systematic identification of BGCs and the discovery of novel SMs. In order to advance exploration of microbial secondary metabolism and its diversity, we developed the largest publicly available database of predicted BGCs combined with experimentally verified BGCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc-public). Here we describe the first major content update of the IMG-ABC knowledgebase, since its initial release in 2015, refreshing the BGC prediction pipeline with the latest version of antiSMASH (v5) as well as presenting the data in the context of underlying environmental metadata sourced from GOLD (https://gold.jgi.doe.gov/). This update has greatly improved the quality and expanded the types of predicted BGCs compared to the previous version.

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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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A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽

10.1128/mbio.00191-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 28

Author(s):

Ye Xu ◽

Andrew Willems ◽

Catherine Au-yeung ◽

Kapil Tahlan ◽

Justin R. Nodwell

Keyword(s):

Secondary Metabolites ◽

Pathogenic Bacteria ◽

Streptomyces Coelicolor ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Resistance Mechanisms ◽

Biosynthetic Gene ◽

Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

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Antiplasmodial activity, biosynthetic gene clusters diversity, and secondary metabolite constituent of selected Indonesian Streptomyces

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220657 ◽

2021 ◽

Vol 22 (6) ◽

Author(s):

Ema Damayanti ◽

Puspita Lisdiyanti ◽

Andini Sundowo ◽

Shanti Ratnakomala ◽

Achmad Dinoto ◽

...

Keyword(s):

Secondary Metabolites ◽

Ethyl Acetate ◽

Secondary Metabolite ◽

Molecular Identification ◽

Gene Clusters ◽

Antiplasmodial Activity ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Genus Streptomyces ◽

Rdna Sequencing

Abstract. Damayanti E, Lisdiyanti P, Sundowo A, Ratnakomala S, Dinoto A, Widada J, Mustofa. 2021. Antiplasmodial activity, biosynthetic gene clusters diversity, and secondary metabolite constituent of selected Indonesian Streptomyces. Biodiversitas 22: 3478-3487. Actinobacteria of the genus Streptomyces are known as the primary candidate antibiotics, but still limited for antiplasmodial drugs. This study aimed to investigate the antiplasmodial activity, the biosynthetic gene clusters (BGCs) diversity, and the secondary metabolites constituent of selected Indonesian Streptomyces. The bacteria were isolated from various habitats: karst soil (GMR22), mangrove sediments (BSE7F and SHP 22-7), and marine sediment (GMY01). Molecular identification by 16S rDNA sequencing were performed for confirmation and morphological characterization by scanning electron microscope (SEM) were performed for identification. In vitro antiplasmodial assay was performed on human Plasmodium falciparum FCR-3. The BGCs which encode secondary metabolites were analysed using antiSMASH version 5 based on available whole genome sequence (WGS) data. The secondary metabolites were obtained from liquid fermentation followed by extraction using methanol and ethyl acetate. The secondary metabolites constituent was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). The molecular identification showed that GMR22 had similarity to Streptomyces lactacystinicus (98.02%), while BSE7F was similar to Streptomyces althioticus (97.06%), SHP 22-7 was similar to Streptomyces rochei (94.84%), and GMY01 to Streptomyces odonnellii (98.57%). All of isolates had morphological characteristics as the genus Streptomyces bacteria. The highest Plasmodium inhibition (81.84 ± 3.5%) was demonstrated by ethyl acetate extract of marine-derived Streptomyces sp. GMY01 (12.5 µg/mL). Non-ribosomal polyketide synthetase (NRPS), polyketide synthase (PKS) and hybrid of NRPS-PKS were the major BGCs in all Streptomyces. Majority of the Streptomyces produced compounds containing CHON elements with molecular weight approximately 100-400 Da. The active extract of GMY01 bacterium had five major detected compounds, namely kuraramine (C12H18N2O2), laminine (C9H20N2O2) 2-ethylacetanilide (C10H13NO), propoxur (C11H15NO3), and 3-methyl-1,2-diphenylbutan-1-one (C17H18O). This Indonesian marine bacterium is potential for bioassay guided isolation of antiplasmodial compounds in the future studies.

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