Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics

AbstractMicrobial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 695 novel, full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night in cyanobacteria but not in other phyla, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

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Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The ISME Journal ◽

10.1038/s41396-021-01052-3 ◽

2021 ◽

Author(s):

Valentin Waschulin ◽

Chiara Borsetto ◽

Robert James ◽

Kevin K. Newsham ◽

Stefano Donadio ◽

...

Keyword(s):

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Full Length ◽

Metagenomic Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Rich Diversity ◽

Long Read ◽

The Rich

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

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Metagenomic exploration of the marine sponge mycale hentscheli uncovers multiple polyketide-producing bacterial symbionts

10.26686/wgtn.12444086 ◽

2020 ◽

Author(s):

Mathew Storey ◽

SK Andreassend ◽

Joe Bracegirdle ◽

Alistair Brown ◽

Robert Keyzers ◽

...

Keyword(s):

Marine Sponge ◽

Bacterial Species ◽

Gene Clusters ◽

Taxonomic Diversity ◽

Marine Sponges ◽

Metagenomic Sequencing ◽

Defensive Symbiosis ◽

Long Read ◽

Comprehensive Picture ◽

Mycale Hentscheli

© 2020 Storey et al. Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metag-enomic investigation of Mycale hentscheli, a chemically gifted marine sponge that pos-sesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short-and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemo-types. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holo-biont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These re-sults suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small mol-ecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing mi-crobe. We also find that the microbiome of M. hentscheli is stably maintained among in-dividuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

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Metagenomic Exploration of the Marine Sponge Mycale hentscheli Uncovers Multiple Polyketide-Producing Bacterial Symbionts

mBio ◽

10.1128/mbio.02997-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 5

Author(s):

Mathew A. Storey ◽

Sarah K. Andreassend ◽

Joe Bracegirdle ◽

Alistair Brown ◽

Robert A. Keyzers ◽

...

Keyword(s):

Marine Sponge ◽

Bacterial Species ◽

Gene Clusters ◽

Taxonomic Diversity ◽

Marine Sponges ◽

Metagenomic Sequencing ◽

Content Type ◽

Defensive Symbiosis ◽

Long Read ◽

Mycale Hentscheli

ABSTRACT Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metagenomic investigation of Mycale hentscheli, a chemically gifted marine sponge that possesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short- and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemotypes. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holobiont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These results suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small molecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing microbe. We also find that the microbiome of M. hentscheli is stably maintained among individuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

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Metagenomic exploration of the marine sponge mycale hentscheli uncovers multiple polyketide-producing bacterial symbionts

10.26686/wgtn.12444086.v1 ◽

2020 ◽

Author(s):

Mathew Storey ◽

SK Andreassend ◽

Joe Bracegirdle ◽

Alistair Brown ◽

Robert Keyzers ◽

...

Keyword(s):

Marine Sponge ◽

Bacterial Species ◽

Gene Clusters ◽

Taxonomic Diversity ◽

Marine Sponges ◽

Metagenomic Sequencing ◽

Defensive Symbiosis ◽

Long Read ◽

Comprehensive Picture ◽

Mycale Hentscheli

© 2020 Storey et al. Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metag-enomic investigation of Mycale hentscheli, a chemically gifted marine sponge that pos-sesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short-and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemo-types. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holo-biont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These re-sults suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small mol-ecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing mi-crobe. We also find that the microbiome of M. hentscheli is stably maintained among in-dividuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

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Automated strain separation in low-complexity metagenomes using long reads

10.1101/2021.02.24.429166 ◽

2021 ◽

Author(s):

R. Vicedomini ◽

C. Quince ◽

A. E. Darling ◽

R. Chikhi

Keyword(s):

Large Scale ◽

Functional Characterization ◽

Low Complexity ◽

Complete Separation ◽

Metagenomic Sequencing ◽

Assembly Method ◽

Starting Point ◽

Long Read ◽

Different Strains ◽

Strain Separation

AbstractHigh-throughput short-read metagenomics has enabled large-scale species-level analysis and functional characterization of microbial communities. Microbiomes often contain multiple strains of the same species, and different strains have been shown to have important differences in their functional roles. Despite this, strain-level resolution from metagenomic sequencing remains challenging. Recent advances on long-read based methods enabled accurate assembly of bacterial genomes from complex microbiomes and an as-yet-unrealized opportunity to resolve strains. Here we present Strainberry, a metagenome assembly method that performs strain separation in single-sample low-complexity metagenomes and that relies uniquely on long-read data. We benchmarked Strainberry on mock communities and showed it consistently produces strain-resolved assemblies with near-complete reference coverage and 99.9% base accuracy. We also applied Strainberry on real datasets for which it improved assemblies generating 27-89% additional genomic material than conventional metagenome assemblies on individual strain genomes. Our results hence demonstrate that strain separation is possible in low-complexity microbiomes using a single regular long read dataset. We show that Strainberry is also able to refine microbial diversity in a complex microbiome, with complete separation of strain genomes. We anticipate this work to be a starting point for further methodological improvements aiming to provide better strain-resolved metagenome assemblies in environments of higher complexities.

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The HAP Complex Governs Fumonisin Biosynthesis and Maize Kernel Pathogenesis in Fusarium verticillioides

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-596 ◽

2016 ◽

Vol 79 (9) ◽

pp. 1498-1507 ◽

Cited By ~ 2

Author(s):

JOHN B. RIDENOUR ◽

JONATHON E. SMITH ◽

BURTON H. BLUHM

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Transcriptional Profiling ◽

Fusarium Verticillioides ◽

Consensus Sequence ◽

Functional Characterization ◽

Gene Clusters ◽

Negative Regulator ◽

Maize Production ◽

Hap Complex

ABSTRACT Contamination of maize (Zea mays) with fumonisins produced by the fungus Fusarium verticillioides is a global concern for food safety. Fumonisins are a group of polyketide-derived secondary metabolites linked to esophageal cancer and neural tube birth defects in humans and numerous toxicoses in livestock. Despite the importance of fumonisins in global maize production, the regulation of fumonisin biosynthesis during kernel pathogenesis is poorly understood. The HAP complex is a conserved, heterotrimeric transcriptional regulator that binds the consensus sequence CCAAT to modulate gene expression. Recently, functional characterization of the Hap3 subunit linked the HAP complex to the regulation of secondary metabolism and stalk rot pathogenesis in F. verticillioides. Here, we determine the involvement of HAP3 in fumonisin biosynthesis and kernel pathogenesis. Deletion of HAP3 suppressed fumonisin biosynthesis on both nonviable and live maize kernels and impaired pathogenesis in living kernels. Transcriptional profiling via RNA sequencing indicated that the HAP complex regulates at least 1,223 genes in F. verticillioides, representing nearly 10% of all predicted genes. Disruption of the HAP complex caused the misregulation of biosynthetic gene clusters underlying the production of secondary metabolites, including fusarins. Taken together, these results reveal that the HAP complex is a central regulator of fumonisin biosynthesis and kernel pathogenesis and works as both a positive and negative regulator of secondary metabolism in F. verticillioides.

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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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Global biogeographic sampling of bacterial secondary metabolism

eLife ◽

10.7554/elife.05048 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 74

Author(s):

Zachary Charlop-Powers ◽

Jeremy G Owen ◽

Boojala Vijay B Reddy ◽

Melinda A Ternei ◽

Denise O Guimarães ◽

...

Keyword(s):

Secondary Metabolites ◽

Genome Sequencing ◽

Geographic Distance ◽

Gene Clusters ◽

Natural World ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Road Map ◽

Two Factors ◽

Natural Products Discovery

Recent bacterial (meta)genome sequencing efforts suggest the existence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters in the environment. Here we use the pyro-sequencing of PCR amplicons derived from both nonribosomal peptide adenylation domains and polyketide ketosynthase domains to compare biosynthetic diversity in soil microbiomes from around the globe. We see large differences in domain populations from all except the most proximal and biome-similar samples, suggesting that most microbiomes will encode largely distinct collections of bacterial secondary metabolites. Our data indicate a correlation between two factors, geographic distance and biome-type, and the biosynthetic diversity found in soil environments. By assigning reads to known gene clusters we identify hotspots of biomedically relevant biosynthetic diversity. These observations not only provide new insights into the natural world, they also provide a road map for guiding future natural products discovery efforts.

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Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽

10.1186/s40168-020-00974-y ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Yusuke Okazaki ◽

Shohei Fujinaga ◽

Michaela M. Salcher ◽

Cristiana Callieri ◽

Atsushi Tanaka ◽

...

Keyword(s):

16S Rrna ◽

Regional Scale ◽

Scale Up ◽

Amplicon Sequencing ◽

Freshwater Ecosystems ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

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