Elucidating the Diversity and Potential Function of Nonribosomal Peptide and Polyketide Biosynthetic Gene Clusters in the Root Microbiome

ABSTRACTPolyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores, and others. Despite their involvement in bacterium-bacterium and bacterium-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics, and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low-similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria, while the remaining were not associated with known bacteria. One Streptomyces-derived BGC was predicted to encode a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic data sets revealed higher abundances of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs.IMPORTANCE We identified distinct secondary-metabolite-encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary-metabolite-encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in interbacterial and bacterium-plant interactions.

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Elucidating the diversity and potential function of nonribosomal peptide and polyketide biosynthetic gene clusters in the root microbiome

10.1101/2020.06.07.138487 ◽

2020 ◽

Author(s):

Barak Dror ◽

Zongqiang Wang ◽

Sean F. Brady ◽

Edouard Jurkevitch ◽

Eddie Cytryn

Keyword(s):

Secondary Metabolite ◽

Gene Clusters ◽

Bulk Soil ◽

Large Set ◽

Biosynthetic Gene ◽

Plant Interactions ◽

Biosynthetic Gene Clusters ◽

Shotgun Metagenomics ◽

Nonribosomal Peptide ◽

Encoding Genes

AbstractPolyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores and others. Despite their involvement in bacteria-bacteria and bacteria-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria, while the remaining were not associated with known bacteria. One Streptomyces-derived BGC was predicted to encode for a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic datasets revealed a higher abundance of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs.ImportanceWe identified distinct secondary metabolite (polyketide and nonribosomal peptide) encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems, yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding for antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode for novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary metabolite encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in inter-bacterial and bacterial-plant interactions.

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Increased abundance of secreted hydrolytic enzymes and secondary metabolite gene clusters define the genomes of latent plant pathogens in the Botryosphaeriaceae

BMC Genomics ◽

10.1186/s12864-021-07902-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Jan H. Nagel ◽

Michael J. Wingfield ◽

Bernard Slippers

Keyword(s):

Secondary Metabolite ◽

Plant Pathogens ◽

Plant Cell Wall ◽

Hydrolytic Enzymes ◽

Gene Clusters ◽

Comparative Genomic ◽

Biosynthetic Gene ◽

Plant Interactions ◽

Biosynthetic Gene Clusters ◽

Genome Analyses

Abstract Background The Botryosphaeriaceae are important plant pathogens, but also have the ability to establish asymptomatic infections that persist for extended periods in a latent state. In this study, we used comparative genome analyses to shed light on the genetic basis of the interactions of these fungi with their plant hosts. For this purpose, we characterised secreted hydrolytic enzymes, secondary metabolite biosynthetic gene clusters and general trends in genomic architecture using all available Botryosphaeriaceae genomes, and selected Dothideomycetes genomes. Results The Botryosphaeriaceae genomes were rich in carbohydrate-active enzymes (CAZymes), proteases, lipases and secondary metabolic biosynthetic gene clusters (BGCs) compared to other Dothideomycete genomes. The genomes of Botryosphaeria, Macrophomina, Lasiodiplodia and Neofusicoccum, in particular, had gene expansions of the major constituents of the secretome, notably CAZymes involved in plant cell wall degradation. The Botryosphaeriaceae genomes were shown to have moderate to high GC contents and most had low levels of repetitive DNA. The genomes were not compartmentalized based on gene and repeat densities, but genes of secreted enzymes were slightly more abundant in gene-sparse regions. Conclusion The abundance of secreted hydrolytic enzymes and secondary metabolite BGCs in the genomes of Botryosphaeria, Macrophomina, Lasiodiplodia, and Neofusicoccum were similar to those in necrotrophic plant pathogens and some endophytes of woody plants. The results provide a foundation for comparative genomic analyses and hypotheses to explore the mechanisms underlying Botryosphaeriaceae host-plant interactions.

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Pangenome Analysis of Enterobacteria Reveals Richness of Secondary Metabolite Gene Clusters and their Associated Gene Sets

10.1101/781328 ◽

2019 ◽

Cited By ~ 1

Author(s):

Omkar S. Mohite ◽

Colton J. Lloyd ◽

Jonathan M. Monk ◽

Tilmann Weber ◽

Bernhard O. Palsson

Keyword(s):

Secondary Metabolite ◽

Soil Bacteria ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Type Vi Secretion System ◽

Biosynthetic Gene Clusters ◽

Nonribosomal Peptide ◽

Peptide Synthetases ◽

Type Vi Secretion ◽

Gene Sets

AbstractThe growing number of sequenced genomes enables the study of secondary metabolite biosynthetic gene clusters (BGC) in phyla beyond well-studied soil bacteria. We mined 2627 enterobacterial genomes to detect 8604 BGCs, including nonribosomal peptide synthetases, siderophores, polyketide-nonribosomal peptide hybrids, and 60 other BGC types, with an average of around 3.3 BGCs per genome. These BGCs represented 212 distinct BGC families, of which only 20 have associated products in the MIBiG standard database with functions such as siderophores, antibiotics, and genotoxins. Pangenome analysis identified genes associated with a specific BGC encoding for colon cancer-related colibactin. In one example, we associated genes involved in the type VI secretion system with the presence of a colibactin BGC inEscherichia. This richness of BGCs in enterobacteria opens up the possibility to discover novel secondary metabolites, their physiological roles and provides a guide to identify and understand PKS associated gene sets.

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Abundant secreted hydrolytic enzymes and secondary metabolite gene clusters in genomes of the Botryosphaeriaceae reflect their role as important plant pathogens

10.1101/2021.01.22.427741 ◽

2021 ◽

Author(s):

JH Nagel ◽

MJ Wingfield ◽

B Slippers

Keyword(s):

Secondary Metabolite ◽

Plant Pathogens ◽

Plant Cell Wall ◽

Hydrolytic Enzymes ◽

Gene Clusters ◽

Comparative Genomic ◽

Biosynthetic Gene ◽

Plant Interactions ◽

Biosynthetic Gene Clusters ◽

Plant Cell Wall Degradation

AbstractThe Botryosphaeriaceae are important plant pathogens, but unique in their ability to establish asymptomatic infections that persist for extended periods in a latent state. In this study, we used comparative analyses to consider elements that might shed light on the genetic basis of the interactions of these fungi with their plant hosts. For this purpose, we characterised secreted hydrolytic enzymes, secondary metabolite biosynthetic gene clusters and considered general trends in genomic architecture using all available Botryosphaeriaceae genomes, and selected Dothideomycetes genomes. The Botryosphaeriaceae genomes were rich in carbohydrate-active enzymes (CAZymes), proteases, lipases and secondary metabolic biosynthetic gene clusters (BGCs) compared to other Dothideomycete genomes. The genomes of Botryosphaeria, Macrophomina, Lasiodiplodia and Neofusicoccum, in particular, had gene expansions of the major constituents of the secretome, notably CAZymes involved in plant cell wall degradation. The Botryosphaeriaceae genomes were shown to have moderate to high GC contents and most had low levels of repetitive DNA. The genomes were not compartmentalized based on gene and repeat densities, but genes of secreted enzymes were slightly more abundant in gene-sparse regions. The abundance of secreted hydrolytic enzymes and secondary metabolite BGCs in the genomes of Botryosphaeria, Macrophomina, Lasiodiplodia, and Neofusicoccum were similar to those in necrotrophic plant pathogens, but also endophytes of woody plants. The results provide a foundation for future comparative genomic analyses and hypothesis to explore the mechanisms underlying Botryosphaeriaceae host-plant interactions.

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Identification of a New Antimicrobial, Desertomycin H, Utilizing a Modified Crowded Plate Technique

Marine Drugs ◽

10.3390/md19080424 ◽

2021 ◽

Vol 19 (8) ◽

pp. 424

Author(s):

Osama G. Mohamed ◽

Sadaf Dorandish ◽

Rebecca Lindow ◽

Megan Steltz ◽

Ifrah Shoukat ◽

...

Keyword(s):

Antibiotic Production ◽

Gene Clusters ◽

Multidrug Resistant ◽

Microbial Interactions ◽

Mass Spectrometry Data ◽

Metagenomic Data ◽

Resistant Bacteria ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Plate Technique

The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny Earth®, we isolated over 1400 antibiotic-producing microbes, including 62, showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.

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Gut microbiome-based secondary metabolite biosynthetic gene clusters detection in Parkinson’s disease

Neuroscience Letters ◽

10.1016/j.neulet.2018.12.021 ◽

2019 ◽

Vol 696 ◽

pp. 93-98 ◽

Cited By ~ 8

Author(s):

Shengqin Wang ◽

Nan Li ◽

Huixi Zou ◽

Mingjiang Wu

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Secondary Metabolite ◽

Gut Microbiome ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters

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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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A Comparative Analysis of Biosynthetic Gene Clusters in Lean and Obese Humans

BioMed Research International ◽

10.1155/2019/6361320 ◽

2019 ◽

Vol 2019 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Shengqin Wang ◽

Na Li ◽

Nan Li ◽

Huixi Zou ◽

Mingjiang Wu

Keyword(s):

Gut Microbiome ◽

Gene Clusters ◽

Human Adipose Tissue ◽

Taxonomic Diversity ◽

Metagenomic Data ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Intestinal Microbes ◽

Microbe Interactions ◽

Treatment Of Obesity

Obesity is intrinsically linked with the gut microbiome, and studies have identified several obesity-associated microbes. The microbe-microbe interactions can alter the composition of the microbial community and influence host health by producing secondary metabolites (SMs). However, the contribution of these SMs in the prevention and treatment of obesity has been largely ignored. We identified several SM-encoding biosynthetic gene clusters (BGCs) from the metagenomic data of lean and obese individuals and found significant association between some BGCs, including those that produce hitherto unknown SM, and obesity. In addition, the mean abundance of BGCs was positively correlated with obesity, consistent with the lower taxonomic diversity in the gut microbiota of obese individuals. By comparing the BGCs of known SM between obese and nonobese samples, we found that menaquinone produced by Enterobacter cloacae showed the highest correlation with BMI, in agreement with a recent study on human adipose tissue composition. Furthermore, an obesity-related nonribosomal peptide synthetase (NRPS) was negatively associated with Bacteroidetes, indicating that the SMs produced by intestinal microbes in obese individuals can change the microbiome structure. This is the first systemic study of the association between gut microbiome BGCs and obesity and provides new insights into the causes of obesity.

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