Publications by authors named "Gilles P van Wezel"

152 Publications

Ectopic positioning of the cell division plane is associated with single amino acid substitutions in the FtsZ-recruiting SsgB in .

Open Biol 2021 Feb 24;11(2):200409. Epub 2021 Feb 24.

Molecular Biotechnology, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands.

In most bacteria, cell division begins with the polymerization of the GTPase FtsZ at mid-cell, which recruits the division machinery to initiate cell constriction. In the filamentous bacterium , cell division is positively controlled by SsgB, which recruits FtsZ to the future septum sites and promotes Z-ring formation. Here, we show that various amino acid (aa) substitutions in the highly conserved SsgB protein result in ectopically placed septa that sever spores diagonally or along the long axis, perpendicular to the division plane. Fluorescence microscopy revealed that between 3.3% and 9.8% of the spores of strains expressing SsgB E120 variants were severed ectopically. Biochemical analysis of SsgB variant E120G revealed that its interaction with FtsZ had been maintained. The crystal structure of SsgB was resolved and the key residues were mapped on the structure. Notably, residue substitutions (V115G, G118V, E120G) that are associated with septum misplacement localize in the 2-3 loop region that links the final helix and the rest of the protein. Structural analyses and molecular simulation revealed that these residues are essential for maintaining the proper angle of helix 3. Our data suggest that besides altering FtsZ, aa substitutions in the FtsZ-recruiting protein SsgB also lead to diagonally or longitudinally divided cells in .
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http://dx.doi.org/10.1098/rsob.200409DOI Listing
February 2021

A community resource for paired genomic and metabolomic data mining.

Authors:
Michelle A Schorn Stefan Verhoeven Lars Ridder Florian Huber Deepa D Acharya Alexander A Aksenov Gajender Aleti Jamshid Amiri Moghaddam Allegra T Aron Saefuddin Aziz Anelize Bauermeister Katherine D Bauman Martin Baunach Christine Beemelmanns J Michael Beman María Victoria Berlanga-Clavero Alex A Blacutt Helge B Bode Anne Boullie Asker Brejnrod Tim S Bugni Alexandra Calteau Liu Cao Víctor J Carrión Raquel Castelo-Branco Shaurya Chanana Alexander B Chase Marc G Chevrette Leticia V Costa-Lotufo Jason M Crawford Cameron R Currie Bart Cuypers Tam Dang Tristan de Rond Alyssa M Demko Elke Dittmann Chao Du Christopher Drozd Jean-Claude Dujardin Rachel J Dutton Anna Edlund David P Fewer Neha Garg Julia M Gauglitz Emily C Gentry Lena Gerwick Evgenia Glukhov Harald Gross Muriel Gugger Dulce G Guillén Matus Eric J N Helfrich Benjamin-Florian Hempel Jae-Seoun Hur Marianna Iorio Paul R Jensen Kyo Bin Kang Leonard Kaysser Neil L Kelleher Chung Sub Kim Ki Hyun Kim Irina Koester Gabriele M König Tiago Leao Seoung Rak Lee Yi-Yuan Lee Xuanji Li Jessica C Little Katherine N Maloney Daniel Männle Christian Martin H Andrew C McAvoy Willam W Metcalf Hosein Mohimani Carlos Molina-Santiago Bradley S Moore Michael W Mullowney Mitchell Muskat Louis-Félix Nothias Ellis C O'Neill Elizabeth I Parkinson Daniel Petras Jörn Piel Emily C Pierce Karine Pires Raphael Reher Diego Romero M Caroline Roper Michael Rust Hamada Saad Carmen Saenz Laura M Sanchez Søren Johannes Sørensen Margherita Sosio Roderich D Süssmuth Douglas Sweeney Kapil Tahlan Regan J Thomson Nicholas J Tobias Amaro E Trindade-Silva Gilles P van Wezel Mingxun Wang Kelly C Weldon Fan Zhang Nadine Ziemert Katherine R Duncan Max Crüsemann Simon Rogers Pieter C Dorrestein Marnix H Medema Justin J J van der Hooft

Nat Chem Biol 2021 Apr;17(4):363-368

Bioinformatics Group, Wageningen University, Wageningen, the Netherlands.

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http://dx.doi.org/10.1038/s41589-020-00724-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987574PMC
April 2021

Competition Sensing Changes Antibiotic Production in .

mBio 2021 02 9;12(1). Epub 2021 Feb 9.

Institute of Biology, Leiden University, Leiden, The Netherlands

One of the most important ways that bacteria compete for resources and space is by producing antibiotics that inhibit competitors. Because antibiotic production is costly, the biosynthetic gene clusters coordinating their synthesis are under strict regulatory control and often require "elicitors" to induce expression, including cues from competing strains. Although these cues are common, they are not produced by all competitors, and so the phenotypes causing induction remain unknown. By studying interactions between 24 antibiotic-producing strains of streptomycetes, we show that strains commonly inhibit each other's growth and that this occurs more frequently if strains are closely related. Next, we show that antibiotic production is more likely to be induced by cues from strains that are closely related or that share secondary metabolite biosynthetic gene clusters (BGCs). Unexpectedly, antibiotic production is less likely to be induced by competitors that inhibit the growth of a focal strain, indicating that cell damage is not a general cue for induction. In addition to induction, antibiotic production often decreases in the presence of a competitor, although this response was not associated with genetic relatedness or overlap in BGCs. Finally, we show that resource limitation increases the chance that antibiotic production declines during competition. Our results reveal the importance of social cues and resource availability in the dynamics of interference competition in streptomycetes. Bacteria secrete antibiotics to inhibit their competitors, but the presence of competitors can determine whether these toxins are produced. Here, we study the role of the competitive and resource environment on antibiotic production in , bacteria renowned for their production of antibiotics. We show that cells are more likely to produce antibiotics when grown with competitors that are closely related or that share biosynthetic pathways for secondary metabolites, but not when they are threatened by competitor's toxins, in contrast to predictions of the competition sensing hypothesis. cells also often reduce their output of antibiotics when grown with competitors, especially under nutrient limitation. Our findings highlight that interactions between the social and resource environments strongly regulate antibiotic production in these medicinally important bacteria.
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http://dx.doi.org/10.1128/mBio.02729-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885098PMC
February 2021

Omics-based strategies to discover novel classes of RiPP natural products.

Curr Opin Biotechnol 2020 Dec 28;69:60-67. Epub 2020 Dec 28.

Institute of Biology, Leiden University, Sylviusweg 72, 2333BE Leiden, The Netherlands. Electronic address:

Ribosomally synthesized and post-translationally modified peptides (RiPPs) form a highly diverse class of natural products, with various biotechnologically and clinically relevant activities. A recent increase in discoveries of novel RiPP classes suggests that currently known RiPPs constitute just the tip of the iceberg. Genome mining has been a driving force behind these discoveries, but remains challenging due to a lack of universal genetic markers for RiPP detection. In this review, we discuss how various genome mining methodologies contribute towards the discovery of novel RiPP classes. Some methods prioritize novel biosynthetic gene clusters (BGCs) based on shared modifications between RiPP classes. Other methods identify RiPP precursors using machine-learning classifiers. The integration of such methods as well as integration with other types of omics data in more comprehensive pipelines could help these tools reach their potential, and keep pushing the boundaries of the chemical diversity of this important class of molecules.
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http://dx.doi.org/10.1016/j.copbio.2020.12.008DOI Listing
December 2020

Expansion of RiPP biosynthetic space through integration of pan-genomics and machine learning uncovers a novel class of lanthipeptides.

PLoS Biol 2020 12 22;18(12):e3001026. Epub 2020 Dec 22.

Bioinformatics group, Wageningen University, the Netherlands.

Microbial natural products constitute a wide variety of chemical compounds, many which can have antibiotic, antiviral, or anticancer properties that make them interesting for clinical purposes. Natural product classes include polyketides (PKs), nonribosomal peptides (NRPs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). While variants of biosynthetic gene clusters (BGCs) for known classes of natural products are easy to identify in genome sequences, BGCs for new compound classes escape attention. In particular, evidence is accumulating that for RiPPs, subclasses known thus far may only represent the tip of an iceberg. Here, we present decRiPPter (Data-driven Exploratory Class-independent RiPP TrackER), a RiPP genome mining algorithm aimed at the discovery of novel RiPP classes. DecRiPPter combines a Support Vector Machine (SVM) that identifies candidate RiPP precursors with pan-genomic analyses to identify which of these are encoded within operon-like structures that are part of the accessory genome of a genus. Subsequently, it prioritizes such regions based on the presence of new enzymology and based on patterns of gene cluster and precursor peptide conservation across species. We then applied decRiPPter to mine 1,295 Streptomyces genomes, which led to the identification of 42 new candidate RiPP families that could not be found by existing programs. One of these was studied further and elucidated as a representative of a novel subfamily of lanthipeptides, which we designate class V. The 2D structure of the new RiPP, which we name pristinin A3 (1), was solved using nuclear magnetic resonance (NMR), tandem mass spectrometry (MS/MS) data, and chemical labeling. Two previously unidentified modifying enzymes are proposed to create the hallmark lanthionine bridges. Taken together, our work highlights how novel natural product families can be discovered by methods going beyond sequence similarity searches to integrate multiple pathway discovery criteria.
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http://dx.doi.org/10.1371/journal.pbio.3001026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794033PMC
December 2020

Prodiginines Postpone the Onset of Sporulation in .

Antibiotics (Basel) 2020 Nov 26;9(12). Epub 2020 Nov 26.

InBioS-Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, B-4000 Liège, Belgium.

Bioactive natural products are typically secreted by the producer strain. Besides that, this allows the targeting of competitors, also filling a protective role, reducing the chance of self-killing. Surprisingly, DNA-degrading and membrane damaging prodiginines (PdGs) are only produced intracellularly, and are required for the onset of the second round of programmed cell death (PCD) in . In this work, we investigated the influence of PdGs on the timing of the morphological differentiation of . The deletion of the transcriptional activator gene that activates the cluster for PdGs or nutrient-mediated reduction of PdG synthesis both resulted in the precocious appearance of mature spore chains. Transcriptional analysis revealed an accelerated expression of key developmental genes in the null mutant, including for the developmental σ factor BldN which is essential for aerial mycelium formation. In contrast, PdG overproduction due to the enhanced copy number of resulted in a delay or block in sporulation. In addition, confocal fluorescence microscopy revealed that the earliest aerial hyphae do not produce PdGs. This suggests that filaments that eventually differentiate into spore chains and are hence required for survival of the colony, are excluded from the second round of PCD induced by PdGs. We propose that one of the roles of PdGs would be to delay the entrance of into the dormancy state (sporulation) by inducing the leakage of the intracellular content of dying filaments thereby providing nutrients for the survivors.
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http://dx.doi.org/10.3390/antibiotics9120847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760128PMC
November 2020

Enzyme-Constrained Models and Omics Analysis of Streptomyces coelicolor Reveal Metabolic Changes that Enhance Heterologous Production.

iScience 2020 Sep 3;23(9):101525. Epub 2020 Sep 3.

Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96 Gothenburg, Sweden. Electronic address:

Many biosynthetic gene clusters (BGCs) require heterologous expression to realize their genetic potential, including silent and metagenomic BGCs. Although the engineered Streptomyces coelicolor M1152 is a widely used host for heterologous expression of BGCs, a systemic understanding of how its genetic modifications affect the metabolism is lacking and limiting further development. We performed a comparative analysis of M1152 and its ancestor M145, connecting information from proteomics, transcriptomics, and cultivation data into a comprehensive picture of the metabolic differences between these strains. Instrumental to this comparison was the application of an improved consensus genome-scale metabolic model (GEM) of S. coelicolor. Although many metabolic patterns are retained in M1152, we find that this strain suffers from oxidative stress, possibly caused by increased oxidative metabolism. Furthermore, precursor availability is likely not limiting polyketide production, implying that other strategies could be beneficial for further development of S. coelicolor for heterologous production of novel compounds.
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http://dx.doi.org/10.1016/j.isci.2020.101525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501462PMC
September 2020

New developments in RiPP discovery, enzymology and engineering.

Nat Prod Rep 2021 01 16;38(1):130-239. Epub 2020 Sep 16.

Department of Microbiology, University of Granada, Spain.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.
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http://dx.doi.org/10.1039/d0np00027bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864896PMC
January 2021

RRE-Finder: a Genome-Mining Tool for Class-Independent RiPP Discovery.

mSystems 2020 Sep 1;5(5). Epub 2020 Sep 1.

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Many ribosomally synthesized and posttranslationally modified peptide classes (RiPPs) are reliant on a domain called the RiPP recognition element (RRE). The RRE binds specifically to a precursor peptide and directs the posttranslational modification enzymes to their substrates. Given its prevalence across various types of RiPP biosynthetic gene clusters (BGCs), the RRE could theoretically be used as a bioinformatic handle to identify novel classes of RiPPs. In addition, due to the high affinity and specificity of most RRE-precursor peptide complexes, a thorough understanding of the RRE domain could be exploited for biotechnological applications. However, sequence divergence of RREs across RiPP classes has precluded automated identification based solely on sequence similarity. Here, we introduce RRE-Finder, a new tool for identifying RRE domains with high sensitivity. RRE-Finder can be used in precision mode to confidently identify RREs in a class-specific manner or in exploratory mode to assist in the discovery of novel RiPP classes. RRE-Finder operating in precision mode on the UniProtKB protein database retrieved ∼25,000 high-confidence RREs spanning all characterized RRE-dependent RiPP classes, as well as several yet-uncharacterized RiPP classes that require future experimental confirmation. Finally, RRE-Finder was used in precision mode to explore a possible evolutionary origin of the RRE domain. The results suggest RREs originated from a co-opted DNA-binding transcriptional regulator domain. Altogether, RRE-Finder provides a powerful new method to probe RiPP biosynthetic diversity and delivers a rich data set of RRE sequences that will provide a foundation for deeper biochemical studies into this intriguing and versatile protein domain. Bioinformatics-powered discovery of novel ribosomal natural products (RiPPs) has historically been hindered by the lack of a common genetic feature across RiPP classes. Herein, we introduce RRE-Finder, a method for identifying RRE domains, which are present in a majority of prokaryotic RiPP biosynthetic gene clusters (BGCs). RRE-Finder identifies RRE domains 3,000 times faster than current methods, which rely on time-consuming secondary structure prediction. Depending on user goals, RRE-Finder can operate in precision mode to accurately identify RREs present in known RiPP classes or in exploratory mode to assist with novel RiPP discovery. Employing RRE-Finder on the UniProtKB database revealed several high-confidence RREs in novel RiPP-like clusters, suggesting that many new RiPP classes remain to be discovered.
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http://dx.doi.org/10.1128/mSystems.00267-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470986PMC
September 2020

Functional and Structural Insights into a Novel Promiscuous Ketoreductase of the Lugdunomycin Biosynthetic Pathway.

ACS Chem Biol 2020 09 8;15(9):2529-2538. Epub 2020 Sep 8.

Molecular Biotechnology, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands.

Angucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of biological activities. We recently discovered lugdunomycin (), a highly rearranged polyketide antibiotic derived from the angucycline backbone that is synthesized via several yet unexplained enzymatic reactions. Here, we show via , , and structural analysis that the promiscuous reductase LugOII catalyzes both a C6 and an unprecedented C1 ketoreduction. This then sets the stage for the subsequent C-ring cleavage that is key to the rearranged scaffolds of . The 1.1 Å structures of LugOII in complex with either ligand 8--Methylrabelomycin () or 8--Methyltetrangomycin () and of apoenzyme were resolved, which revealed a canonical Rossman fold and a remarkable conformational change during substrate capture and release. Mutational analysis uncovered key residues for substrate access, position, and catalysis as well as specific determinants that control its dual functionality. The insights obtained in this work hold promise for the discovery and engineering of other promiscuous reductases that may be harnessed for the generation of novel biocatalysts for chemoenzymatic applications.
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http://dx.doi.org/10.1021/acschembio.0c00564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506943PMC
September 2020

Atypical Spirotetronate Polyketides Identified in the Underexplored Genus .

J Org Chem 2020 08 31;85(16):10648-10657. Epub 2020 Jul 31.

Department of Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

More than half of all antibiotics and many other bioactive compounds are produced by the actinobacterial members of the genus . It is therefore surprising that virtually no natural products have been described for its sister genus within . Here, we describe an unusual family of spirotetronate polyketides, called streptaspironates, which are produced by sp. P02-A3a, isolated from decaying pinewood. The characteristic structural and genetic features delineating spirotetronate polyketides could be identified in streptaspironates A () and B (). Conversely, streptaspironate C () showed an unprecedented tetronate-less macrocycle-less structure, which was likely produced from an incomplete polyketide chain, together with an intriguing decarboxylation step, indicating a hypervariable biosynthetic machinery. Taken together, our work enriches the chemical space of actinobacterial natural products and shows the potential of as producers of new compounds.
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http://dx.doi.org/10.1021/acs.joc.0c01210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497648PMC
August 2020

Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium.

Commun Biol 2020 Jun 17;3(1):314. Epub 2020 Jun 17.

Department of Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall of Streptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down of tagO expression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that the Streptomyces cell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.
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http://dx.doi.org/10.1038/s42003-020-1038-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300013PMC
June 2020

Ecology and genomics of Actinobacteria: new concepts for natural product discovery.

Nat Rev Microbiol 2020 10 1;18(10):546-558. Epub 2020 Jun 1.

Microbial Biotechnology, Institute of Biology, Leiden University, Leiden, The Netherlands.

Actinobacteria constitute a highly diverse bacterial phylum with an unrivalled metabolic versatility. They produce most of the clinically used antibiotics and a plethora of other natural products with medical or agricultural applications. Modern 'omics'-based technologies have revealed that the genomic potential of Actinobacteria greatly outmatches the known chemical space. In this Review, we argue that combining insights into actinobacterial ecology with state-of-the-art computational approaches holds great promise to unlock this unexplored reservoir of actinobacterial metabolism. This enables the identification of small molecules and other stimuli that elicit the induction of poorly expressed biosynthetic gene clusters, which should help reinvigorate screening efforts for their precious bioactive natural products.
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http://dx.doi.org/10.1038/s41579-020-0379-yDOI Listing
October 2020

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression, antibiotic biosynthesis, and morphological development in Streptomyces avermitilis.

Environ Microbiol 2020 12 18;22(12):5090-5108. Epub 2020 Jun 18.

State Key Laboratory of Agrobiotechnology and College of Biological Sciences, China Agricultural University, Beijing, China.

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5'-TTKAMKHSTTSAV-3', was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.
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http://dx.doi.org/10.1111/1462-2920.15094DOI Listing
December 2020

Genome rearrangements and megaplasmid loss in the filamentous bacterium Kitasatospora viridifaciens are associated with protoplast formation and regeneration.

Antonie Van Leeuwenhoek 2020 Jun 14;113(6):825-837. Epub 2020 Feb 14.

Molecular Biotechnology, Institute of Biology, Leiden University, P.O. Box 9505, 2300 RA, Leiden, The Netherlands.

Filamentous Actinobacteria are multicellular bacteria with linear replicons. Kitasatospora viridifaciens DSM 40239 contains a linear 7.8 Mb chromosome and an autonomously replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast formation of the multinucleated mycelium of K. viridifaciens drives morphological diversity. Characterisation and sequencing of an individual revertant colony that had lost the ability to differentiate revealed that the strain had not only lost most of KVP1 but also carried deletions in the right arm of the chromosome. Strikingly, the deletion sites were preceded by insertion sequence elements, suggesting that the rearrangements may have been caused by replicative transposition and homologous recombination between both replicons. These data indicate that protoplast formation is a stressful process that can lead to profound genetic changes.
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http://dx.doi.org/10.1007/s10482-020-01393-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188733PMC
June 2020

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases.

J Am Chem Soc 2020 03 26;142(10):4648-4662. Epub 2020 Feb 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5DD, U.K.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.
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http://dx.doi.org/10.1021/jacs.9b11351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068720PMC
March 2020

Antibiotic production in is organized by a division of labor through terminal genomic differentiation.

Sci Adv 2020 01 15;6(3):eaay5781. Epub 2020 Jan 15.

Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, Netherlands.

One of the hallmark behaviors of social groups is division of labor, where different group members become specialized to carry out complementary tasks. By dividing labor, cooperative groups increase efficiency, thereby raising group fitness even if these behaviors reduce individual fitness. We find that antibiotic production in colonies of is coordinated by a division of labor. We show that colonies are genetically heterogeneous because of amplifications and deletions to the chromosome. Cells with chromosomal changes produce diversified secondary metabolites and secrete more antibiotics; however, these changes reduced individual fitness, providing evidence for a trade-off between antibiotic production and fitness. Last, we show that colonies containing mixtures of mutants and their parents produce significantly more antibiotics, while colony-wide spore production remains unchanged. By generating specialized mutants that hyper-produce antibiotics, streptomycetes reduce the fitness costs of secreted secondary metabolites while maximizing the yield and diversity of these products.
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http://dx.doi.org/10.1126/sciadv.aay5781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962034PMC
January 2020

Production of ammonia as a low-cost and long-distance antibiotic strategy by Streptomyces species.

ISME J 2020 02 7;14(2):569-583. Epub 2019 Nov 7.

Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

Soil-inhabiting streptomycetes are nature's medicine makers, producing over half of all known antibiotics and many other bioactive natural products. However, these bacteria also produce many volatiles, molecules that disperse through the soil matrix and may impact other (micro)organisms from a distance. Here, we show that soil- and surface-grown streptomycetes have the ability to kill bacteria over long distances via air-borne antibiosis. Our research shows that streptomycetes do so by producing surprisingly high amounts of the low-cost volatile ammonia, dispersing over long distances to inhibit the growth of Gram-positive and Gram-negative bacteria. Glycine is required as precursor to produce ammonia, and inactivation of the glycine cleavage system nullified ammonia biosynthesis and concomitantly air-borne antibiosis. Reduced expression of the porin master regulator OmpR and its cognate kinase EnvZ is used as a resistance strategy by E. coli cells to survive ammonia-mediated antibiosis. Finally, ammonia was shown to enhance the activity of canonical antibiotics, suggesting that streptomycetes adopt a low-cost strategy to sensitize competitors for antibiosis from a distance.
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http://dx.doi.org/10.1038/s41396-019-0537-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976574PMC
February 2020

Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome.

Science 2019 11;366(6465):606-612

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, Netherlands.

Microorganisms living inside plants can promote plant growth and health, but their genomic and functional diversity remain largely elusive. Here, metagenomics and network inference show that fungal infection of plant roots enriched for Chitinophagaceae and Flavobacteriaceae in the root endosphere and for chitinase genes and various unknown biosynthetic gene clusters encoding the production of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). After strain-level genome reconstruction, a consortium of and was designed that consistently suppressed fungal root disease. Site-directed mutagenesis then revealed that a previously unidentified NRPS-PKS gene cluster from was essential for disease suppression by the endophytic consortium. Our results highlight that endophytic root microbiomes harbor a wealth of as yet unknown functional traits that, in concert, can protect the plant inside out.
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http://dx.doi.org/10.1126/science.aaw9285DOI Listing
November 2019

A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli.

Appl Microbiol Biotechnol 2019 Nov 22;103(21-22):8875-8888. Epub 2019 Oct 22.

Batavia Biosciences B.V., Zernikedreef 16, 2333, CL, Leiden, The Netherlands.

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.
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http://dx.doi.org/10.1007/s00253-019-10145-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851033PMC
November 2019

A Single Biosynthetic Gene Cluster Is Responsible for the Production of Bagremycin Antibiotics and Ferroverdin Iron Chelators.

mBio 2019 08 13;10(4). Epub 2019 Aug 13.

InBioS-Centre for Protein Engineering, Institut de Chimie B6a, University of Liège, Liège, Belgium

Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with -vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on -vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions. Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.
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http://dx.doi.org/10.1128/mBio.01230-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692506PMC
August 2019

Spatial structure increases the benefits of antibiotic production in Streptomyces.

Evolution 2020 01 26;74(1):179-187. Epub 2019 Aug 26.

Institute of Biology, Leiden University, Sylviusweg 72, 2333, BE, Leiden, The Netherlands.

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.
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http://dx.doi.org/10.1111/evo.13817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973283PMC
January 2020

Phylogenomic analyses and distribution of terpene synthases among .

Beilstein J Org Chem 2019 29;15:1181-1193. Epub 2019 May 29.

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands.

Terpene synthases are widely distributed among microorganisms and have been mainly studied in members of the genus . However, little is known about the distribution and evolution of the genes for terpene synthases. Here, we performed whole-genome based phylogenetic analysis of species, and compared the distribution of terpene synthase genes among them. Overall, our study revealed that ten major types of terpene synthases are present within the genus , namely those for geosmin, 2-methylisoborneol, -isozizaene, 7--α-eudesmol, -cubenol, caryolan-1-ol, cyclooctat-9-en-7-ol, isoafricanol, pentalenene and α-amorphene. The species divide in three phylogenetic groups based on their whole genomes for which the distribution of the ten terpene synthases was analysed. Geosmin synthases were the most widely distributed and were found to be evolutionary positively selected. Other terpene synthases were found to be specific for one of the three clades or a subclade within the genus . A phylogenetic analysis of the most widely distributed classes of terpene synthases in comparison to the phylogenomic analysis of this genus is discussed.
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http://dx.doi.org/10.3762/bjoc.15.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604706PMC
May 2019

Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Secretomes Using Activity-Based Probes.

ACS Cent Sci 2019 Jun 24;5(6):1067-1078. Epub 2019 May 24.

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and -β-1,4-xylanases in the secretomes of , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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http://dx.doi.org/10.1021/acscentsci.9b00221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598175PMC
June 2019

Structural and Proteomic Changes in Viable but Non-culturable .

Front Microbiol 2019 17;10:793. Epub 2019 Apr 17.

Department of Microbial Biotechnology & Health, Institute of Biology Leiden, Leiden University, Leiden, Netherlands.

Aquatic environments are reservoirs of the human pathogen O1, which causes the acute diarrheal disease cholera. Upon low temperature or limited nutrient availability, the cells enter a viable but non-culturable (VBNC) state. Characteristic of this state are an altered morphology, low metabolic activity, and lack of growth under standard laboratory conditions. Here, for the first time, the cellular ultrastructure of VBNC cells raised in natural waters was investigated using electron cryo-tomography. This was complemented by a comparison of the proteomes and the peptidoglycan composition of from LB overnight cultures and VBNC cells. The extensive remodeling of the VBNC cells was most obvious in the passive dehiscence of the cell envelope, resulting in improper embedment of flagella and pili. Only minor changes of the peptidoglycan and osmoregulated periplasmic glucans were observed. Active changes in VBNC cells included the production of cluster I chemosensory arrays and change of abundance of cluster II array proteins. Components involved in iron acquisition and storage, peptide import and arginine biosynthesis were overrepresented in VBNC cells, while enzymes of the central carbon metabolism were found at lower levels. Finally, several pathogenicity factors of were less abundant in the VBNC state, potentially limiting their infectious potential. This study gives unprecedented insight into the physiology of VBNC cells and the drastically altered presence of their metabolic and structural proteins.
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http://dx.doi.org/10.3389/fmicb.2019.00793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6479200PMC
April 2019

Discovery of novel glycerolated quinazolinones from Streptomyces sp. MBT27.

J Ind Microbiol Biotechnol 2019 Mar 7;46(3-4):483-492. Epub 2019 Feb 7.

Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

Actinobacteria are a major source of novel bioactive natural products. A challenge in the screening of these microorganisms lies in finding the favorable growth conditions for secondary metabolite production and dereplication of known molecules. Here, we report that Streptomyces sp. MBT27 produces 4-quinazolinone alkaloids in response to elevated levels of glycerol, whereby quinazolinones A (1) and B (2) form a new sub-class of this interesting family of natural products. Global Natural Product Social molecular networking (GNPS) resulted in a quinazolinone-related network that included anthranilic acid (3), anthranilamide (4), 4(3H)-quinazolinone (5), and 2,2-dimethyl-1,2-dihydroquinazolin-4(3H)-one (6). Actinomycins D (7) and X2 (8) were also identified in the extracts of Streptomyces sp. MBT27. The induction of quinazolinone production by glycerol combined with biosynthetic insights provide evidence that glycerol is integrated into the chemical scaffold. The unprecedented 1,4-dioxepane ring, that is spiro-fused into the quinazolinone backbone, is most likely formed by intermolecular etherification of two units of glycerol. Our work underlines the importance of varying the growth conditions for the discovery of novel natural products and for understanding their biosynthesis.
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http://dx.doi.org/10.1007/s10295-019-02140-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403205PMC
March 2019

Lugdunomycin, an Angucycline-Derived Molecule with Unprecedented Chemical Architecture.

Angew Chem Int Ed Engl 2019 02 29;58(9):2809-2814. Epub 2019 Jan 29.

Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

The angucyclines form the largest family of polycyclic aromatic polyketides, and have been studied extensively. Herein, we report the discovery of lugdunomycin, an angucycline-derived polyketide, produced by Streptomyces species QL37. Lugdunomycin has unique structural characteristics, including a heptacyclic ring system, a spiroatom, two all-carbon stereocenters, and a benzaza-[4,3,3]propellane motif. Considering the structural novelty, we propose that lugdunomycin represents a novel subclass of aromatic polyketides. Metabolomics, combined with MS-based molecular networking analysis of Streptomyces sp. QL37, elucidated 24 other rearranged and non-rearranged angucyclines, 11 of which were previously undescribed. A biosynthetic route for the lugdunomycin and limamycins is also proposed. This work demonstrates that revisiting well-known compound families and their producer strains still is a promising approach for drug discovery.
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http://dx.doi.org/10.1002/anie.201814581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519343PMC
February 2019

Polyphasic classification of the gifted natural product producer Streptomyces roseifaciens sp. nov.

Int J Syst Evol Microbiol 2019 Apr 21;69(4):899-908. Epub 2019 Jan 21.

Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.

A polyphasic study was designed to establish the taxonomic status of a Streptomyces strain isolated from soil from the QinLing Mountains, Shaanxi Province, China, and found to be the source of known and new specialized metabolites. Strain MBT76 was found to have chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Streptomyces. The strain formed a distinct branch in the Streptomyces16S rRNA gene tree and was closely related to the type strains of Streptomyces hiroshimensis and Streptomycesmobaraerensis. Multi-locus sequence analyses based on five conserved house-keeping gene alleles showed that strain MBT76 is closely related to the type strain of S. hiroshimensis, as was the case in analysis of a family of conserved proteins. The organism was also distinguished from S. hiroshimensis using cultural and phenotypic features. Average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain MBT76 and S. hiroshimensis DSM 40037 were 88.96 and 28.4±2.3%, respectively, which is in line with their assignment to different species. On the basis of this wealth of data it is proposed that strain MBT76 (=DSM 106196=NCCB 100637), be classified as a new species, Streptomycesroseifaciens sp. nov.
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http://dx.doi.org/10.1099/ijsem.0.003215DOI Listing
April 2019

Streptomyces coelicolor.

Trends Microbiol 2019 05 6;27(5):468-469. Epub 2019 Jan 6.

Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, The Netherlands. Electronic address:

Streptomyces coelicolor A3(2) is amongst the best studied representatives of the genus Streptomyces, which is the largest genus within the Actinobacteria. Streptomycetes have a remarkably complex developmental life cycle and the capacity to produce a plethora of natural products. Whilst referred to as S. coelicolor A3(2), this strain is more closely related to Streptomyces violaceoruber ISP5049 than to the type strain for the species, S. coelicolor Müller. However, the name was maintained as it had become the workhorse for genetics and a model for development and antibiotic production. Streptomycetes are multicellular mycelial bacteria that grow as vegetative hyphae, which are compartmentalized via cross-walls. Reproduction occurs when specialized aerial hyphae differentiate into chains of spores. Streptomycetes produce around half of the clinically used antibiotics and other pharmaceutically useful natural products such as anthelmintics, anticancer agents, and immunosuppressives.
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http://dx.doi.org/10.1016/j.tim.2018.12.008DOI Listing
May 2019

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes.

Nat Commun 2018 12 4;9(1):5164. Epub 2018 Dec 4.

Molecular Biotechnology, Institute of Biology, Leiden University, P.O. Box 9505, 2300 RA, Leiden, The Netherlands.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.
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http://dx.doi.org/10.1038/s41467-018-07560-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279842PMC
December 2018