April 21, 2020  |  

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.


April 21, 2020  |  

Natural product drug discovery in the genomic era: realities, conjectures, misconceptions, and opportunities.

Natural product discovery from microorganisms provided important sources for antibiotics, anti-cancer agents, immune-modulators, anthelminthic agents, and insecticides during a span of 50 years starting in the 1940s, then became less productive because of rediscovery issues, low throughput, and lack of relevant new technologies to unveil less abundant or not easily detected drug-like natural products. In the early 2000s, it was observed from genome sequencing that Streptomyces species encode about ten times as many secondary metabolites as predicted from known secondary metabolomes. This gave rise to a new discovery approach-microbial genome mining. As the cost of genome sequencing dropped, the numbers of sequenced bacteria, fungi and archaea expanded dramatically, and bioinformatic methods were developed to rapidly scan whole genomes for the numbers, types, and novelty of secondary metabolite biosynthetic gene clusters. This methodology enabled the identification of microbial taxa gifted for the biosynthesis of drug-like secondary metabolites. As genome sequencing technology progressed, the realities relevant to drug discovery have emerged, the conjectures and misconceptions have been clarified, and opportunities to reinvigorate microbial drug discovery have crystallized. This perspective addresses these critical issues for drug discovery.


April 21, 2020  |  

Complete genome sequence unveiled cellulose degradation enzymes and secondary metabolic potentials in Streptomyces sp. CC0208.

Marine Streptomyces sp. CC0208 isolated from the Bohai Bay showed high efficiency of cellulose degradation under optimized fermentation parameters. Also, as one of the bioinformatics-based approaches for the discovery of novel natural product and enzyme effectively, genome mining has been developed and applied widely. Herein, we reported the complete genome sequence of Streptomyces sp. CC0208.Whole-genome sequencing analysis revealed a genome size of 9,325,981?bp with a linear chromosome, GC content of 70.59% and 8487 protein-coding genes. Abundant genes have predicted functions in antibiotic metabolism and enzymes. A 20 enzymes closely associated with cellulose degradation were discovered. A total of 25 biosynthetic gene clusters (BGCs) of secondary metabolites were identified, including diverse classes of natural products. The availability of genome sequence of Streptomyces sp. CC0208 not only will assist in cracking the mechanism of cellulose degradation but also will provide the insights into the significant secondary metabolic potentials for the production of diverse compound classes based on rational strategies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


April 21, 2020  |  

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.


April 21, 2020  |  

Evolution of Antibiotic Synthesis Gene Clusters in the Streptomyces globisporus TFH56, Isolated from Tomato Flower.

Streptomyces species are known to produce various bioactive metabolites that can prevent plant diseases. Previously, the Streptomyces strain TFH56 was found to inhibit the gray mold pathogen, Botrytis cinerea, in tomato flower. In this study, the genome sequence of strain TFH56 was acquired using the Pacific Biosciences RS II platform. Three linear sequences (7.67 Mbp in total) were obtained. Based on average nucleotide identity, strain TFH56 was classified as Streptomyces globisporus, which is consistent with the presence of a linear chromosome and linear plasmids. Moreover, as with other examples of S. globisporus, the genome of strain TFH56 included a caryolan-1-ol synthase gene, a conprimycin synthetic gene cluster, and a lidamycin synthetic gene cluster.Copyright © 2019 Cho, Kwak.


April 21, 2020  |  

Comparative genome analysis provides novel insight into the interaction of Aquimarina sp. AD1, BL5 and AD10 with their macroalgal host.

The Aquimarina genus is widely distributed throughout the marine environment, however little is understood regarding its ecological role, particularly when in association with eukaryotic hosts. Here, we examine the genomes of two opportunistic pathogens, Aquimarina sp. AD1 and BL5, and a non-pathogenic strain Aquimarina sp. AD10, that were isolated from diseased individuals of the red alga Delisea pulchra. Each strain encodes multiple genes for the degradation of marine carbohydrates and vitamin biosynthesis. These traits are hypothesised to promote nutrient exchange between the Aquimarina strains and their algal host, facilitating a close symbiotic relationship. Moreover, each strain harbours the necessary genes for the assembly of a Type 9 Secretion System (T9SS) and the associated gliding motility apparatus. In addition to these common features, pathogenic strains AD1 and BL5, encode genes for the production of flexirubin type pigments and a number of unique non-ribosomal peptide synthesis (NRPS) gene clusters, suggesting a role for these uncharacterised traits in virulence. This study provides valuable insight into the potential ecological role of Aquimarina in the marine environment and the complex factors driving pathogenesis and symbiosis in this genus.Copyright © 2019 Elsevier B.V. All rights reserved.


April 21, 2020  |  

The genome sequence of Streptomyces rochei 7434AN4, which carries a linear chromosome and three characteristic linear plasmids.

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211?kb), -M (113?kb), and -S (18?kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G?+?C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406?bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.


April 21, 2020  |  

Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences.

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools to identify chromosomally clustered domains. We show that a single soil sample can contain more than a thousand uncharacterized biosynthetic gene clusters, most of which originate from low frequency genomes which are practically inaccessible through untargeted sequencing. CONKAT-seq allows scalable exploration of largely untapped biosynthetic diversity across multiple soils, and can guide the discovery of novel secondary metabolites from rare members of the soil microbiome.


April 21, 2020  |  

Comparative Genomics of Marine Sponge-Derived Streptomyces spp. Isolates SM17 and SM18 With Their Closest Terrestrial Relatives Provides Novel Insights Into Environmental Niche Adaptations and Secondary Metabolite Biosynthesis Potential.

The emergence of antibiotic resistant microorganisms has led to an increased need for the discovery and development of novel antimicrobial compounds. Frequent rediscovery of the same natural products (NPs) continues to decrease the likelihood of the discovery of new compounds from soil bacteria. Thus, efforts have shifted toward investigating microorganisms and their secondary metabolite biosynthesis potential, from diverse niche environments, such as those isolated from marine sponges. Here we investigated at the genomic level two Streptomyces spp. strains, namely SM17 and SM18, isolated from the marine sponge Haliclona simulans, with previously reported antimicrobial activity against clinically relevant pathogens; using single molecule real-time (SMRT) sequencing. We performed a series of comparative genomic analyses on SM17 and SM18 with their closest terrestrial relatives, namely S. albus J1074 and S. pratensis ATCC 33331 respectively; in an effort to provide further insights into potential environmental niche adaptations (ENAs) of marine sponge-associated Streptomyces, and on how these adaptations might be linked to their secondary metabolite biosynthesis potential. Prediction of secondary metabolite biosynthetic gene clusters (smBGCs) indicated that, even though the marine isolates are closely related to their terrestrial counterparts at a genomic level; they potentially produce different compounds. SM17 and SM18 displayed a better ability to grow in high salinity medium when compared to their terrestrial counterparts, and further analysis of their genomes indicated that they possess a pool of 29 potential ENA genes that are absent in S. albus J1074 and S. pratensis ATCC 33331. This ENA gene pool included functional categories of genes that are likely to be related to niche adaptations and which could be grouped based on potential biological functions such as osmotic stress, defense; transcriptional regulation; symbiotic interactions; antimicrobial compound production and resistance; ABC transporters; together with horizontal gene transfer and defense-related features.


April 21, 2020  |  

Genome Features and Secondary Metabolites Biosynthetic Potential of the Class Ktedonobacteria.

The prevalence of antibiotic resistance and the decrease in novel antibiotic discovery in recent years necessitates the identification of potentially novel microbial resources to produce natural products. Ktedonobacteria, a class of deeply branched bacterial lineage in the ancient phylum Chloroflexi, are ubiquitous in terrestrial environments and characterized by their large genome size and complex life cycle. These characteristics indicate Ktedonobacteria as a potential active producer of bioactive compounds. In this study, we observed the existence of a putative “megaplasmid,” multiple copies of ribosomal RNA operons, and high ratio of hypothetical proteins with unknown functions in the class Ktedonobacteria. Furthermore, a total of 104 antiSMASH-predicted putative biosynthetic gene clusters (BGCs) for secondary metabolites with high novelty and diversity were identified in nine Ktedonobacteria genomes. Our investigation of domain composition and organization of the non-ribosomal peptide synthetase and polyketide synthase BGCs further supports the concept that class Ktedonobacteria may produce compounds structurally different from known natural products. Furthermore, screening of bioactive compounds from representative Ktedonobacteria strains resulted in the identification of broad antimicrobial activities against both Gram-positive and Gram-negative tested bacterial strains. Based on these findings, we propose the ancient, ubiquitous, and spore-forming Ktedonobacteria as a versatile and promising microbial resource for natural product discovery.


April 21, 2020  |  

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.


April 21, 2020  |  

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum.

Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood.In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects.This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.


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