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April 21, 2020  |  

A microbial factory for defensive kahalalides in a tripartite marine symbiosis.

Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont (“Candidatus Endobryopsis kahalalidefaciens”) uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. “Ca E. kahalalidefaciens” has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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April 21, 2020  |  

Complete Genome Sequence of Enterococcus faecalis Strain SGAir0397, Isolated from a Tropical Air Sample Collected in Singapore.

Enterococcus faecalis strain SGAir0397 was isolated from a tropical air sample collected in Singapore. Its genome was assembled using single-molecule real-time sequencing data and comprises one circular chromosome with a length of 2.69 Mbp. The genome contains 2,595 protein-coding genes, 59 tRNAs, and 12 rRNAs.Copyright © 2019 Purbojati et al.

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April 21, 2020  |  

The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor.

The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpPADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.Copyright © 2019 American Society for Microbiology.

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April 21, 2020  |  

Plantibacter flavus, Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens Endophytes Provide Host-Specific Growth Promotion of Arabidopsis thaliana, Basil, Lettuce, and Bok Choy Plants.

A collection of bacterial endophytes isolated from stem tissues of plants growing in soils highly contaminated with petroleum hydrocarbons were screened for plant growth-promoting capabilities. Twenty-seven endophytic isolates significantly improved the growth of Arabidopsis thaliana plants in comparison to that of uninoculated control plants. The five most beneficial isolates, one strain each of Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens and two strains of Plantibacter flavus were further examined for growth promotion in Arabidopsis, lettuce, basil, and bok choy plants. Host-specific plant growth promotion was observed when plants were inoculated with the five bacterial strains. P. flavus strain M251 increased the total biomass and total root length of Arabidopsis plants by 4.7 and 5.8 times, respectively, over that of control plants and improved lettuce and basil root growth, while P. flavus strain M259 promoted Arabidopsis shoot and root growth, lettuce and basil root growth, and bok choy shoot growth. A genome comparison between P. flavus strains M251 and M259 showed that both genomes contain up to 70 actinobacterial putative plant-associated genes and genes involved in known plant-beneficial pathways, such as those for auxin and cytokinin biosynthesis and 1-aminocyclopropane-1-carboxylate deaminase production. This study provides evidence of direct plant growth promotion by Plantibacter flavusIMPORTANCE The discovery of new plant growth-promoting bacteria is necessary for the continued development of biofertilizers, which are environmentally friendly and cost-efficient alternatives to conventional chemical fertilizers. Biofertilizer effects on plant growth can be inconsistent due to the complexity of plant-microbe interactions, as the same bacteria can be beneficial to the growth of some plant species and neutral or detrimental to others. We examined a set of bacterial endophytes isolated from plants growing in a unique petroleum-contaminated environment to discover plant growth-promoting bacteria. We show that strains of Plantibacter flavus exhibit strain-specific plant growth-promoting effects on four different plant species.Copyright © 2019 American Society for Microbiology.

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April 21, 2020  |  

Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.

The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting non-ribosomal peptides (NRPS) and Type I polyketides (PKS). NRPS-encoding genes were more widespread across the Antarctic, whereas PKS genes were only recoverable from a handful of sites. Many recovered sequences were deemed novel due to their low amino acid sequence similarity to known protein sequences, particularly throughout the east Antarctic sites. Phylogenetic analysis revealed that a high proportion were most similar to antifungal and biosurfactant-type clusters. Multivariate analysis showed that soil fertility factors of carbon, nitrogen and moisture displayed significant negative relationships with natural product gene richness. Our combined results suggest that secondary metabolite production is likely to play an important physiological component of survival for microorganisms inhabiting arid, nutrient-starved soils. © FEMS 2019.

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April 21, 2020  |  

Comparative genomic analysis of eight novel haloalkaliphilic bacteriophages from Lake Elmenteita, Kenya.

We report complete genome sequences of eight bacteriophages isolated from Haloalkaline Lake Elmenteita found on the floor of Kenyan Rift Valley. The bacteriophages were sequenced, annotated and a comparative genomic analysis using various Bioinformatics tools carried out to determine relatedness of the bacteriophages to each other, and to those in public databases. Basic genome properties like genome size, percentage coding density, number of open reading frames, percentage GC content and gene organizations revealed the bacteriophages had no relationship to each other. Comparison to other nucleotide sequences in GenBank database showed no significant similarities hence novel. At the amino acid level, phages of our study revealed mosaicism to genes with conserved domains to already described phages. Phylogenetic analyses of large terminase gene responsible for DNA packaging and DNA polymerase gene for replication further showed diversity among the bacteriophages. Our results give insight into diversity of bacteriophages in Lake Elmenteita and provide information on their evolution. By providing primary sequence information, this study not only provides novel sequences for biotechnological exploitation, but also sets stage for future studies aimed at better understanding of virus diversity and genomes from haloalkaline lakes in the Rift Valley.

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April 21, 2020  |  

Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria.

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B.?ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B.?ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B.?ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia?cepacia complex bacteria. Removal of the third replicon reduced B.?ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.

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April 21, 2020  |  

Genomic and transcriptomic insights into the survival of the subaerial cyanobacterium Nostoc flagelliforme in arid and exposed habitats.

The cyanobacterium Nostoc flagelliforme is an extremophile that thrives under extraordinary desiccation and ultraviolet (UV) radiation conditions. To investigate its survival strategies, we performed whole-genome sequencing of N. flagelliforme CCNUN1 and transcriptional profiling of its field populations upon rehydration in BG11 medium. The genome of N. flagelliforme is 10.23 Mb in size and contains 10 825 predicted protein-encoding genes, making it one of the largest complete genomes of cyanobacteria reported to date. Comparative genomics analysis among 20 cyanobacterial strains revealed that genes related to DNA replication, recombination and repair had disproportionately high contributions to the genome expansion. The ability of N. flagelliforme to thrive under extreme abiotic stresses is supported by the acquisition of genes involved in the protection of photosynthetic apparatus, the formation of monounsaturated fatty acids, responses to UV radiation, and a peculiar role of ornithine metabolism. Transcriptome analysis revealed a distinct acclimation strategy to rehydration, including the strong constitutive expression of genes encoding photosystem I assembly factors and the involvement of post-transcriptional control mechanisms of photosynthetic resuscitation. Our results provide insights into the adaptive mechanisms of subaerial cyanobacteria in their harsh habitats and have important implications to understand the evolutionary transition of cyanobacteria from aquatic environments to terrestrial ecosystems. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Complete genome sequence of Streptomyces spongiicola HNM0071T, a marine sponge-associated actinomycete producing staurosporine and echinomycin

Streptomyes spongiicola HNM0071T is a novel marine sponge-associated actinomycete with potential to produce antitumor agents including staurosporine and echinomycin. Here, we present the complete genome sequence of S. spongiicola HNM0071, which consists of a linear chromosome of 7,180,417?bp, 5669 protein coding genes, 18 rRNA genes, and 66 tRNA genes. Twenty-seven putative secondary metabolite biosynthetic gene clusters were found in the genome. Among them, the staurosporine and echinomycin gene clusters have been described completely. The complete genome information presented here will enable us to investigate the biosynthetic mechanism of two well-known antitumor antibiotics and to discover novel secondary metabolites with potential antitumor activities.

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April 21, 2020  |  

Whole genome sequence of Auricularia heimuer (Basidiomycota, Fungi), the third most important cultivated mushroom worldwide.

Heimuer, Auricularia heimuer, is one of the most famous traditional Chinese foods and medicines, and it is the third most important cultivated mushroom worldwide. The aim of this study is to develop genomic resources for A. heimuer to furnish tools that can be used to study its secondary metabolite production capability, wood degradation ability and biosynthesis of polysaccharides. The genome was obtained from single spore mycelia of the strain Dai 13782 by using combined high-throughput Illumina HiSeq 4000 system with the PacBio RSII long-read sequencing platform. Functional annotation was accomplished by blasting protein sequences with different public available databases to obtain their corresponding annotations. It is 49.76Mb in size with a N50 scaffold size of 1,350,668bp and encodes 16,244 putative predicted genes. This is the first genome-scale assembly and annotation for A. heimuer, which is the third sequenced species in Auricularia. Copyright © 2018 Elsevier Inc. All rights reserved.

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April 21, 2020  |  

Genome mining and metabolic profiling illuminate the chemistry driving diverse biological activities of Bacillus siamensis SCSIO 05746.

Bacillus spp. are important producers of bioactive natural products with potential applications in medicine and agriculture. Bacillus sp. SCSIO 05476 from a deep-sea sediment exhibits broad-spectrum antimicrobial activities and strong cytotoxic activity. Here, an integrative approach combining genome mining and metabolic profiling has been applied to decipher the chemical origins of this strain’s varied and significant biological activities. First, genome mining revealed 19 candidate gene clusters encoding the biosynthesis of diverse secondary metabolites. Then, a series of bacillibactins, fengycins, bacillomycins, surfactins, bacillaenes, macrolactins, and related species were found by LC-DAD-MS. Finally, three new linear bacillibactins, linbacillibactins A-C (1-3), along with 11 known secondary metabolites, bacillibactin (4), normal-C13 Val7 surfactin (5), anteiso-C13 Leu7 surfactin (6), iso-C14 Leu7 surfactin (7), normal-C14 Leu7 surfactin (8), anteiso-C14 Leu7 surfactin (9), macrolactin D (10), normal-C14 bacillomycin D (11), iso-C16 bacillomycin D (12), normal-C17 bacillomycin D (13), and iso-C17 bacillomycin D (14), were obtained and elucidated by bioactivity and structure-guided isolation from the fermentation of strain SCSIO 05746. Among them, new compounds 1-3 show significant siderophore activities comparable to that of bacillibactin (4), compounds 13 and 14 exhibit strong cytotoxic activity. At the same time, the strain classification status was confirmed by genomic analyses, and the complete genome sequence of Bacillus siamensis was presented firstly. This study provides a foundation for understanding the mechanisms driving SCSIO 05746’s multiple bioactivities and demonstrates a successful way of discovering bioactive metabolites using a combination of genome mining and metabolic profiling methods.

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April 21, 2020  |  

Antimicrobial, plant growth-promoting and genomic properties of the peanut endophyte Bacillus velezensis LDO2.

Peanut suffer from a number of fungal and bacterial pathogens, while plant endophytes were considered excellent candidates as biocontrol agents. In this study, the peanut endophytic bacterium LDO2 was evaluated for the potential of peanut pathogens inhibition and growth-promotion, and the genetic mechanisms were explored by genome mining. Strain LDO2 significantly inhibited the growth of peanut pathogenic fungi and pathogenic bacteria, and specifically, it showed pronounced inhibition on mycelia growth of Aspergillus flavus mycelia and caused mycelial deformity. Gene clusters responsible for antifungal metabolites (fengycin, surfactin, bacilysin) and antibacterial metabolites (butirosin, bacillaene, difficidin, macrolactin, surfactin, bacilysin) were identified. Strain LDO2 also exhibited several growth-promoting related features including phosphate solubilization, siderophore production and growth promotion of peanut root. Genes associated with plant growth promotion were also identified and analyzed, as well as genes related to secreted proteins. These findings suggested that this peanut endophyte could be a potential biocontrol agent in peanut production and a source of antimicrobial compounds for further exploitation. Copyright © 2018 Elsevier GmbH. All rights reserved.

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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.

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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.

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