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July 7, 2019  |  

Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity.

The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil-derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole-genome sequencing and linker-mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


July 7, 2019  |  

Genome sequence of the filamentous actinomycete Kitasatospora viridifaciens.

The vast majority of antibiotics are produced by filamentous soil bacteria called actinomycetes. We report here the genome sequence of the tetracycline producer “Streptomyces viridifaciens” DSM 40239. Given that this species has the hallmark signatures characteristic of the Kitasatospora genus, we previously proposed to rename this organism Kitasatospora viridifaciens. Copyright © 2017 Ramijan et al.


July 7, 2019  |  

Complete genome sequence of Stenotrophomonas sp. KACC 91585, an efficient bacterium for unsaturated fatty acid hydration.

Hydroxy fatty acids (HFAs) such as 10-hydroxystearic acid (10-HSA) and 10-hydroxy-12(Z)-octadecenoic acid (10-HOD), which are similar to ricinoleic acid, are important starting materials and intermediates for the industrial manufacture of many commodities. Stenotrophomonas sp. KACC 91585, which was isolated from lake sediment, is an efficient bacterium for unsaturated fatty acid hydration that produces 10-HSA and 10-HOD from oleic acid and linoleic acid, respectively, with high conversion rates. The complete genome of this strain is 4,541,729bp with 63.83% GC content and devoid of plasmids. Sets of genes involved in the fatty acid biosynthesis and modification as well as modified lipids were identified in the genome, and these genes were concerned with HFA production. This genome sequence provides molecular information and elucidation for HFA production, and will be used as an efficient biocatalyst source for the biotechnological production of HFA. Copyright © 2016 Elsevier B.V. All rights reserved.


July 7, 2019  |  

Draft genome sequence of the rhizobacterium Pseudomonas chlororaphis PCL1601, displaying biocontrol against soilborne phytopathogens.

In this study, we present the draft genome sequence of the bacterial strain Pseudomonas chlororaphis PCL1601. This bacterium was isolated from the rhizosphere of healthy avocado trees and displayed antagonistic and biological control activities against different soilborne phytopathogenic fungi and oomycetes. Copyright © 2017 Vida et al.


July 7, 2019  |  

Complete genome of Brachybacterium sp. P6-10-X1 isolated from deep-sea sediments of the Southern Ocean

Brachybacterium sp. P6-10-X1 is a rare actinobacterium isolated from deep-sea sediments in the Southern Ocean. To explore the potential of natural product biosynthesis, the genome was completely sequenced. It contained a circular chromosome of 4,385,603 bp with an average GC content of 70.9%. Genome mining revealed four biosynthetic gene clusters potentially producing new natural products.


July 7, 2019  |  

Complete genome sequence of Amycolatopsis orientalis CPCC200066, the producer of norvancomycin.

Amycolatopsis orientalis CPCC200066 is an actinomycete exploited commercially in China for the production of norvancomycin, an important glycopeptide antibiotic structurally close to the well-known vancomycin. The availability of the complete genome sequence of CPCC200066 would greatly strengthen our understanding of the regulation pattern of norvancomycin biosynthesis and ultimately improve its production, as well as potentiate discoveries of novel bioactive compounds. Here we report the complete genome sequence of A. orientalis CPCC200066, a circular chromosome consisting of 9,490,992bp. Forty putative secondary metabolite biosynthetic gene clusters, including norvancomycin, were predicted, covering 20.3% of the whole genome. To facilitate genetic manipulation of this strain, an efficient transformation system was established by constructing a novel integrative vector pIMBT1, which could be transferred into CPCC200066 by electroporation with high efficiency. FBT1 attB sites were also identified in other known Amycolatopsis genomes, indicating pIMBT1’s prospect to be a novel vector for genus Amycolatopsis. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019  |  

Complete genome sequence of Lysinibacillus sphaericus LMG 22257, a strain with ureolytic activity inducing calcium carbonate precipitation.

Microbiologically induced calcium carbonate precipitation shows the potential for use in bioremediation and construction consolidation, but the efficiency of this process must be improved. Lysinibacillus sphaericus LMG 22257 is a gram-positive ureolytic strain that has recently been applied for consolidating construction by mediating calcium carbonate precipitation. The complete genome sequence of L. sphaericus LMG 22257 is 3,436,578 base pairs with a GC content of 38.99%. The urea degradation pathway and genes related to extracellular polymeric substance biosynthesis were also identified. The strain can tolerate high alkalinity (pH up to 10) and high urea concentration (up to 3M). These findings provide insights into the microbiologically induced carbonate precipitation and extend the application of the metabolic potential of L. sphaericus LMG 22257 for bioremediation. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019  |  

The evolutionary life cycle of the polysaccharide biosynthetic gene cluster based on the Sphingomonadaceae.

Although clustering of genes from the same metabolic pathway is a widespread phenomenon, the evolution of the polysaccharide biosynthetic gene cluster remains poorly understood. To determine the evolution of this pathway, we identified a scattered production pathway of the polysaccharide sanxan by Sphingomonas sanxanigenens NX02, and compared the distribution of genes between sphingan-producing and other Sphingomonadaceae strains. This allowed us to determine how the scattered sanxan pathway developed, and how the polysaccharide gene cluster evolved. Our findings suggested that the evolution of microbial polysaccharide biosynthesis gene clusters is a lengthy cyclic process comprising cluster 1???scatter???cluster 2. The sanxan biosynthetic pathway proved the existence of a dispersive process. We also report the complete genome sequence of NX02, in which we identified many unstable genetic elements and powerful secretion systems. Furthermore, nine enzymes for the formation of activated precursors, four glycosyltransferases, four acyltransferases, and four polymerization and export proteins were identified. These genes were scattered in the NX02 genome, and the positive regulator SpnA of sphingans synthesis could not regulate sanxan production. Finally, we concluded that the evolution of the sanxan pathway was independent. NX02 evolved naturally as a polysaccharide producing strain over a long-time evolution involving gene acquisitions and adaptive mutations.


July 7, 2019  |  

Genomic and phenotypic analyses of Pseudomonas psychrotolerans PRS08-11306 reveal a turnerbactin biosynthesis gene cluster that contributes to nitrogen fixation.

Plant-microbe interactions can provide agronomic benefits, such as enhancing nutrient uptake and providing fixed nitrogen. The Pseudomonas psychrotolerans strain PRS08-11306 was isolated from rice seeds and can enhance plant growth. Here, we analyzed the P. psychrotolerans genome, which is ~5Mb, with 4389 coding sequences, 77 tRNAs, and 7 rRNAs. Genome analysis identified a cluster of turnerbactin biosynthetic genes, which are responsible for the production of a catecholate siderophore and contribute to nitrogen fixation for the host. Analysis of the transcription factor mutant ?rpoS, which does not express this gene cluster, confirmed the relationship between the gene cluster and siderophore production. The nitrogen fixation characteristics of the cluster were confirmed in a plant growth-promoting experiment. The annotated full genome sequence of this strain sheds light on the role of P. psychrotolerans PRS08-11306 as a plant beneficial bacterium. Copyright © 2017. Published by Elsevier B.V.


July 7, 2019  |  

Complete genome sequence of Bacillus subtilis J-5, a potential biocontrol agent.

Bacillus subtilis J-5 was isolated from tomato rhizosphere soil and exhibited strong inhibitory activity against Botrytis cinerea To shed light on the molecular mechanism underlying the biological control on phytopathogens, the whole genome of this strain was sequenced. Genes encoding antimicrobial compounds and the regulatory systems were identified in the genome. Copyright © 2017 Jia et al.


July 7, 2019  |  

Identification and characterization of a biosynthetic gene cluster for tryptophan dimers in deep sea-derived Streptomyces sp. SCSIO 03032.

Tryptophan dimers (TDs) are an important class of natural products with diverse bioactivities and share conserved biosynthetic pathways. We report the identification of a partial gene cluster (spm) responsible for the biosynthesis of a class of unusual TDs with non-planar skeletons including spiroindimicins (SPMs), indimicins (IDMs), and lynamicins (LNMs) from the deep-sea derived Streptomyces sp. SCSIO 03032. Bioinformatics analysis, targeted gene disruptions, and heterologous expression studies confirmed the involvement of the spm gene cluster in the biosynthesis of SPM/IDM/LNMs, and revealed the indispensable roles for the halogenase/reductase pair SpmHF, the amino acid oxidase SpmO, and the chromopyrrolic acid (CPA) synthase SpmD, as well as the positive regulator SpmR and the putative transporter SpmA. However, the spm gene cluster was unable to confer a heterologous host the ability to produce SPM/IDM/LNMs. In addition, the P450 enzyme SpmP and the monooxygenase SpmX2 were found to be non-relevant to the biosynthesis of SPM/IDM/LNMs. Sequence alignment and structure modeling suggested the lack of key conserved amino acid residues in the substrate-binding pocket of SpmP. Furthermore, feeding experiments in the non-producing ?spmO mutant revealed several biosynthetic precursors en route to SPMs, indicating that key enzymes responsible for the biosynthesis of SPMs should be encoded by genes outside of the identified spm gene cluster. Finally, the biosynthetic pathways of SPM/IDM/LNMs are proposed to lay a basis for further insights into their intriguing biosynthetic machinery.


July 7, 2019  |  

Formicamycins, antibacterial polyketides produced by Streptomyces formicae isolated from African Tetraponera plant-ants.

We report a new Streptomyces species named S. formicae that was isolated from the African fungus-growing plant-ant Tetraponera penzigi and show that it produces novel pentacyclic polyketides that are active against MRSA and VRE. The chemical scaffold of these compounds, which we have called the formicamycins, is similar to the fasamycins identified from the heterologous expression of clones isolated from environmental DNA, but has significant differences that allow the scaffold to be decorated with up to four halogen atoms. We report the structures and bioactivities of 16 new molecules and show, using CRISPR/Cas9 genome editing, that biosynthesis of these compounds is encoded by a single type 2 polyketide synthase biosynthetic gene cluster in the S. formicae genome. Our work has identified the first antibiotic from the Tetraponera system and highlights the benefits of exploring unusual ecological niches for new actinomycete strains and novel natural products.


July 7, 2019  |  

Genomics-driven discovery of the gliovirin biosynthesis gene cluster in the plant beneficial fungus Trichoderma virens

Gliovirin is a strong anti-oomycete and a candidate anticancer compound. It is produced by “P” strains of the plant disease biocontrol fungus Trichoderma virens and is involved in biological control of certain plant pathogens. Even though the compound is known for more than three decades, neither the genes involved nor the biosynthetic pathway are known. We have sequenced the whole genome of a gliovirin producing strain of T. virens and discovered a novel gene cluster comprising of 22 genes. Disruption of the non-ribosomal peptide synthetase eliminated biosynthesis of gliovirin. The gene cluster is very similar to a hitherto un-described gene cluster of Aspergillus udagawae, a human pathogen. Our findings open-up the possibility of strain improvement of T. virens for improved biocontrol of plant diseases through enhanced production of gliovirin. Research also can now be initiated on the role of this gene cluster in pathogenicity of the human pathogen A. udagawae.


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