Slemc, Lucija, Jakše, Jernej, Filisetti, Alessandro et al. (19 more authors) (2022) Reference-Grade Genome and Large Linear Plasmid of Streptomyces rimosus:Pushing the Limits of Nanopore Sequencing. Microbiology spectrum. e0243421. ISSN 2165-0497
Abstract
Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 Slemc et al. |
Keywords: | Genome, Bacterial,High-Throughput Nucleotide Sequencing/methods,Nanopore Sequencing,Oxytetracycline/metabolism,Plasmids/genetics,Streptomyces rimosus/genetics,Transposases/genetics |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) |
Funding Information: | Funder Grant number EUROPEAN COMMISSION 720793 |
Depositing User: | Pure (York) |
Date Deposited: | 10 May 2022 12:50 |
Last Modified: | 27 Dec 2024 00:21 |
Published Version: | https://doi.org/10.1128/spectrum.02434-21 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1128/spectrum.02434-21 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:186678 |