Dataset Information

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces.

ABSTRACT: Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces .

SUBMITTER: Gao H

PROVIDER: S-EPMC8749152 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces.

Gao Hong H Smith Margaret C M MCM

Access microbiology 20211208 12

Some major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces< ...[more]

PMID: 35024553

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Project description:UnlabelledConjugative DNA transfer in mycelial Streptomyces is a unique process involving the transfer of a double-stranded plasmid from the donor into the recipient and the subsequent spreading of the transferred plasmid within the recipient mycelium. This process is associated with growth retardation of the recipient and manifested by the formation of circular inhibition zones, named pocks. To characterize the unique Streptomyces DNA transfer machinery, we replaced each gene of the conjugative 12.1-kbp Streptomyces venezuelae plasmid pSVH1, with the exception of the rep gene required for plasmid replication, with a hexanucleotide sequence. Only deletion of traB, encoding the FtsK-like DNA translocase, affected efficiency of the transfer dramatically and abolished pock formation. Deletion of spdB3, spd79, or spdB2 had a minor effect on transfer but prevented pock formation and intramycelial plasmid spreading. Biochemical characterization of the encoded proteins revealed that the GntR-type regulator TraR recognizes a specific sequence upstream of spdB3, while Orf108, SpdB2, and TraR bind to peptidoglycan. SpdB2 promoted spheroplast formation by T7 lysozyme and formed pores in artificial membranes. Bacterial two-hybrid analyses and chemical cross-linking revealed that most of the pSVH1-encoded proteins interacted with each other, suggesting a multiprotein DNA translocation complex of TraB and Spd proteins which directs intramycelial plasmid spreading.ImportanceMycelial soil bacteria of the genus Streptomyces evolved specific resistance genes as part of the biosynthetic gene clusters to protect themselves from their own antibiotic, making streptomycetes a huge natural reservoir of antibiotic resistance genes for dissemination by horizontal gene transfer. Streptomyces conjugation is a unique process, visible on agar plates with the mere eye by the formation of circular inhibition zones, called pocks. To understand the Streptomyces conjugative DNA transfer machinery, which does not involve a type IV secretion system (T4SS), we made a thorough investigation of almost all genes/proteins of the model plasmid pSVH1. We identified all genes involved in transfer and intramycelial plasmid spreading and showed that the FtsK-like DNA translocase TraB interacts with multiple plasmid-encoded proteins. Our results suggest the existence of a macromolecular DNA translocation complex that directs intramycelial plasmid spreading.

Dataset Information

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces.

Publications

Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from <i>E. coli</i> into <i>Streptomyces</i>.

Similar Datasets

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