Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus

Juan Pablo Gomez-Escribano, Lis Algora Gallardo, Kenan A. J. Bozhüyük, Steven G. Kendrew, Benjamin D. Huckle, Nicola A. Crowhurst, Mervyn J. Bibb, Andrew J. Collis, Jason Micklefield, Paul R. Herron, Barrie Wilkinson

Research output: Contribution to journalArticlepeer-review

3 Downloads (Pure)

Abstract

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid's parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

Original languageEnglish
Article number000669
Number of pages12
JournalMicrobial Genomics
Volume7
Issue number11
DOIs
Publication statusPublished - 8 Nov 2021

Keywords

  • actinomycetes genomics
  • Bionano optical mapping
  • CRISPR-Cas9
  • next-generation sequencing
  • PacBio and plasmid curing

Cite this