Genome editing reveals that pSCL4 is required for chromosome linearity in 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...

Full description

Saved in:
Bibliographic Details
Published in:Microbial genomics 2021-11, Vol.7 (11)
Main Authors: Gomez-Escribano, Juan Pablo, Algora Gallardo, Lis, Bozhüyük, Kenan A J, Kendrew, Steven G, Huckle, Benjamin D, Crowhurst, Nicola A, Bibb, Mervyn J, Collis, Andrew J, Micklefield, Jason, Herron, Paul R, Wilkinson, Barrie
Format: Article
Language:English
Subjects:
Chromosomes
Gene Editing - methods
Plasmids - genetics
Streptomyces - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 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.
ISSN:2057-5858
2057-5858
DOI:10.1099/mgen.0.000669