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

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.

ActinoBase: tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria

“…However, advances in genome sequencing and analysis tools, as well as expanded genetic toolkits, have opened up new opportunities and reduced or removed some of these barriers. For instance, the development of tools such as CRISPR-Cas9 genome editing for Streptomyces species [39][40][41][42] alongside useful (albeit not actinobacterial-specific) bioinformatic tools, such as antiSMASH [43,44], autoMLST [45], and ClusterFinder [46] have proven to be major contributions to this scientific field. The community driven ActinoBase Wiki aims to draw on the experience of researchers worldwide and bring it together into a free and easy-to-use online interface.…”

Streptomyces clavuligerus: The Omics Era

“…These authors published an assembly with single contigs for the chromosome (6.75 Mb) and pSCL4 (1.8 Mb), and annotated 7,163 genes including 6,880 protein-encoding genes, 196 pseudogenes, 66 tRNAs, 18 rRNAs and 3 ncRNAs; this assembly however does not contain any data on any of the other three plasmids. An additional genome assembly (GCA_015708605.1) has been recently made public from an S. clavuligerus ATCC 27064 isolate studied for more than 25 years in the Microbiology laboratory at the University of León in Spain (Gomez-Escribano et al, 2021 ). This is a refined sequence (200× PacBio coverage and optical mapping) in which plasmids pSCL1, pSCL2 and pSCL4 were sequenced close to completion but plasmid pSCL3 was not found.…”

“…This is a refined sequence (200× PacBio coverage and optical mapping) in which plasmids pSCL1, pSCL2 and pSCL4 were sequenced close to completion but plasmid pSCL3 was not found. The authors, intrigued by the presence of pSCL3 only in the NRRL 3585 genome assembly from Song and colleagues ( 2010 ), further assessed the presence of plasmids in five different genuine type strain in culture collection by Illumina deep whole-genome sequencing, and found all four plasmids in all type strain deposits; this finding prompted the authors to suggest that pSCL3 might have been lost in the León isolate during subculturing of the original ATCC 27064 culture (Gomez-Escribano et al, 2021 ). Finally, the genomes of two industrial strains sequenced with PacBio/Ilumina have been described: S. clavuligerus F613-1 from which was reported a chromosome of 6.88 Mb and one plasmid of 0.7 Mb with sequence partially identical to pSCL4 (Cao et al, 2016 ) and S. clavuligerus F1D-5.…”

“…Recently CRISPR-cas9-mediated targeting of pSCL4 parB gene resulted in new pSCL4-free strains (Gomez-Escribano et al, 2021 ). Five pSCL4-cured isolates were sequenced and all of them showed deletions in one or both chromosomal arms, and other rearrangements, although they kept plasmids pSCL1 and pSCL2.…”

“…For two of the mutants enough sequence data were obtained to show circularisation of the chromosome concomitant to the loss of around 100 kb of each end. The deletions are not site specific as they range from 88 kb to 303 kb (Álvarez-Álvarez et al, 2017 ; Gomez-Escribano et al, 2021 ) in the right arm of the chromosome, but probably chromosome circularisation relies on the deletion of fragments at both arm ends (Chen et al, 2002 ; Tidjani et al, 2020 ). From these results it can be concluded that pSCL4 is not essential for S. clavuligerus viability since the pSCL4-less mutant growth is only slightly delayed in relation to the wild-type strain; however, pSCL4 appears to be essential for the stability and maintenance of the linear chromosome, a genetic hallmark of streptomycetes, and it is still to be elucidated whether the lack of morphological development of pSCL4-cured strains is due to the loss of specific genes located in pSCL4 or the loss of chromosomal sequence.…”