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.

Keywords: Bionano optical mapping; CRISPR-Cas9; PacBio; actinomycetes genomics; next-generation sequencing; plasmid curing.

Fig. 1.

Workflow and strategies leading to the genome sequence reported in this work. The linear plasmid pSCL1 was not fully sequenced by PacBio nor completely assembled by the Illumina pipeline; the read data for both were mapped to the previously published sequence for pSCL1 (accession no. X54107) indicating the full published sequence is present in, and equivalent to, the León isolate.

Fig. 2.

Map of gene editing vector pIJ13104 showing relevant genetic features. Positions of the sequencing primers M13-rev (5′-TCACACAGGAAACAGCTATGAC-3′), CRPMY2_F (5′-ATAAGGCTTGCAGCATCTGG-3′) and CRPMY2_R (5′-CGGTGCCACTTTTTCAAGTT-3′) are shown. Details of the genetic elements inherited from pBluescript II KS+ (ColE1 origin of replication, AmpR ampicillin-resistance gene, F1 origin of ssDNA replication), pCRISPomyces2 [SpCas9 gene, transcriptional promoter gapdhp(EL) and terminators fd-ter and oop ter, LacZ cassette, and gRNA-tracr], ermEp* promoter, and theophylline riboswitch can be found in the relevant references [21, 49, 65, 66]. Sites SpeI and NdeI can be used to exchange the SpCas9 gene promoter; sites XbaI or XbaI/SpeI can be used to excise the full CRISPR cassette.

Fig. 3.

Analysis of chromosome circularization in pSCL4-cured strain BW0216. (a) Plot of Illumina reads for the BW0216 chromosome sequence aligned against the León isolate reference showing a loss of coverage at both termini (top panel); the alignments for the two termini are expanded in middle and bottom panels. (b) Comparison of sequence alignments for sections of the reference chromosome sequence ends versus two Illumina reads and one assembled contig that cover the intersection resulting in circularization for BW0216; the bold letters indicate the first and last chromosome positions present in the mutants (a detailed analysis including for all five mutants can be found in the Supplementary Material). (c) Interpretation of the blastn results of Illumina contigs and reads overlapping both ends of the chromosome of strains BW0216 and BW0219 along with nucleotide positions and deletion sizes inferred from the analysis.