Streptomyces clavuligerus is the producer of clavulanic acid; a β-lactamase inhibitor that isused in combination with the antibiotic amoxicillin to treat many bacterial infections. The unique genome of S. clavuligerus includes a chromosome, and four linear plasmids pSCL1, pSCL2, pSCL3 and the megaplasmid pSCL4, which is the largest linear plasmid ever identified at 1.8 Mb in size. In order to study the genomic architecture of S. clavuligerus strains, we established an optimised method of pulsed-field gel electrophoresis, which allowed visualisation of the three giant linear plasmids pSCL2, pSCL3 and pSCL4, as well as chromosomal fragments in order to establish the physical map of the S. clavuligerus genome.Furthermore, to establish a foundation on which the genomic analyses of industrial strains of this species could be carried out, we closed the genome sequence of the S. clavuligerus type strain DSM 738, using a combination of short-read Illumina sequencing and long-read PacBio technology. Additionally, the telomeres of the five replicons were purified by a self ligation method, and identification of the sequences confirmed that the five replicons carry archetypal telomeres, with the chromosome and megaplasmid sharing identical telomeric sequences. Streptomycetes telomeres are maintained by the terminal proteins Tap and Tpg, however, while copies of the genes encoding these proteins were identified in pSCL4, pSCL3 and pSCL2, the chromosome carries none, making these the only known essential elements missing from the chromosome.Moreover, in order to investigate the essentiality of the megaplasmid in S. clavuligerus, we cured pSCL4 by targeting parB with CRISPR/Cas9. This caused deletion of the chromosomal ends and circularisation, confirming that pSCL4 Tap-Tpg are required for maintaining the chromosome telomeres, and cannot be complemented by the proteins encoded on the other two plasmids. In addition, pSCL4-free strains were confirmed to be non-sporulating as well as presenting a phenotype of short and thin hyphae, in addition to higher branching frequency. Some mutant strains also exhibited reduced clavulanic acid production which confirmed that the loss of pSCL4 has important effects onmorphological development and secondary metabolism of S. clavuligerus. Overall, with this study we have closed the genome sequence of S. clavuligerus type strain and optimised a method for rapid screening of the S. clavuligerus mutant strains that will greatly benefit the industry for development of production strains. Additionally, we have confirmed that pSCL4 is a dispensable plasmid, but is yet necessary for maintaining optimum fitness of this bacterium.
|Date of Award||11 Nov 2020|
- University Of Strathclyde
|Sponsors||University of Strathclyde|
|Supervisor||Paul Herron (Supervisor) & Paul Hoskisson (Supervisor)|