Construction of S. islandicus strains

The type I-A CRISPR defence module including seven genes i.e. cas3b, csa5, cas7, cas5, cas3’, cas3”, and casX ³¹ was in-frame deleted from the genetic host S. islandicus RJW007, derived from wild-type strain S. islandicus M.16.4 carrying a double pyrEF and argD deletion ³², by employing a modified Plasmid Integration and Segregation knockout strategy ³³, in line with the methodology developed by the She laboratory ³⁴. The resultant type I-A deletion mutant (RJW007∆type I-A) was then used as a parental strain to further delete csx1 gene, generating the mutant strain RJW007∆type I-A∆csx1. Mutant strains were confirmed by PCR analysis using primers that bind outside of the homologous flanking arms of genes to be deleted.

Synthesised SIRV1 gp29 gene was purchased from IDT as a g-block and was cloned into a Sulfolobus-E.coli shuttle vector pSeSd-SsoargD ³² (referred to as pOE hereafter) at the NdeI and NotI sites, generating the gp29 expression plasmid pOE-gp29 in which the gp29 gene was placed under the control of the arabinose promoter. The pOE-gp29 and pOE plasmids were then transformed into competent cells of the ∆type I-A mutant and ∆type I-A∆csx1 mutant via electroporation as described previously ³², generating strains expressing and not expressing SIRV1 gp29, respectively.