FUNCTIONAL CHARACTERIZATION OF THE TYPE I-F CRISPR-CAS SYSTEM FROM THE CLINICAL ISOLATE Shewanella xiamenensis Sh95

HANNAH HAMPTON; PETER FINERAN; GISELA PARMECIANO DI NOTO; DANIELA CENTRÓN; TEOLINCACIHUATL AYALA NUÑEZ; CECILIA QUIROGA

Mont Orford

Congreso; 20th Riboclub 2019; 2019

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociatedproteins (Cas) are considered the adaptive immune system of bacteria andarchaea. They play a fundamental role in defending the host against phage and plasmidinvasion. The CRISPR-Cas mechanism involves acquisition of foreign DNA as spacers,target recognition and cleavage of exogenous DNA or RNA. Shewanella spp. are gramnegativesthat thrive in aquatic environments and are known for their potential inbioremediation and microbial fuel cells. More recently, they have been consideredemergent opportunistic pathogens. We previously sequenced the complete genome of S.xiamenensis Sh95 isolated from an ocular infection. Genome analysis revealed thepresence of a CRISPR-Cas system. The aim of this work was to study the activity of theCRISPR-Cas system of S. xiamenensis Sh95 and evaluate the factors involved in itsregulation. Strain Sh95 CRISPR array was comprised of 153 repeats (28 bp-long) and 152spacers (32 bp-long). Upstream of the array we found genes encoding a type I-F system:cas1, cas2-3, cas8f (csy1), cas5 (csy2), cas7 (csy3) and cas6f (csy4). Spacer analysisshowed that while some spacers contain the information to target bacteriophages fromLambda and Mu families, most of them did not have an identifiable target. Specifictargeting of the resistance gene dfrA15 with a designed crRNA sequence was used toassess its interference activity. As a result, it was possible to eliminate not only theresistance gene but also the genomic island that harbored it, reverting the strain to anantibiotic sensitive phenotype. Furthermore, we assessed the effect of external factors onthe expression of cas genes by RT-qPCR. As a result, we observed that exposure of strainSh95 to UV light increases cas2-3 expression by 8-fold whereas high sucroseconcentration (20%) in the medium increases on average 4-fold cas1, cas8fc and cas6fexpression. Our results confirmed that S. xiamenensis Sh95 contains an active type I-FCRISPR-Cas immune system capable of endogenous genome editing and that differentenvironmental conditions may exert an impact on its activity.