CONICET | Buscador de Institutos y Recursos Humanos

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas)areconsidered the adaptive immune system ofbacteria and archaea. They play a fundamental role in defending the host against plasmid and phageinvasion. CRISPR-Cas unique defense mechanism involves acquisition of foreign DNA as spacers in order to gain immunity,target recognition and cleavage of exogenous DNA or RNA. There is still much to be revealedof the external factors that activate these systems.The aim of this work was to understand the impact thatthese factorsplay in the expression of the type I-F CRISPR-Cas system from Shewanella sp. Sh95.This strainis a gram-negative rodisolated from a patient with an ocular infection from a public hospital of Argentina. Its CRISPR-Cas type I-F system comprises the cas operon(cas1, cas2/3, csy1, csy2, csy3 and csy4), a leader region and CRISPR array with 152 elements. Under standard growth conditions we observed by RT-PCR that thecas operon and the whole CRISPR array were transcribed suggesting that this element is active. We then assessed the effect of sucrose (20%) and NaCl (3.5 and 10%) on CRISPR-Cassystem activity. qRT-PCR assays showed that NaCl did not have significant impact on the system activity, however sucrose stressresulted in a 4-fold increase ofcsy1 and csy4 after 1h exposureand no significant fold-variations for cas2/3.Our results confirm that Shewanella sp. Sh95 has an active CRISPR-Cas system. Furthermore, in the presenceof high sucrose concentrations the expression of the Cascade complexwas increased, which could be theresult ofcell membrane integrity weakening. This work provides new insights on the external factors that activate CRISPR-Cassystems, which could be used asa tool to control immunity against mobile genetic elements.