UNVEILING CRISPR-CAS SYSTEMS DIVERSITY AND LOCALIZATION IN Shewanella GENOMES

AYALA NUÑEZ, TEOLINCACIHUATL; CERBINO GABRIELA N.; CENTRON, DANIELA; QUIROGA, CECILIA

Chapadmalal

Congreso; XVIII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL - SAMIGE 2023; 2023

SAMIGE

CRISPR-Cas systems are adaptive immunity mechanisms found in prokaryotes, classified into 2classes and 6 types with several subtypes. They are present in ~40% of bacteria and ~90% ofarchaea. A typical CRISPR-cas locus includes cas genes adjacent to at least one CRISPR array,which consist of direct repeats separated by spacers, some of which offer defense against mobilegenetic elements (MGEs), such as phages and plasmids. The aim of this work was to characterizethese systems within our model bacteria, Shewanella. We searched CRISPR-Cas systems within349 complete and draft genomes of Shewanella spp. retrieved from the curated NCBI RefSeqdatabase. Cas proteins and CRISPR arrays were identified using the bioinformatic toolsCRISPRCasFinder, CRISPRloci, and CRISPR Recognition Tool v1.0; supplemented by a localBLASTX search for Cas and I-F3-specific proteins. We analyzed CRISPR-Cas systems geneticcontext with ACT v18.1.0 and MAUVE v2 by comparing CRISPR-Cas (+) and CRISPR-Cas (-)genomes. Shewanella spp. identification was done by constructing a phylogenomic tree usingMaximum Likelihood (TIM2e+R10 model, 1000 bootstraps) with IQ-Tree. Our findings revealed 168CRISPR-Cas systems in 157 Shewanella spp. genomes, showing an occurrence rate of 45%.These systems were distributed among 10 subtypes, with I-F1 (n=87), I-F3 (n=32), and I-E (n=33)being the most prevalent. Only 11 genomes harbored two systems, mainly from class 1. Analyzingthe structural organization, subtype I systems showed highly conserved cas gene arrangements,whereas subtype III displayed a greater diversity. CRISPR analysis showed that subtypes I-F1, IF2 and I-E contain larger arrays (up to 154, 147, 127 spacers, respectively), which may provide awider protection against phages and plasmids. Analysis of the genetic context of these systemsallowed us to identify different CRISPR-Cas modules, showcasing gene diversity, and cooccurrences with other defense systems (i.e., restriction-modification, toxin-antitoxin) and MGEs(i.e., a multidrug-resistant transposon Tn6297 with 3 class 1 integrons). This analysis also unveiled21 specific hotspot sites, where subtypes I-F1 and I-E were found at ric-yicC (S. xiamenensislineage) or at IFP-pbpC (S. algae lineage); whereas subtypes I-F3 were adjacent to the rsmJ gene.In conclusion, our comprehensive study showed that Shewanella spp. hosts diverse CRISPR-Cassubtypes, some of them with a large number of CRISPRs protecting against different MGEs,highlighting their pivotal role in shaping the survival strategies of this bacterium. Also, the cooccurrences with various defense systems and MGEs exemplify bacterial evolution’s ongoingdynamics. The variability of hotspots and the genes that constitute these modules not onlyhighlights evolution within Shewanella spp. but also emphasizes the fundamental role of thesehotspots in driving new adaptations