UNVEILING CRISPR-CAS SYSTEMS DIVERSITY AND LOCALIZATION IN Shewanella GENOMES

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

CHAPADMALAL

Congreso; XVIII CONGRESO DE LA SAMIGE 2023; 2023

CRISPR-Cas systems are adaptive immunity mechanisms found in prokaryotes,classified into 2 classes and 6 types with several subtypes. They are present in ~40% ofbacteria and ~90% of archaea. A typical CRISPR-cas locus includes cas genes adjacent toat least one CRISPR array, which consist of direct repeats separated by spacers, some ofwhich offer defense against mobile genetic elements (MGEs), such as phages andplasmids. The aim of this work was to characterize these systems within our model bacteria,Shewanella. We searched CRISPR-Cas systems within 349 complete and draft genomesof Shewanella spp. retrieved from the curated NCBI RefSeq database. Cas proteins andCRISPR arrays were identified using the bioinformatic tools CRISPRCasFinder,CRISPRloci, and CRISPR Recognition Tool v1.0; supplemented by a local BLASTX searchfor Cas and I-F3-specific proteins. We analyzed CRISPR-Cas systems genetic context withACT 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 findingsrevealed 168 CRISPR-Cas systems in 157 Shewanella spp. genomes, showing anoccurrence 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 harboredtwo systems, mainly from class 1. Analyzing the structural organization, subtype I systemsshowed highly conserved cas gene arrangements, whereas subtype III displayed a greaterdiversity. CRISPR analysis showed that subtypes I-F1, I-F2 and I-E contain larger arrays(up to 154, 147, 127 spacers, respectively), which may provide a wider protection againstphages and plasmids. Analysis of the genetic context of these systems allowed us to identifydifferent CRISPR-Cas modules, showcasing gene diversity, and co-occurrences with otherdefense systems (i.e., restriction-modification, toxin-antitoxin) and MGEs (i.e., a multidrugresistant transposon Tn6297 with 3 class 1 integrons). This analysis also unveiled 21specific 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 thersmJ gene. In conclusion, our comprehensive study showed that Shewanella spp. hostsdiverse CRISPR-Cas subtypes, some of them with a large number of CRISPRs protectingagainst different MGEs, highlighting their pivotal role in shaping the survival strategies ofthis bacterium. Also, the co-occurrences with various defense systems and MGEs exemplifybacterial evolution’s ongoing dynamics. The variability of hotspots and the genes thatconstitute these modules not only highlights evolution within Shewanella spp. but alsoemphasizes the fundamental role of these hotspots in driving new adaptations.