UNVEILING CRISPR-CAS I-F1 SYSTEM MOBILIZATION MECHANISM IN Shewanella xiamenensis Sh95

AYALA NUÑEZ, TEOLINCACIHUATL; CERBINO GABRIELA N.; PARMECIANO DI NOTO, GISELA; CENTRÓN, DANIELA; QUIROGA, CECILIA

Chapadmalal

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

SAMIGE

CRISPR-Cas systems are adaptive immunity mechanisms in prokaryotes, classified into 2 classesand 6 types with several subtypes, and reported in diverse mobile genetic elements (MGEs), suchas transposon-like elements, plasmids, integrative and conjugative elements, genomic islands, andprophages, which may be responsible for their lateral transfer. On the other hand, other defensesystems were found within phage satellites. These satellites harbor key genes, including anintegrase (Int) for chromosome integration and AlpA a putative excisionase, necessary for satelliteexcision. The aim of this work was to untangle the role of MGEs in the mobilization of type I-F1CRISPR-Cas systems. Analysis of the genetic surroundings using blastp of the subtype I-F1 in S.xiamenensis Sh95, a clinical isolate that harbors a large CRISPR array, revealed the presence ofgenes int-P4-like (a site-specific integrase) and alpA, frequently encoded in phage satellites.Comparative analysis between 87 subtype I-F1 CRISPR-Cas systems from Shewanella spp. withACT v18.1.0 and MAUVE v2 confirmed that the Sh95 CRISPR-Cas module was found at ric-yicClocus. We then identified via nucleotide-level sequence analysis the attL/attR sites, which arecommonly recognized by int-P4-like integrases and delimit the boundaries of genomic islands. Thepresence of both att sites suggest that this module may be acquired by an integration/excisionmechanism. Therefore, we looked for the excised circular form of the CRISPR-Cas module byPCR. We obtained a product of 758 bp and confirmed the excision event and the identification ofthe attP recombination site (5'-ATTCAACGTTTTGGATCTG-3') by sequencing. Analysis of ric-yicClocus in other S. xiamenensis genomes lacking a CRISPR-Cas system revealed a probable attBsite similar to attL and attR (94.7%; 5'-ATTCCACGTTTTGGATCTG-3'). Comparison of otherCRISPR-Cas modules in S. xiamenensis (strains NUITM-VS1 and ZYW6) showed that they werelocated at the same insertion site (ric-yicC) and had identical sequences for the attL/attR sites.While NUITM-VS1 contained a subtype I-F1 CRISPR-Cas module at ric-yicC, ZYW6 had a subtypeI-E system along with other defense systems at the same locus. These modules also encoded anint-P4-like gene similar to the one found in Sh95 (98.02% for NUITM-VS1 and 51.27% for ZYW6).alpA was also detected in both modules. The presence of int-P4-like and alpA genes together withthe attL/attR recombination sites, in the CRISPR-Cas modules of certain S. xiamenensis genomesallow us to infer their potential mobilization. Varied CRISPR-Cas types and co-occurrence withother defense systems in the analyzed S. xiamenensis modules hint at strategic defenseacquisition and show how phage satellites and defense systems shape microorganism genomes.Last, we propose the mobility of the subtype I-F1 CRISPR-Cas module from S. xiamenensis Sh95via a phage satellite-like mechanism