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 DE LA SAMIGE 2023; 2023

CRISPR-Cas systems are adaptive immunity mechanisms in prokaryotes, classified into2 classes and 6 types with several subtypes, and reported in diverse mobile geneticelements (MGEs), such as transposon-like elements, plasmids, integrative and conjugativeelements, genomic islands, and prophages, which may be responsible for their lateraltransfer. On the other hand, other defense systems were found within phage satellites.These satellites harbor key genes, including an integrase (Int) for chromosome integrationand AlpA a putative excisionase, necessary for satellite excision. The aim of this work wasto untangle the role of MGEs in the mobilization of type I-F1 CRISPR-Cas systems. Analysisof the genetic surroundings using blastp of the subtype I-F1 in S. xiamenensis Sh95, aclinical isolate that harbors a large CRISPR array, revealed the presence of genes int-P4-like (a site-specific integrase) and alpA, frequently encoded in phage satellites. Comparativeanalysis between 87 subtype I-F1 CRISPR-Cas systems from Shewanella spp. with ACTv18.1.0 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, whichare commonly recognized by int-P4-like integrases and delimit the boundaries of genomicislands. The presence of both att sites suggest that this module may be acquired by anintegration/excision mechanism. Therefore, we looked for the excised circular form of theCRISPR-Cas module by PCR. We obtained a product of 758 bp and confirmed the excisionevent and the identification of the attP recombination site (5´-ATTCAACGTTTTGGATCTG3´) by sequencing. Analysis of ric-yicC locus in other S. xiamenensis genomes lacking aCRISPR-Cas system revealed a probable attB site similar to attL and attR (94.7%; 5´-ATTCCACGTTTTGGATCTG-3´). Comparison of other CRISPR-Cas modules in S.xiamenensis (strains NUITM-VS1 and ZYW6) showed that they were located at the sameinsertion site (ric-yicC) and had identical sequences for the attL/attR sites. While NUITMVS1 contained a subtype I-F1 CRISPR-Cas module at ric-yicC, ZYW6 had a subtype I-Esystem along with other defense systems at the same locus. These modules also encodedan int-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 alpAgenes together with the attL/attR recombination sites, in the CRISPR-Cas modules ofcertain S. xiamenensis genomes allow us to infer their potential mobilization. VariedCRISPR-Cas types and co-occurrence with other defense systems in the analyzed S.xiamenensis modules hint at strategic defense acquisition and show how phage satellitesand defense systems shape microorganism genomes. Last, we propose the mobility of thesubtype I-F1 CRISPR-Cas module from S. xiamenensis Sh95 via a phage satellite-likemechanism.