Genomic Exploration of CRISPR-Cas Systems in Raoultella species: Diversity and Architecture.

Abstract

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems are adaptive immune mechanisms that protect bacteria against invading mobile genetic elements. They can influence horizontal gene transfer, phage resistance, and antimicrobial resistance (AMR) dynamics. Although these systems have been investigated in several Klebsiella species, information on historically defined Raoultella species remains limited. In this study, 103 complete genomes representing Raoultella species, currently placed within the broader Klebsiella lineage, were analyzed to characterize the distribution, architecture, and diversity of CRISPR-Cas systems. CRISPR-Cas loci were identified using CRISPRCasFinder and CRISPRone, followed by subtype classification, direct repeat and leader sequence analyses, spacer matching, Cas1-based phylogenetic analysis, and AMR determinant screening. Complete CRISPR-Cas systems were detected in 23 genomes, predominantly in R. ornithinolytica strains. All complete systems were chromosomally located, whereas plasmid-associated orphan CRISPR arrays were observed in selected R. ornithinolytica strains. Two major architectures were identified, with Type I-E being dominant and Type I-F restricted to fewer strains. Direct repeat analysis revealed six consensus sequences, while leader regions displayed conserved AT-rich motifs and strain-specific divergence. Spacer analyses suggested historical interactions with phages and plasmids, particularly mobile genetic elements associated with Klebsiella and other Enterobacteriaceae. Cas1 phylogeny showed subtype-dependent clustering, indicating that CRISPR-Cas evolution does not strictly follow core genome phylogeny. The coexistence of predicted CRISPR-Cas loci and AMR determinants indicates that the presence of these loci does not necessarily preclude the carriage of resistance determinants. Overall, this study expands current knowledge of CRISPR-Cas diversity among the analyzed Raoultella genomes and provides a genomic framework for future functional studies.