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bioRxiv · 10.1101/2025.03.26.645530

Insights on the regulation and function of the CRISPR/Cas transposition system located in the pathogenicity island VpaI-7 from Vibrio parahaemolyticus RIMD2210633.

Alejandre-Sixtos, J. E.·Aguirre-Martinez, K.·Cruz-Lopez, J.·Mares-Rivera, A.·Alvarez-Martinez, S.·Zamorano-Sanchez, D.
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Abstract

CRISPR/Cas mediated transposition is a recently recognized strategy for horizontal gene transfer in a variety of bacterial species. However, our understanding of the factors that control their function in their natural hosts is still limited. In this work we report our initial genetic characterization of the elements associated with the CRISPR/Cas-transposition machinery (CASTm) from Vibrio parahaemolyticus (VpaCASTm), which are encoded within the pathogenicity island VpaI-7. Our results revealed that the components of the VpaCASTm and their associated CRISPR arrays (VpaCAST system) are transcriptionally active in their native genetic context. Furthermore, we were able to detect the presence of polycistrons and several internal promoters within the loci that compose the VpaCAST system. Our results also suggest that the activity of the promoter of the atypical CRISPR array is not repressed by the baseline activity of its known regulator VPA1391 in V. parahaemolyticus. Additionally, we found that the activity of the promoter of tniQ was modulated by a regulatory cascade involving ToxR, LeuO and H-NS. Since it was previously reported that the activity of the VpaCAST system was less efficient than that of the VchCAST system at promoting transposition of a miniaturized CRISPR-associated transposon (mini-CAST) in Escherichia coli, we analyzed if the transposition efficiency mediated by the VpaCAST system could be enhanced inside its natural host V. parahaemolyticus. We provide evidence that this might be the case suggesting that there could be host induction factors in V. parahaemolyticus that could enable more efficient transposition of CASTs. ImportanceMobile genetic elements such as transposons play important roles on the evolutionary trajectories of bacterial genomes. The success of transposon dissemination depends on their ability to carry selectable markers that improve the fitness of the host cell or loci with addictive traits such as the toxin-antitoxin systems. Here we aimed to characterize a transposon from Vibrio parahaemolyticus that carries and could disseminate multiple virulence factors. This transposon belongs to a recently discovered family of transposons whose transposition is guided by crRNA. We showed that the transposition machinery of this transposon is transcribed in V. parahaemolyticus and that there are likely host associated factors that favor transposition in the natural host V. parahaemolyticus over transposition in Escherichia coli.

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Alejandre-Sixtos, J. E., Aguirre-Martinez, K., Cruz-Lopez, J., Mares-Rivera, A., Alvarez-Martinez, S., Zamorano-Sanchez, D.. 2025-03-27. Insights on the regulation and function of the CRISPR/Cas transposition system located in the pathogenicity island VpaI-7 from Vibrio parahaemolyticus RIMD2210633.. https://doi.org/10.1101/2025.03.26.645530

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