Genomic and functional dissection of natural transformation-related genes in.

Abstract

the etiological agent of piscirickettsiosis, represents the main health challenge for Chilean salmon farming and an emerging threat to global salmonid production. Inspection of allgenomes available shows that thegeneencoding the DNA uptake channel required for natural transformation (NT) in competent bacteria, is interrupted by various transposable elements, whereas other NT determinants (,,,, and) remain intact and highly conserved. UsingPsal-103 (EM-genogroup) and Psal-104b (LF-genogroup) as representatives of the two most prevalent genogroups in Chile, we combined comparative genomics, gene expression analysis, heterologous expression ofusing homologs from naturally competent bacteria, and CRISPRi-mediated knockdown of the remaining NT-related genes to examine their functionality. According to our results, expression ofwith homologs fromandimpairedviability in axenic culture, indicating a fitness burden. CRISPRi-mediated repression ofrevealed its essentiality in Psal-104b and a critical role during infection of the SHK-1 cell line in Psal-103. Repression oftended to reduce cytopathogenicity in both strains, while repression ofwas associated with a modest delay in cytopathic damage in Psal-104b. Repression ofandcaused moderate reductions in cytopathic activity in Psal-103. By contrast,showed no detectable phenotypes. Together, our results indicate that despite the irreversible interruption of,has retained NT-related genes that contribute to fitness in a gene- and genogroup-specific manner, providing a framework to investigate how determinants of horizontal gene transfer function beyond DNA uptake.IMPORTANCEDespite its major impact on salmon aquaculture,remains poorly characterized at the functional level, largely due to long-standing limitations in genetic tractability. Here, we implement and combine multiple genetic approaches, including CRISPR interference, site-specific chromosomal integration, and heterologous gene expression, to functionally interrogate natural transformation (NT)-related genes in this pathogen. Using representative strains from the two most prevalent Chilean genogroups, we show that conserved NT-associated genes contribute to bacterial physiology and infection in a genogroup-dependent manner. Moreover, and beyond the specific biological findings, this work establishes a versatile genetic platform for functional studies in, expanding the experimental toolbox available to study this pathogen and supporting future efforts aimed at understanding its biology and the development of novel biotechnological approaches.