Genomic and functional dissection of natural transformation-related genes in <i>Piscirickettsia salmonis</i>

<i>Piscirickettsia salmonis,</i> the etiological agent of piscirickettsiosis, represents the main health challenge for Chilean salmon farming and an emerging threat to global salmonid production. Inspection of all <i>P. salmonis</i> genomes available shows that the <i>comEC</i> gene<i>,</i> encoding the DNA uptake channel required for natural transformation (NT) in competent bacteria, is interrupted by various transposable elements, whereas other NT determinants (<i>comEA</i>, <i>comFB</i>, <i>comM</i>, <i>comL/bamD</i>, and <i>dprA</i>) remain intact and highly conserved. Using <i>P. salmonis</i> Psal-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 of <i>comEC</i> using homologs from naturally competent bacteria, and CRISPRi-mediated knockdown of the remaining NT-related genes to examine their functionality. According to our results, expression of <i>comEC</i> with homologs from <i>Legionella pneumophila</i> and <i>Vibrio cholerae</i> impaired <i>P. salmonis</i> viability in axenic culture, indicating a fitness burden. CRISPRi-mediated repression of <i>comL/bamD</i> revealed its essentiality in Psal-104b and a critical role during infection of the SHK-1 cell line in Psal-103. Repression of <i>comM</i> tended to reduce cytopathogenicity in both strains, while repression of <i>comFB</i> was associated with a modest delay in cytopathic damage in Psal-104b. Repression of <i>recA</i> and <i>comEA</i> caused moderate reductions in cytopathic activity in Psal-103. By contrast, <i>dprA</i> showed no detectable phenotypes. Together, our results indicate that despite the irreversible interruption of <i>comEC</i>, <i>P. salmonis</i> 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, <i>Piscirickettsia salmonis</i> 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 <i>P. salmonis</i>, 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.