Genome degradation in the c-di-GMP signaling pathways and serovar-specific YhjH-YciG interactions rewired motility regulation in Salmonella Paratyphi A

Salmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause clinically distinct diseases, yet the molecular bases for their different lifestyles remain incompletely understood. Genome degradation, a hallmark of typhoidal Salmonella, results in extensive pseudogenization across multiple functional pathways. Here, we investigate how such gene inactivation rewires cyclic-di-GMP (c-di-GMP) signaling and flagellar motility regulation in SPA vs. STM. We show that YhjH, a conserved phosphodiesterase (PDE), is required for motility in STM but not in SPA, despite retaining PDE activity in both serovars. We demonstrate that this functional divergence is caused by pseudogenization of ycgR in SPA, which truncates the flagellar brake protein YcgR to a nonfunctional peptide, severing the link between c-di-GMP levels and flagellar motor control. Site directed mutagenesis in the YhjH active site and ectopic expression of intact YcgR from STM that restored YhjH-dependent motility regulation in SPA, confirmed this molecular mechanism. Additionally, using a bacterial two-hybrid (BACTH) genetic screen, we identified a serovar-specific interaction between YhjH and the general stress protein YciG in SPA, but not in STM. Computational RNA folding analysis predicted substantial differences in mRNA secondary structure and stability between the SPA and STM yhjH alleles, suggesting a potential role for synonymous mutations in this serovar-specific interaction. Together, these findings reveal how genome degradation can rewire regulatory networks, uncovering a fundamental difference in motility control between typhoidal and non-typhoidal Salmonella and suggest that these differences allow SPA motility under conditions that suppress motility in STM. IMPORTANCESalmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause fundamentally different diseases in humans, yet the molecular basis for their distinct lifestyles and pathogenicity remains poorly understood. Genome degradation is a hallmark of typhoidal Salmonella, but its functional consequences for regulatory networks are largely unexplored. Here, we demonstrate that pseudogenization of ycgR in SPA dismantles c-di-GMP-mediated flagellar motor control, liberating SPA from an inhibitory brake that suppresses motility in STM. Additionally, we uncover a serovar-specific interaction between the phosphodiesterase YhjH and the general stress protein YciG in SPA, demonstrating that genome degradation can drive regulatory network rewiring beyond simple gene loss. These differences in motility regulation may facilitate the systemic pathogenesis and unique lifestyle of SPA.