Cooperative mechanisms of LexA and HtpG in the regulation of virulence gene expression in Pseudomonas plecoglossicida

LexA is a well-known transcriptional repressor of DNA repair genes induced by DNA damage in Escherichia coli and other bacterial species. Recently, this paradigm—that LexA solely regulates the SOS response—has been challenged as studies reveal its involvement in various biological functions linked to virulence. Pseudomonas plecoglossicida, a major pathogen in mariculture, causes substantial economic losses annually in China. Our previous research suggested that LexA might collaboratively regulate virulence gene expression with HtpG during infection. This study aims to elucidate the molecular mechanism by which LexA controls virulence gene expression. We employed an array of methods including molecular dynamics simulations, molecular docking, ChIP-seq, RNA-seq, mass spectrometry, gene mutagenesis, LacZ reporter assays, electrophoretic mobility shift assays, co-immunoprecipitation, and in vitro LexA degradation experiments. Our findings identified 36 downstream virulence genes regulated by LexA, define three critical LexA binding motifs, and provide an in-depth analysis of LexA's recognition and binding to promoters, thereby regulating virulence gene expression. Additionally, we confirm the cooperative regulatory roles of HtpG, RecA, and LexA in virulence gene modulation. This is the first report of an endogenous accessory factor aiding in the binding of LexA to DNA. This study enhances our understanding of LexA's role in virulence regulation and offers a valuable theoretical and practical foundation for disease prevention and control.