Biofilm formation is important for infection by many pathogens. <i>Bordetella bronchiseptica</i> causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in <i>B. bronchiseptica</i>. In the present work, based on their previously reported function in <i>Pseudomonas fluorescens</i>, we identified three genes in the <i>B. bronchiseptica</i> genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. <i>In vitro</i> and <i>in vivo</i> studies showed that the protease LapG of <i>B. bronchiseptica</i> cleaves the N-terminal domain of BrtA, as well as the LapA protein of <i>P. fluorescens</i>, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a <i>B. bronchiseptica</i> strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by <i>B. bronchiseptica</i>.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecular