Abstract
Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to epidemic strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovers that PdcB, a phosphodiesterase, enhances sporulation in C. difficile strain UK1. Through genetic and biochemical assays, we show that phase-variable expression of pdcB results in hypo- and hyper-sporulation phenotypes. In the "ON" orientation, the identified promotor is in the right orientation to drive the expression of pdcB. Production of the PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP (c-di-GMP) concentration, resulting in a hyper-sporulation phenotype. Loss of PdcB due to the pdcB promoter being in the OFF orientation or mutation of pdcB results in increased c-di-GMP levels and a hypo-sporulation phenotype. Additionally, we demonstrate that CodY binds to the upstream region of pdcB. DNA inversion reorients the CodY binding site so that in the OFF orientation, CodY binds a site that is upstream of the pdcB promoter and can further repress gene expression.