Oxygen-responsive KoBdcA/KoBdcB/KoBpdA system regulates c-di-GMP levels to control bacterial cellulose biosynthesis and motility in <i>Kosakonia oryzendophytica</i> FY-07

Cyclic di-GMP (c-di-GMP) acts as a bacterial second messenger that regulates various physiological processes, including biofilm formation, motility, and virulence. It is synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs), with precise regulatory effects typically requiring the coordinated action of multiple DGCs and PDEs. <i>Kosakonia oryzendophytica</i> FY-07 (FY-07) exhibits high bacterial cellulose (BC) yield, rapid growth, and robustness and can produce BC under varying oxygen conditions; however, the underlying mechanism driving the phenotypic transition from cellulose-producing to motile states during fermentation has yet to be elucidated. In this study, we aimed to identify two DGCs, KoBdcA and KoBdcB, and one PDE, KoBpdA, whose encoding genes are located adjacent to the cellulose synthesis operon in FY-07. KoBdcA and KoBpdA form an antagonistic DGC-PDE pair that maintains intracellular c-di-GMP homeostasis. Deletion of either gene alters BC production and motility. In contrast, KoBdcB acts as an auxiliary component, and its deletion alone causes no phenotypic changes. KoBdcA regulates BC synthesis via an indirect interaction with BcsA mediated by KoBdcB. The Per-Arnt-Sim (PAS) domains of KoBdcA and KoBpdA bind hemin, enabling them to sense oxygen and modulate their enzymatic activities. Elevated DGC activity during early fermentation promotes BC production, while increased PDE activity later suppresses BC synthesis, shifting cells to a motile state.IMPORTANCEIn this study, we provide novel mechanistic insights into how synergistic interactions among multiple DGCs and PDEs, in response to oxygen sensing, control the dynamic switch between the sessile and motile states of biofilm-producing bacteria. The study findings provide theoretical insights into the localized regulation of c-di-GMP signaling in <i>K. oryzendophytica</i> and have significant implications for elucidating the metabolic regulatory network of c-di-GMP. These findings also provide valuable insights into the molecular interactions and mechanisms governing the association between <i>Kosakonia</i> species and their host plants.