Xylella fastidiosa (Xf), a xylem-limited insect-vectored pathogenic bacterium, is the causative agent of devastating plant diseases in many economically important crops in the Americas, Europe and Asia. Diseases caused by this pathogen are associated with the blockage of the host xylem system by biofilm formation, as well as twitching motility. Previous studies of our group found that the mineral nutrient calcium can affect these traits in Xf, but mechanisms involved in these processes are poorly understood, especially molecular mechanisms responsible for sensing environmental Ca. We focused on two component regulatory systems, which are important for bacteria to sense and respond to environmental signals. In silico, we found that PD0576, a histidine kinase and response regulator hybrid protein, had two putative Ca-dependent phosphotriesterase domains which are located in the N terminal sensor part. Taking the advantage of Xf natural competence, a novel gene knockout protocol was used to construct a gene deletion mutant. The gene deletion did not affect the growth of Xf, but the mutation increased twitching motility of the WT strain WM1-1 on PW plates, and decreased cell to cell aggregation and biofilm formation by WM1-1 under flow conditions in microfluidic chambers. We proposed that PD0576 in Xf may sense the environmental Ca to regulate switching from planktonic to biofilm growth. Further characterization of this mutation is ongoing.