Biochemical and structural insights of a new endolysin encoded by the Xanthomonas citri lytic phage CP2.

The Gram-negative bacterium Xanthomonas citri subsp. citri (X. citri) is a phytopathogen causal agent of citrus canker, a severe disease affecting citrus crops worldwide. Disease control relies primarily on copper biocides, which pose toxicity risks to humans and the environment. Endolysins are enzymes that catalyze bacterial cell wall degradation and could serve as environmentally sustainable alternatives for the control of X. citri. Here, we conducted structural in silico studies with the endolysin CP2-07 encoded by the X. citri lytic phage CP2, followed by its heterologous expression and purification for biochemical characterization. Structural analysis revealed that CP2-07 is a globular endolysin that putatively lacks the typical β-hairpin motif found in other lysozymes, while retaining conserved residues in its catalytic cleft. This new muramidase exhibited activity against purified peptidoglycan extracted from X. citri, demonstrating good thermostability (Tm of 52 °C), activity across a broad pH range (7-10), and optimal performance in the presence of 60 mM NaCl. Furthermore, CP2-07 displayed bactericidal action against X. citri at 100 μg/mL and effectively promoted bacterial cell lysis during the mid-log phase, but only in the presence of EDTA (a membrane permeabilizer). This study lays the groundwork for exploring and developing novel biocatalysis-based strategies for controlling economically significant bacterial phytopathogens.