Essential genes are crucial for bacterial growth and the pathogenicity. However, how gene essentiality varies under distinct growth conditions and its role in virulence remains poorly understood. In this study, we constructed high-saturation Tn5 transposon mutant libraries for Xanthomonas citri subsp. citri (Xac), the causative agent of citrus canker disease, which results in substantial economic losses to the citrus industry. These libraries were generated under two growth conditions: nutrient-rich medium (NB) and plant-mimicking medium (XVM2). The libraries achieved an average insertion density of 6.85 base pairs, ensuring high-quality coverage. Using these libraries, we identified 568 genes in both bacterial populations as essential genes of Xac, while the 61 (NB) and 54 (XVM2) as conditional essential genes. To validate the findings, two allele-replacement mutants, ∆ublA and ∆pgl, were generated based on variations in insertion density and gap ratios between two growth conditions. These mutants exhibited significant growth defects in XVM2 and compromised virulence in citrus plants, as evidenced by weaker canker symptoms and reduced bacterial populations in planta. Additionally, we performed a comparative analysis of essential gene conservation across the Xanthomonas genus and other species within the class Gammaproteobacteria. A pan-essentialome dataset was assembled from four Xanthomonas strains, revealing a broad pan-essentialome and a precise core-essentialome. To facilitate further research, we developed XanthoBrowser (http://xacdb.lumoxuan.cn/), an online resource that provides user-friendly interface for searching and comparison of essential genes across four Xanthomonas strains. In conclusion, this study provides comprehensive insights into bacterial adaptation mechanisms and highlight potential targets for bactericide development.

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