Wang, H.; Gu, Y.; He, W.; Yang, J.; Liang, H.; Wang, M.; Wu, Z.; Wen, Y.; Wang, J.; Rao, X.; Fan, Y.; Ma, J.; Yang, X.; Tong, X.; Yang, L.; Xu, Y.-s.; Zhao, J.; Zeng, T.; Zhang, Y.; Zhong, Y.; Zhang, H.; Liu, C.; Shen, X.; Kristiansen, K.; Yang, J.; Liu, F.; Yang, Z.; Li, W.; Gao, P.; Xu, J.; Li, S.; He, N.; Wang, B.; Jin, X.; Liu, C.; Xu, X.; Dong, Y.; Zhou, H.; Zhao, F.; Xiao, L.; Zou, Y.

Cultivated bacterial isolates are essential for elucidating gut microbiota functions, yet reliance on fragmented draft genomes and limited mobile genetic element (MGE) annotation has constrained high-resolution analyses. Here, we present the Cultivated Complete Genome Reference (CCGR), a comprehensive compendium of 1,150 fully circularized human gut bacterial genomes. Overcoming draft limitations, CCGR achieves chromosome-scale resolution and reveals distinct evolutionary strategies shaping gene topological organization in response to bacterial growth. Furthermore, we decode the dynamic landscape of MGEs, demonstrating that broad-host-range phages follow strict positional patterns by integrating at conserved chromosomal hotspots flanked by nutrient transporter loci. Importantly, we provide mechanistic in vivo evidence that accessory genomic elements drive strain-level functional heterogeneity. Specifically, the plasmid-encoded scrK gene in Levilactobacillus brevis involved in fructose catabolism is required to mitigate fructose-diet-exacerbated colitis. Collectively, CCGR establishes a framework linking chromosomal architecture and MGEs to bacterial evolution and host health, enabling precise, strain-resolved functional investigations.