A structural backbone with sequestered plasticity organizes the Escherichia coli pangenome

The exponential growth of microbial genomic data has made computational scalability the primary bottleneck in pangenome analysis because traditional alignment-based methods have quadratic complexity. We developed CVNet, an alignment-free orthology inference framework that uses composition vectors and Markov clustering. CVNet achieves near-linear scalability and high accuracy, enabling pangenome analysis across thousands of genomes. Applying it to 1,200 complete Escherichia coli genomes, we moved beyond the "bag-of-genes" approach to investigate the pangenome's spatial architecture. By constructing a genome-wide core gene synteny network from CVNet orthogroups, we revealed that core genes form a modular (Q = 0.9851) and asymmetric structural backbone, strongly biased toward the replication origin (oriC; KS test, D = 0.9133). Accessory genes and genomic islands are non-randomly sequestered within specific integration hotspots, with over 99% located between core genes connected by extremely weak syntenic links. These findings establish a structural backbone and sequestered plasticity model, demonstrating how E. coli maintains chromosomal integrity through a rigid scaffold that physically compartmentalizes genetic plasticity. This study thus presents CVNet as a scalable computational solution and introduces a spatial paradigm for understanding how bacterial genomes balance evolutionary stability with adaptive flexibility.

Pangenome analysis has been constrained by alignment-based tools that do not scale and a "bag of genes" perspective that ignores chromosomal organization. We present CVNet, an alignment-free framework that enables near-linear scalability for orthology inference across thousands of genomes. Applying CVNet to 1,200 complete E. coli genomes, we discover that the chromosome is organized by a rigid core gene backbone, with accessory genes sequestered into discrete integration hotspots. This structural backbone and sequestered plasticity model reveals bacterial genomes as spatially organized systems in which stability and flexibility are physically compartmentalized, thereby establishing a framework for topological pangenomics.