Abstract
<title>Abstract</title> <p> Background <italic>Pantoea agglomerans</italic> is a Gram-negative bacterium with remarkable ecological versatility, functioning as a plant symbiont, opportunistic pathogen, and environmental epiphyte. This adaptability is strongly influenced by horizontal gene transfer (HGT), yet its sources, vehicles, and key partners involved in these gene exchanges remain unclear. Results We conducted a multi-faceted genomic analysis of 165 <italic>P. agglomerans</italic> genomes that resulted in the construction of a pangenome that revealing an "open" structure driven by the continuous acquisition of shell and cloud genes enriched in mobile genetic element (MGE)-related functions. Comparative analysis of antimicrobial resistance (AMR) and virulence genes across chromosomes, plasmids, and prophages showed that plasmids, particularly non-ubiquitous ones, were the primary vehicles for horizontal AMR gene acquisition, while prophages contributed more modestly. Phylogenetic analysis of key gene clusters, including Pantocin A and the Type III Secretion System (T3SS), revealed both horizontal acquisition from other species and stable vertical inheritance within <italic>P. agglomerans</italic> . A large-scale gene-sharing network analysis across <italic>Enterobacterales</italic> identified <italic>Pantoea vagans</italic> as the most influential gene donor to <italic>P. agglomerans</italic> , with network structure revealing strong ecological niche-based clustering. Conclusions This study maps the landscape of HGT in <italic>P. agglomerans</italic> and highlights the combined roles of MGEs and ecological context in shaping genome evolution in this adaptable species. These findings provide deeper insight into the evolutionary forces driving diversification within <italic>P. agglomerans</italic> and establish a framework for studying HGT dynamics across other environmentally flexible bacteria. </p>