Pantoea agglomerans T6SS effectors reside in hotspots with combinatorial antibacterial and antiviral arsenals

Gram-negative bacteria deploy type VI secretion systems (T6SSs) to mediate interbacterial competition. Although numerous T6SS effectors have been identified, their pan-genomic repertoires and evolutionary dynamics remain poorly understood. Here, we combine proteomics and comparative genomics to map the T6SS effector landscape across Pantoea agglomerans , a species that includes pathogenic and beneficial strains. We uncover an extensive pan-genomic arsenal in which most effectors are encoded outside the T6SS gene cluster, within dynamic hotspots distributed across the chromosome and megaplasmids. Analysis of these hotspots reveals combinatorial arrangements of shuffled genetic cargo. Remarkably, these loci act as versatile “genomic armories” that co-localize offensive antibacterial weapons with protective anti-phage defense systems. By investigating uncharacterized genes within these variable regions, we discovered and validated a novel T6SS effector and a previously unknown anti-phage defense system, named Juno. Collectively, our findings demonstrate that bacterial warfare arsenals are highly modular and reside in dynamic genomic hubs that alternate or combine interbacterial aggression with viral defense. This evolutionary association reveals a functional blurring between offensive and defensive strategies within the bacterial accessory genome. IMPORTANCE Bacteria inhabiting competitive environments must simultaneously contend with rival bacteria and bacteriophage predators. Here, we show that Pantoea agglomerans , a species with both pathogenic and biocontrol lifestyles, meets this challenge through dynamic genomic loci we term “genomic armories.” By mapping the type VI secretion system (T6SS) secreted effector arsenal of P. agglomerans , we uncover the largest T6SS effector arsenal associated with a single T6SS described to date. Strikingly, nearly all effectors reside in variable genomic regions, most of which are hotspots that cycle through diverse genetic cargoes across strains. These hotspots combinatorially co-localize antibacterial T6SS effectors with anti-phage defense systems, providing bacteria with integrated offensive and defensive capabilities within a single locus. These findings reframe our understanding of how bacteria organize and diversify their arsenals, with implications for bacterial competition and phage–bacteria co-evolution.