Diversification of the Type VI Secretion System in Agrobacteria

Abstract

The type VI secretion system (T6SS) is used by many Gram-negative bacteria to deploy toxic effectors for interbacterial competition. This system provides a competitive advantage in planta to agrobacteria, a diverse group with phytopatho-genic members capable of genetically transforming plants. To inform on the ecology and evolution of agrobacteria, we revealed processes that diversify their effector gene collections. From genome sequences of diverse strains, we identified T6SS loci, functionally validated associated effector genes for toxicity, and predicted genes ho-mologous to those that encode proteins known to interact with effectors. The gene loci were analyzed in a phylogenetic framework, and results show that strains of some species-level groups have different patterns of T6SS expression and are enriched in specific sets of T6SS loci. Findings also demonstrate that the modularity of T6SS loci and their associated genes engenders dynamicity, promoting reshuffling of entire loci, fragments therein, and domains to swap toxic effector genes across species. However, diversification is constrained by the need to maintain specific com-binations of gene subtypes, congruent with observations that certain genes function together to regulate T6SS loading and activation. Data are consistent with a scenario where species can acquire unique T6SS loci that are then reshuffled across the genus in a restricted manner to generate new combinations of effector genes. IMPORTANCE The T6SS is used by several taxa of Gram-negative bacteria to secrete toxic effector proteins to attack others. Diversification of effector collections shapes bacterial interactions and impacts the health of hosts and ecosystems in which bac-teria reside. We uncovered the diversity of T6SS loci across a genus of plant -associ-ated bacteria and show that diversification is driven by the acquisition of new loci and reshuffling among species. However, linkages between specific subtypes of genes need to be maintained to ensure that proteins whose interactions are neces-sary to activate the T6SS remain together. Results reveal how organization of gene loci and domain structure of genes provides flexibility to diversify under the con-straints imposed by the system. Findings inform on the evolution of a mechanism that influences bacterial communities.