第六型分泌系統在 Azorhizobium 與 Agrobacterium 之功能與機制研究

Abstract

The type VI secretion system (T6SS) is a protein secretion system composed of 13-14 core proteins, which structurally resembles an inverse phage tail. The T6SS spans the inner and outer membranes of a Gram-negative bacterium and can penetrate the membranes of the eukaryotic or prokaryotic recipient cells. The biological functions of T6SS are versatile, ranging from anti-eukaryotic activity, virulence, interbacterial competition, metal ion sequestering, and facilitating horizontal gene transfer, many of which display in animal-associated bacteria. Despite the existence in more than a quarter of the sequenced Gram-negative bacteria, the biological functions of T6SS in plant-associated bacteria, including mutualistic and pathogenic bacteria, remained mostly unknown. This study used a mutualistic bacterium, Azorbizobium caulinodans, and a pathogenic bacterium, Agrobacterium tumefaciens, to elucidate the functional and mechanistic aspects of T6SS in rhizobacteria. With the discovery of a T6SS gene cluster in A. caulinodans ORS571, a series of T6SS gene deletion mutants were generated, and their phenotypes were analyzed in free-living and symbiotic states. The results showed that whether the T6SS exists or not, there was no detectable effect on vegetative growth, morphology, free-living nitrogen-fixing ability, interbacterial competition, plant colonization, or symbiotic effectiveness. On the other hand, the strains lacking T6SS showed a reduction in the symbiotic competitiveness when co-infected with a wild-type strain on the stem of the leguminous host plant Sesbania rostrata. A. tumefaciens is a causative agent of crown gall disease in a wide range of plants and harbors T6SS. The T6SS of A. tumefaciens strain C58 is an antibacterial weapon capable of killing both inter- and intra-species bacteria. Thus, A. tumefaciens C58 is selected to study the mode of action of T6SS during interbacterial competition. While the molecular mechanisms and structural organization of the T6SS have been extensively studied, little was known about the recipient cell factor(s) needed or subverted for an attack to be successful. Thus, a high-throughput interbacterial competition screening platform was established in search of the recipient strains that were resistant to T6SS killing. A. tumefaciens strain C58 served as a model attacker, and the Escherichia coli devoid of T6SS served as a recipient cell. From the screening, 16 mutants with less susceptibility to A. tumefaciens C58 T6SS-dependent killing were identified. Among them, four genes (clpP, gltA, ydhS, ydaE) that participate in enhancing the recipient susceptibility to A. tumefaciens T6SS killing were confirmed. Further studies demonstrated that the ClpP protease and its adapter protein ClpA forming the ClpAP complex in the E. coli recipient cell act in enhancing the recipient’s susceptibility to the A. tumefaciens T6SS attack. In summary, this study discovered that the T6SS of legume symbiont A. caulinodans ORS571 functions to ensure the competitiveness of nodule occupancy and that multiple recipient factors are required to maximize T6SS killing efficiency of A. tumefaciens C58. The findings expand our understanding of T6SS in plant-associated bacteria, and the knowledge obtained could be applied to other T6SS harboring bacteria.