BACKGROUND Pseudomonas syringae is a γ-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems. RESULTS The three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10- 3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10- 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo. CONCLUSIONS Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.

中文翻译：

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毒性守护者-多重毒素-抗毒素系统提供稳定性、避免缺失和维持丁香假单胞菌毒力质粒的毒力基因。

背景技术 丁香假单胞菌是一种γ-变形菌，在几乎所有栽培植物中引起经济相关疾病。这种病原体的大多数分离株含有天然质粒，共同携带许多致病性和毒力基因。然而，丁香假单胞菌通常是一种机会性病原体，主要栖息在环境宿主中，对毒力质粒的选择压力较低。此外，这些质粒通常包含大部分重复序列，这可能会损害质粒的完整性。因此，鉴定质粒稳定性决定因素和保存毒力基因的机制对于了解这种病原体的进化及其对农业生态系统的适应性至关重要。结果 丁香假单胞菌的三个毒力质粒。savastanoi NCPPB 3335 包含一到七个功能稳定性决定因素，包括 pPsv48A 和 pPsv48C 中的三个高活性毒素-抗毒素系统 (TA)。TA 系统将 pPsv48A 的丢失频率降低了两个数量级，而 pPsv48C 的两个复制子之一可能自身具有稳定的遗传。值得注意的是，来自 pPsv48C 的 TA 系统的失活使质粒暴露于由移动遗传元件促进的高频缺失。因此，MITEPsy2 的两个拷贝之间的重组导致一个 8.3 kb 片段的缺失，频率为 3.8 ± 0.3 × 10-3。同样，IS801 的单端转座产生包含可变大小缺失的质粒，频率为 5.5 ± 2.1 × 10- 4，其中 80% 失去了毒力基因 idi。这些缺失衍生物通过与 IS801 相邻的 repJ 介导的复制在群体中稳定维持。IS801 还通过重组或单端转座促进质粒 pPsv48A 中的缺失。在所有情况下，功能性 TA 系统显着有助于减少体内质粒缺失的发生。结论 来自丁香假单胞菌的毒力质粒具有多种稳定性决定因素，对质粒持久性的贡献各不相同。重要的是，我们证明了多个质粒携带的 TA 系统在保持质粒完整性和确保在自由生存条件下维持毒力基因方面具有重要作用。这种策略可能在丁香假单胞菌和其他细菌的天然质粒中普遍存在。IS801 还通过重组或单端转座促进质粒 pPsv48A 中的缺失。在所有情况下，功能性 TA 系统显着有助于减少体内质粒缺失的发生。结论 来自丁香假单胞菌的毒力质粒具有多种稳定性决定因素，对质粒持久性的贡献各不相同。重要的是，我们证明了多个质粒携带的 TA 系统在保持质粒完整性和确保在自由生存条件下维持毒力基因方面具有重要作用。这种策略可能在丁香假单胞菌和其他细菌的天然质粒中普遍存在。IS801 还通过重组或单端转座促进质粒 pPsv48A 中的缺失。在所有情况下，功能性 TA 系统显着有助于减少体内质粒缺失的发生。结论 来自丁香假单胞菌的毒力质粒具有多种稳定性决定因素，对质粒持久性的贡献各不相同。重要的是，我们证明了多个质粒携带的 TA 系统在保持质粒完整性和确保在自由生存条件下维持毒力基因方面具有重要作用。这种策略可能在丁香假单胞菌和其他细菌的天然质粒中普遍存在。结论 来自丁香假单胞菌的毒力质粒具有多种稳定性决定因素，对质粒持久性的贡献各不相同。重要的是，我们证明了多个质粒携带的 TA 系统在保持质粒完整性和确保在自由生存条件下维持毒力基因方面具有重要作用。这种策略可能在丁香假单胞菌和其他细菌的天然质粒中普遍存在。结论 来自丁香假单胞菌的毒力质粒具有多种稳定性决定因素，对质粒持久性的贡献各不相同。重要的是，我们证明了多个质粒携带的 TA 系统在保持质粒完整性和确保在自由生存条件下维持毒力基因方面具有重要作用。这种策略可能在丁香假单胞菌和其他细菌的天然质粒中普遍存在。