Complete sequence of the three plasmid complement of Pseudomonas syringae pv. savastanoi NCPPB 3335. savastanoi_plasmids

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules (pPsv48A, pPsv48B and pPsv48C), all belonging to the pPT23A-like family of plasmids (PFP) widely distributed in the P. syringae complex. Plasmid pPsv48A contains an incomplete Type IVB secretion system, the type III secretion system (T3SS) effector gene hopAF1, gene ptz, involved in cytokinin biosynthesis, and three copies of a gene highly conserved in plant-associated proteobacteria, which is preceded by a hrp box motif. A complete Type IVA secretion system, a well conserved origin of transfer (oriT), and a homolog of the T3SS effector gene hopAO1 are present in pPsv48B, while pPsv48C contains a gene with significant homology to isopentenyl-diphosphate delta-isomerase, type 1. Several potential mobile elements were found on the three plasmids, including three types of MITE, a derivative of IS801, and a new transposon effector, ISPsy30. Although the replication regions of these three plasmids are phylogenetically closely related, their structure is diverse, suggesting that the plasmid architecture results from an active exchange of sequences. Plasmid pPsv48C contains a second replicon, which is a chimera between the gene encoding a newly described replication protein and the putative replication control region preceeding gene repA from the widespread family of PFP virulence plasmids. Artificial inoculations of olive plants with mutants cured of plasmids pPsv48A and pPsv48B showed that pPsv48A is necessary for full virulence and for the development of mature xylem vessels within the knots; we were unable to obtain mutants cured of pPsv48C, which contains five putative toxin-antitoxin gene.

橄榄假单胞菌 savastanoi 致病型 NCPPB 3335（Pseudomonas savastanoi pv. savastanoi NCPPB 3335）是研究木本宿主病害发生与肿瘤形成分子机制的模式菌株，其草图基因组序列已于近期完成测定。本研究完成了该菌株质粒组的序列闭合组装，其质粒组包含三个环状DNA分子（pPsv48A、pPsv48B与pPsv48C），均隶属于广泛分布于丁香假单胞菌（P. syringae）复合群中的pPT23A类质粒家族（pPT23A-like family of plasmids, PFP）。
质粒pPsv48A携带有一套不完整的IVB型分泌系统、III型分泌系统（Type III Secretion System, T3SS）效应基因hopAF1、参与细胞分裂素生物合成的ptz基因，以及三个在植物相关变形菌中高度保守的基因拷贝，上述基因的上游均带有hrp框基序。
pPsv48B则携带有一套完整的IVA型分泌系统、一个高度保守的转移起始区（origin of transfer, oriT），以及III型分泌系统效应基因hopAO1的同源基因；而pPsv48C携带一个与1型异戊烯基二磷酸Δ-异构酶具有显著同源性的基因。
在这三个质粒上共发现多种潜在可移动元件，包括三类微型反向重复转座子（Miniature Inverted-repeat Transposable Elements, MITE）、一种插入序列IS801（Insertion Sequence 801, IS801）的衍生元件，以及一类新型转座子效应元件ISPsy30。尽管这三个质粒的复制区域在系统发育上紧密相关，但其结构存在多样性，表明该质粒的架构源自频繁的序列交换事件。
质粒pPsv48C还携带第二个复制子，该复制子为嵌合结构，由一个新鉴定的复制蛋白编码基因与广泛分布的PFP致病性质粒的推定复制控制区（位于repA基因上游区域）嵌合而成。
对移除pPsv48A与pPsv48B质粒的突变株开展橄榄植株人工接种试验表明，pPsv48A是菌株实现完全致病力以及在瘤结内形成成熟木质部导管所必需的；我们未成功获得移除pPsv48C的突变株，该质粒携带有五个推定的毒素-抗毒素基因。