Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids

Abstract

Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.

FIG. 1.

Representative plasmid macroarray hybridization images for a P. savastanoi pv. savastanoi PFP and n-PFP. A total of 135 genes were printed in duplicate on a 7.5- by 11.5-cm membrane. The position of each gene on the array was labeled as a combination of P (plate), C (column), and R (row). (A) Array hybridized with PFP pPsv31C from strain Psv31 containing the type IVA secretion system genes; (B) array hybridized with n-PFP pPsv62C from strain Psv62 containing type IVA and type IVB secretion system genes. Closed and open arrowheads point to spots containing water and chromosomal genes, respectively, used as negative controls. repA gene hybridization spots are boxed. Positions C2 R7 P3 and C5 R3 P3 correspond to ISPsy21 and IS801, respectively.

FIG. 2.

Detection of PFP plasmids in P. savastanoi pv. savastanoi strains. (A) Gel electrophoresis of native plasmids isolated from the indicated P. savastanoi pv. savastanoi strains (Psv29 [29] to Psv416 [416]). (B) Southern blot analysis of plasmid profiles shown in panel A using a digoxigenin-labeled repA probe from plasmid pPT23A (Table 2). Plasmids giving a negative hybridization signal (n-PFPs) are indicated by black arrows. Hybridization results were confirmed using the PFP DNA macroarray. The positions of molecular size markers (in kilobases) are indicated to the left of the gels. C, chromosomal DNA.

FIG. 3.

Detection of phytohormone biosynthetic genes (iaa and ptz) on P. savastanoi pv. savastanoi plasmids. (A and B) Gel electrophoresis of native plasmids isolated from the indicated P. savastanoi pv. savastanoi strains. (C and D) Southern blot analysis of plasmid profiles shown in panels A and B using iaaM and ptz probes, respectively. Southern blot analysis of plasmid profiles shown in panel A using iaaH and iaaL probes (Table 2) resulted in hybridization patterns identical to those in panel C. Plasmids giving positive hybridizations are indicated by white arrows. The positions of molecular size markers (in kilobases) are indicated to the left of the gels. C, chromosomal DNA.