Isabel Pérez-Martínez

Global Genomic Analysis of Pseudomonas savastanoi pv. savastanoi Plasmids

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.

Fate of a Pseudomonas savastanoi pv. savastanoi Type III Secretion System Mutant in Olive Plants (Olea europaea L.)

ABSTRACT Pseudomonas savastanoi pv. savastanoi strain NCPPB 3335 is a model bacterial pathogen for studying the molecular basis of disease production in woody hosts. We report the sequencing of the hrpS-to-hrpZ region of NCPPB 3335, which has allowed us to determine the phylogenetic position of this pathogen with respect to previously sequenced Pseudomonas syringae hrp clusters. In addition, we constructed a mutant of NCPPB 3335, termed T3, which carries a deletion from the 3′ end of the hrpS gene to the 5′ end of the hrpZ operon. Despite its inability to multiply in olive tissues and to induce tumor formation in woody olive plants, P. savastanoi pv. savastanoi T3 can induce knot formation on young micropropagated olive plants. However, the necrosis and formation of internal open cavities previously reported in knots induced by the wild-type strain were not observed in those induced by P. savastanoi pv. savastanoi T3. Tagging of P. savastanoi pv. savastanoi T3 with green fluorescent protein (GFP) allowed real-time monitoring of its behavior on olive plants. In olive plant tissues, the wild-type strain formed aggregates that colonized the intercellular spaces and internal cavities of the hypertrophic knots, while the mutant T3 strain showed a disorganized distribution within the parenchyma of the knot. Ultrastructural analysis of knot sections revealed the release of extensive outer membrane vesicles from the bacterial cell surface of the P. savastanoi pv. savastanoi T3 mutant, while the wild-type strain exhibited very few vesicles. This phenomenon has not been described before for any other bacterial phytopathogen during host infection.

Sequence and Role in Virulence of the Three Plasmid Complement of the Model Tumor-Inducing Bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335

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 of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex. A total of 152 coding sequences were predicted in the plasmid complement, of which 38 are hypothetical proteins and seven correspond to putative virulence genes. 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. 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 genes.

Pseudomonas savastanoi pv. savastanoi Contains Two iaaL Paralogs, One of Which Exhibits a Variable Number of a Trinucleotide (TAC) Tandem Repeat†

ABSTRACT In this study, Pseudomonas savastanoi pv. savastanoi isolates were demonstrated to contain two iaaL paralogs, which are both chromosomally located in most strains. Comparative analysis of iaaL nucleotide sequences amplified from these two paralogs revealed that one paralog, iaaLPsn, is 100% identical to iaaL from P. savastanoi pv. nerii, while the other paralog, iaaLPsv, exhibited 93% identity to iaaL from Pseudomonas syringae pv. tomato (iaaLPto). A 3-nucleotide motif (TAC) comprised of 3 to 15 repeats, which remained stable after propagation of the strains in olive plants, was found in iaaLPsv. Based on the observed nucleotide sequence variations, a restriction fragment length polymorphism assay was developed that allowed differentiation among iaaLPsn, iaaLPsv, and iaaLPto. In addition, reverse transcriptase PCR on total RNA from P. savastanoi pv. savastanoi strains demonstrated that both iaaLPsv and iaaLPsn containing 14 or fewer TAC repeats are transcribed. Capillary electrophoresis analysis of PCR-amplified DNA fragments containing the TAC repeats from iaaLPsv allowed the differentiation of P. savastanoi pv. savastanoi isolates.

IS53: an insertion element for molecular typing of Pseudomonas savastanoi pv. savastanoi

A worldwide collection of Pseudomonas savastanoi pv. savastanoi strains from olive knots was examined for the distribution, variation in position and copy numbers of the IS53 insertion element. Southern hybridization analysis of plasmid DNA from six olive strains using IS53 and repA probes revealed that this insertion element was present in the chromosomal replicon and not in a plasmid, as had been originally described in an oleander strain. Southern hybridization analysis also revealed that IS53 was present in multiple copies in all analyzed strains. Copy numbers of IS53 elements ranged from 4 up to 10. Although all strains displayed a remarkably high degree of restriction fragment length polymorphism, we demonstrated that transposition of this element is extremely rare in bacteria grown in vitro for up to 390 generations. The genetic diversity of 62 olive strains based on 47 different IS53 RFLP fingerprints and UPGMA analysis enabled all strains to be clustered into eight groups with 60% similarity. IS53 typing can be considered a suitable marker for epidemiological and ecological studies, given its widespread distribution on P. savastanoi pv. savastanoi olive populations, its high stability and the high degree of polymorphism generated.