Genomic analysis of eight native plasmids of the phytopathogen Pseudomonas syringae

Comunicación a conferenciaThe pPT23A family of plasmids (PFPs) appears to be indigenous to the plant pathogen Pseudomonas syringae and these plasmids are widely distributed and widely transferred among pathovars of P. syringae and related species. PFPs are sources of accessory genes for their hosts that can include genes important for virulence and epiphytic colonization of plant leaf surfaces. Further understanding of the evolution of the pPT23A plasmid family and the role of these plasmids in P. syringae biology and pathogenesis, requires the determination and analysis of additional complete, closed plasmid genome sequences. Therefore, our main objective was to obtain complete genome sequences from PFPs from three different P. syringae pathovars and perform a comparative genomic analysis. In this work plasmid DNA isolation, purification by CsCl-EtBr gradients, and sequencing using 454 platform, were used to obtain the complete sequence of P. syringae plasmids. Different bioinformatic tools were used to analyze the plasmid synteny, to identify virulence genes (i.e. type 3 effectors) and to unravel the evolutionary history of PFPs. Our sequence analysis revealed that PFPs from P. syringae encode suites of accessory genes that are selected at different levels (universal, interpathovar and intrapathovar). The conservation of type IVSS encoding conjugation functions also contributes to the distribution of these plasmids within P. syringae populations. Thus, this study contributes to unravel the genetic basis of the role of PFPs in different P. syringae lifestyles.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

HrcU and HrpP are pathogenicity factors in the fire blight pathogen Erwinia amylovora required for the type III secretion of DspA/E

Table S1. Description of data: Sequences of oligonucleotide primers used in this study. (DOCX 109 kb

Comparative genomic analysis of native pseudomonas syringae plasmids belonging to the ppt23 a family reveals their role in p. Syringae epiphytic and pathogenic lifestyles

Backgrounds The pPT23A family of plasmids (PFPs) appears to be indigenous to the plant pathogen Pseudomonas syringae and these plasmids are widely distributed and widely transferred among pathovars of P. syringae and related species. PFPs are sources of accessory genes for their hosts that can include genes important for virulence and epiphytic colonization of plant leaf surfaces. Objectives Further understanding of the evolution of the pPT23A plasmid family and the role of these plasmids in P. syringae biology and pathogenesis, requires the determination and analysis of additional complete, closed plasmid genome sequences. Therefore, our main objective was to obtain complete genome sequences of PFPs from three different P. syringae pathovars and perform a comprehensive comparative genomic analysis. Methods In this work plasmid DNA isolation, purification by CsCl-EtBr gradients, and sequencing using 454 platform, were carried out to obtain the complete sequence of P. syringae plasmids. Different bioinformatic tools were used to analyze the plasmid synteny, to identify virulence genes (i.e. type 3 effectors) and to unravel the evolutionary history of PFPs. Conclusions Our sequence analysis revealed that PFPs from P. syringae encode suites of accessory genes that are selected at different levels (universal, interpathovar and intrapathovar). The conservation of type IVSS encoding conjugation functions also contributes to the distribution of these plasmids within P. syringae populations. Thus, this study contributes to unravel the genetic bases of the role of PFPs in different P. syringae lifestyles. This work was supported by grants Proyecto de Excelencia, Junta de Andalucía (P07-AGR-02471; P12-AGR-1473) and by Michigan State University AgBioResearch.This work was supported by grants Proyecto de Excelencia, Junta de Andalucía (P07-AGR-02471; P12-AGR-1473) and by Michigan State University AgBioResearch; Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Systems level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility

<p>Abstract</p> <p>Background</p> <p>Two-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in <it>Erwinia amylovora </it>have either not been identified or have not yet been studied.</p> <p>Results</p> <p>We used a systems approach to identify TCST and related signal transduction genes in the genome of <it>E. amylovora</it>. Comparative genomic analysis of TCSTs indicated that <it>E. amylovora </it>TCSTs were closely related to those of <it>Erwinia tasmaniensis</it>, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in <it>E. amylovora </it>including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in <it>E. amylovora </it>control amylovoran biosynthesis, one of two major virulence factors in <it>E. amylovora</it>. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in <it>E. amylovora </it>were also identified.</p> <p>Conclusion</p> <p>Our results demonstrated that TCSTs in <it>E. amylovora </it>played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in <it>E. amylovora</it>.</p

Physiological and Microscopic Characterization of Cyclic-di-GMP-Mediated Autoaggregation in Erwinia amylovora

The second messenger cyclic-di-GMP (c-di-GMP) is a critical regulator of biofilm formation in the plant pathogen Erwinia amylovora. Phosphodiesterase (PDE) enzymes are responsible for the degradation of intracellular c-di-GMP. Previously, we found that the deletion of one or more of the three PDE enzyme encoding genes (pdeA, pdeB, and pdeC) in E. amylovora Ea1189 led to an increase in biofilm formation. However, in mutants Ea1189ΔpdeAC and Ea1189ΔpdeABC, biofilm formation was reduced compared to the other single and double deletion mutants. Here, we attribute this to an autoaggregation phenotype observed in these two mutants. Examination of Ea1189ΔpdeABC cellular aggregates using scanning electron microscopy indicated that a subset of cells were impaired in cell separation post cell division. Concomitant with this phenotype, Ea1189ΔpdeABC also exhibited increased transcription of the cell-division inhibitor gene sulA and reduced transcription of ftsZ. Ea1189ΔpdeABC showed a significant reduction in biofilm formation, and biofilms formed by Ea1189ΔpdeABC exhibited a distinctive morphology of sparsely scattered aggregates rather than an evenly distributed biofilm as observed in WT Ea1189. Our results suggest that highly elevated levels of c-di-GMP lead to increased cell–cell interactions that contribute to autoaggregation and impair cell-surface interaction, negatively affecting biofilm formation

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

Growth Parameter Components of Adaptive Specificity during Experimental Evolution of the UVR-Inducible Mutator Pseudomonas cichorii 302959

BACKGROUND: Mutagenic DNA repair (MDR) transiently increases mutation rate through the activation of low-fidelity repair polymerases in response to specific, DNA-damaging environmental stress conditions such as ultraviolet radiation (UVR) exposure. These repair polymerases also confer UVR tolerance, intimately linking mutability and survival in bacteria that colone habitats subject to regular UVR exposure. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate adaptive specificity in experimental lineages of the highly UVR-mutable epiphytic plant pathogen Pseudomonas cichorii 302959. Relative fitness measurements of isolates and population samples from replicate lineages indicated that adaptive improvements emerged early in all lineages of our evolution experiment and specific increases in relative fitness correlated with distinct improvements in doubling and lag times. Adaptive improvements gained under UVR and non-UVR conditions were acquired preferentially, and differentially contributed to relative fitness under varied growth conditions. CONCLUSIONS: These results support our earlier observations that MDR activation may contribute to gains in relative fitness without impeding normal patterns of adaptive specificity in P. cichorii 302959

Development of a Method to Monitor Gene Expression in Single Bacterial Cells During the Interaction With Plants and Use to Study the Expression of the Type III Secretion System in Single Cells of Dickeya dadantii in Potato

Dickeya dadantii is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS) is an important virulence factor in D. dadantii. Expression of the T3SS is induced in the plant apoplast or in hrp-inducing minimal medium (hrp-MM), and is repressed in nutrient-rich media. Despite the understanding of induction conditions, how individual cells in a clonal bacterial population respond to these conditions and modulate T3SS expression is not well understood. In our previous study, we reported that in a clonal population, only a small proportion of bacteria highly expressed T3SS genes while the majority of the population did not express T3SS genes under hrp-MM condition. In this study, we developed a method that enabled in situ observation and quantification of gene expression in single bacterial cells in planta. Using this technique, we observed that the expression of the T3SS genes hrpA and hrpN is restricted to a small proportion of D. dadantii cells during the infection of potato. We also report that the expression of T3SS genes is higher at early stages of infection compared to later stages. This expression modulation is achieved through adjusting the ratio of T3SS ON and T3SS OFF cells and the expression intensity of T3SS ON cells. Our findings not only shed light into how bacteria use a bi-stable gene expression manner to modulate an important virulence factor, but also provide a useful tool to study gene expression in individual bacterial cells in planta