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

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

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

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

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

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

Functional characterization of a global virulence regulator Hfq and identification of Hfq-dependent sRNAs in the plant pathogen Pantoea ananatis

To successfully infect plant hosts, the collective regulation of virulence factors in a bacterial pathogen is crucial. Hfq is an RNA chaperone protein that facilitates the small RNA (sRNA) regulation of global gene expression at the post-transcriptional level. In this study, the functional role of Hfq in a broad host range phytopathogen Pantoea ananatis was determined. Inactivation of the hfq gene in P. ananatis LMG 2665T resulted in the loss of pathogenicity and motility. In addition, there was a significant reduction of quorum sensing signal molecule acyl-homoserine lactone (AHL) production and biofilm formation. Differential sRNA expression analysis between the hfq mutant and wild-type strains of P. ananatis revealed 276 sRNAs affected in their abundance by the loss of hfq at low (OD600 = 0.2) and high cell (OD600 = 0.6) densities. Further analysis identified 25 Hfq-dependent sRNAs, all showing a predicted Rho-independent terminator of transcription and mapping within intergenic regions of the P. ananatis genome. These included known sRNAs such as ArcZ, FnrS, GlmZ, RprA, RyeB, RyhB, RyhB2, Spot42, and SsrA, and 16 novel P. ananatis sRNAs. The current study demonstrated that Hfq is an important component of the collective regulation of virulence factors and sets a foundation for understanding Hfq-sRNA mediated regulation in the phytopathogen P. ananatis.Figure S1 : Southern blot validation of hfq knock-out mutation in Pantoea ananatis. Genomic DNA of the wild-type (WT) and hfq mutant (1hfq) strains of P. ananatis LMG 2665T digested with EcoRI and HindIII restriction enzymes was hybridized to a DIG-labeled probe (a partial amplicon of kanamycin resistance gene). Positive detection of the antibiotic marker was observed in the 1hfq strains of P. ananatis LMG 2665T (lanes 2–8). WT of P. ananatis LMG 2665T DNA was used as a negative control (lane 1) whereas unlabeled probe was used as a positive control (lane 9).Figure S2 : Colony PCR verification of hfq knock-out mutation in Pantoea ananatis. A colony PCR confirmation of insertion of kanamycin resistance gene in the hfq gene region using Test primers (Table 2) hfq mutant (1hfq) strains of P. ananatis LMG 2665T. L represents a molecular ladder and the sizes of its prominent bands 1, 3, and 6 kilo basepairs (kb) are indicated below. A wild-type (WT) colony of P. ananatis LMG 2665T was used as a negative control (lane 1; 500 bp). Insertion of kanamycin resistance marker is shown in colony PCRs of hfq mutant (1hfq) strains of P. ananatis LMG 2665T (lanes 2, 3, and 4; 1.5 kb).Figure S3 : In vitro growth assay. Growths of wild-type (WT), hfq mutant (1hfq), and hfq complementing (1hfq pBBR1MCS::hfq) strains of Pantoea ananatis LMG 2665T in LB broth at 28 C. The growth was monitored for 20 h at optical density 600 nm (OD600) and the mean OD600 readings of the three replicates for each P. ananatis LMG 2665T strains were plotted. Solid line (yellow) represents WT, dashed line (purple) 1hfq, and dotted line (green) 1hfq pBBR1MCS::hfq. Asterisks denote significance differences (P < 0.05) in the absorbance of 1hfq relative to WT P. ananatis LMG 2665T.Figure S4 : In planta growth assay. (A) Disease progression in onion scales inoculated with wild-type (WT), hfq mutant (1hfq), and hfq complementing [1hfq (pBBR1MCS::hfq)] strains of P. ananatis LMG 2665T, and incubated for 5 days post inoculation (dpi). (B) In planta populations of WT, 1hfq, and 1hfq (pBBR1MCS::hfq) strains of P. ananatis LMG 2665T in onion scales measured for 5 dpi. The mean CFUs of three replicates for each strain from two independent experiments were plotted. Solid line (yellow) represents WT, dashed line (purple) 1hfq, and dotted line (green) 1hfq (pBBR1MCS::hfq).Figure S5 : Logarithmic plot of the number of putative small RNAs (sRNAs) identified in Pantoea ananatis LMG 2665T (pPAR sRNA) as a function of the threshold selected for calling sRNAs. This was generated by calling putative sRNAs across a range of thresholds using the custom script (see Supplementary Data Sheet S1 in the section “peak_ID.py”).Figure S6 : In silico prediction of selected Pantoea ananatis sRNAs (pPAR sRNA) secondary structure. Secondary structures of P. ananatis LMG 2665T sRNAs (A) FnrS, (B) GlmZ, (C) pPAR 237, (D) pPAR 238, and (E) pPAR 395 were predicted based on a minimum free energy model provided by RNAfold (http://rna.tbi.univie.ac.at).Figure S7 : Putative interaction of pPAR237 and pPAR238 to eanIR in Pantoea ananatis LMG 2665T. (A) Location of pPAR237 and pPAR238. In silico predicted interaction of pPAR237 (red) to eanIR (black): (B) eanI upstream sequence (energy: 8.62323 kcal/mol; hybridization energy: 23.5). (C) eanR coding sequence (energy: 13.63700 kcal/mol, hybridization energy: 39.4) and (D) in silico predicted interaction of pPAR238 (red) to eanI (black) upstream sequence (energy: 7.83954 kcal/mol, hybridization energy: 12.0).Table S1 : Summary of sRNA sequencing reads obtained and filtered for use in sRNA identification.Table S2 : A list of sRNAs identified, their genomic coordinates, sequences, and selected characteristics.Table S3 : A list of sRNAs that has significant abundance difference between WT and hfq mutant strains of Pantoea ananatis.Table S4 : A list of predicted targets of selected sRNAs.Data Sheet S1 : A custom phython script compiled for bioinformatic analyses of sRNA sequencing data.The National Research Foundation (NRF) of South Africa, the University of Pretoria and the MSU AgBioResearch.http://www.frontiersin.org/Microbiologyam2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

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