Hacia la identificación del regulón hrpL en Pseudomonas savastanoi

La especie Pseudomonas savastanoi, perteneciente al complejo Pseudomonas syringae, incluye cepas bacterianas aisladas de diversos huéspedes. Hasta la fecha, se han descrito 4 patovares de P. savastanoi capaces de infectar plantas leñosas: pv. savastanoi (aislados de olivo), pv. nerii (aislados de adelfa), pv. fraxini (aislados de fresno) y pv. retacarpa (aislados de retama). Además, recientemente se ha identificado a P. savastanoi como el agente causal de la necrosis bacteriana de la dipladenia (Mandevilla spp.), cuyo rango de huésped es diferente al de los patovares ya establecidos dentro de esta especie (ver comunicación presentada por E. Caballo-Ponce et al.). Con el objetivo de avanzar en el conocimiento de los factores genéticos determinantes del rango de huésped en P. savastanoi, hemos obtenido los borradores de los genomas de varios aislados pertenecientes a los diferentes patovares de esta especie (incluyendo un aislado de dipladenia). Este trabajo se ha centrado en el análisis bioinformático del repertorio de efectores (T3Es) del sistema de secreción tipo III (T3SS) de los diversos aislados secuenciados. Se ha identificado el conjunto de efectores comunes a todos los patovares de P. savastanoi, así como los efectores específicos de cada uno de ellos. Por otro lado, hemos construido mutantes de pérdida de función del gen hrpL en cepas modelo de cada patovar y en el aislado de dipladenia. Este gen, cuya implicación en patogenicidad se ha estudiado ampliamente en aislados de olivo, codifica un regulador positivo de la transcripción de la mayoría de los T3Es y de otros factores de virulencia. Actualmente estamos analizando el papel del gen hrpL en 1) la patogenicidad de todos los aislados seleccionados y, 2) la expresión de varios T3Es seleccionados.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.661025 and 1.161026, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria

Análisis bioinformático y funcional del sistema de secreción tipo III en cepas de Pseudomonas savastanoi aisladas de diversos huéspedes

La especie Pseudomonas savastanoi se engloba dentro del complejo Pseudomonas syringae, constituido por un conjunto de bacterias fitopatógenas Gram (-) de gran interés agrícola y económico. Dentro de esta especie, hay descritos actualmente 4 patovares capaces de infectar plantas leñosas: pv. savastanoi (aislados de olivo), pv. nerii (aislados de adelfa), pv. fraxini (aislados de fresno) y pv. retacarpa (aislados de retama). Además, se han aislado cepas de P. savastanoi de otros huéspedes, entre los que se encuentra la dipladenia (Mandevilla spp.), aunque los aislados de esta planta no se han asignado aún a un patovar concreto. Dentro del complejo P. syringae, uno de los factores más importantes para el establecimiento de la enfermedad es el sistema de secreción tipo III (T3SS), así como su repertorio de efectores (T3E), los cuales han sido identificados como uno de los factores más relevantes en la determinación del rango de huésped. Actualmente, los genomas de varios aislados de los patovares savastanoi, nerii, fraxini y retacarpa están disponibles en NCBI. Además, hemos obtenido el borrador de la secuencia de aislados adicionales de P. savastanoi, incluyendo el de una cepa patógena en dipladenia. En la actualidad, estamos llevando a cabo ensayos de patogenicidad cruzada de todas estas cepas en diferentes huéspedes de P. savastanoi, con el objetivo de poder relacionar su especificidad de huésped con sus diferencias genómicas. Análisis bioinformáticos comparativos del T3SS de todas estas cepas y de su repertorio de T3E, nos ha permitido identificar el conjunto de T3E compartido entre todas las cepas, así como los específicos de cada una de ellas. Además, hemos construido mutantes en cepas modelo de cada patovar, así como en el aislado de la dipladenia, del gen hrpA, que codifica la principal proteína estructural del pilus del T3SS, y del gen hrpL, que codifica un activador transcripcional de los genes del T3SS y de la mayoría de sus T3E. En la actualidad, estamos analizando el papel de ambos genes en la patogenicidad de las cepas de P. savastanoi seleccionadas y la expresión de varios T3E que podrían estar implicados en el rango de huésped.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Plasmid replicons from Pseudomonas are natural chimeras of functional, exchangeable modules

14 p.-5 figPlasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.This work was funded by the Spanish Plan Nacional I+D+i grant AGL2014-53242-C2-2-R, fromthe Ministerio de Economía y Competitividad (MINECO), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER). M.A. was supported by an FPI fellowship (reference BES-2012-054016, Ministerio de Ciencia e Innovación/Ministerio de Economía y Competitividad, Spain).Peer reviewe

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

The toxic guardians — multiple toxin-antitoxin systems provide stability, avoid deletions and maintain virulence genes of Pseudomonas syringae virulence plasmids

Abstract Background Pseudomonas syringae is a γ-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems. Results The three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10− 3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10− 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo. Conclusions Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria

Pseudomonas savastanoi pv. savastanoi: Some like it knot

Pseudomonas savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease and an unorthodox member of the P. syringae complex, causing aerial tumours instead of the foliar necroses and cankers characteristic of most members of this complex. Olive knot is present wherever olive is grown; although losses are difficult to assess, it is assumed that olive knot is one of the most important diseases of the olive crop. The last century witnessed a large number of scientific articles describing the biology, epidemiology and control of this pathogen. However, most P. savastanoi pv. savastanoi strains are highly recalcitrant to genetic manipulation, which has effectively prevented the pathogen from benefitting from the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A number of studies in recent years have made significant advances in the biology, ecology and genetics of P. savastanoi pv. savastanoi, paving the way for the molecular dissection of its interaction with other nonpathogenic bacteria and their woody hosts. The selection of a genetically pliable model strain was soon followed by the development of rapid methods for virulence assessment with micropropagated olive plants and the analysis of cellular interactions with the plant host. The generation of a draft genome of strain NCPPB 3335 and the closed sequence of its three native plasmids has allowed for functional and comparative genomic analyses for the identification of its pathogenicity gene complement. This includes 34 putative type III effector genes and genomic regions, shared with other pathogens of woody hosts, which encode metabolic pathways associated with the degradation of lignin-derived compounds. Now, the time is right to explore the molecular basis of the P. savastanoi pv. savastanoi–olive interaction and to obtain insights into why some pathovars like it necrotic and why some like it knot.This work was supported by the Spanish Plan Nacional I Di grants AGL2008-05311-C02-01, AGL2008-05311-C02-02, AGL2011-30343-C02-01 and AGL2011-30343-C02-02 (Ministerio de Economía y Competitividad), co-financed by Fondo Europeo de Desarrollo Regional (FEDER), and by grant P08-CVI-03475 from the Junta de Andalucía, Spain (http://www.juntadeandalucia.es). IMM and IMA were supported by the Ramón Areces Foundation (Spain) and by an FPU fellowship from the Ministerio de Economía y Competitividad (Spain), respectively. We thank L. Rodríguez-Moreno for confocal and electron microscopy images and T. Osinga for help with the English language.Peer Reviewe

Two Homologues of the Global Regulator Csr/Rsm Redundantly Control Phaseolotoxin Biosynthesis and Virulence in the Plant Pathogen Pseudomonas amygdali pv. phaseolicola 1448A

The widely conserved Csr/Rsm (carbon storage regulator/repressor of stationary-phase metabolites) post-transcriptional regulatory system controls diverse phenotypes involved in bacterial pathogenicity and virulence. Here we show that Pseudomonas amygdali pv. phaseolicola 1448A contains seven rsm genes, four of which are chromosomal. In RNAseq analyses, only rsmE was thermoregulated, with increased expression at 18 °C, whereas the antagonistic sRNAs rsmX1, rsmX4, rsmX5 and rsmZ showed increased levels at 28 °C. Only double rsmA-rsmE mutants showed significantly altered phenotypes in functional analyses, being impaired for symptom elicitation in bean, including in planta growth, and for induction of the hypersensitive response in tobacco. Double mutants were also non-motile and were compromised for the utilization of different carbon sources. These phenotypes were accompanied by reduced mRNA levels of the type III secretion system regulatory genes hrpL and hrpA, and the flagellin gene, fliC. Biosynthesis of the phytotoxin phaseolotoxin by mutants in rsmA and rsmE was delayed, occurring only in older cultures, indicating that these rsm homologues act as inductors of toxin synthesis. Therefore, genes rsmA and rsmE act redundantly, although with a degree of specialization, to positively regulate diverse phenotypes involved in niche colonization. Additionally, our results suggest the existence of a regulatory molecule different from the Rsm proteins and dependent on the GacS/GacA (global activator of antibiotic and cyanide production) system, which causes the repression of phaseolotoxin biosynthesis at high temperatures

Annotation and overview of the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 draft genome reveals the virulence gene complement of a tumour-inducing pathogen of woody hosts

Pseudomonas savastanoi pv. savastanoi is a tumour-inducing pathogen of Olea europaea L. causing oliveknot disease. Bioinformatic analysis of the draftgenome sequence of strain NCPPB 3335, whichencodes 5232 predicted coding genes on a totallength of 5856 998 bp and a 57.12% G + C, revealed alarge degree of conservation with Pseudomonassyringae pv. phaseolicola 1448A and P. syringae pv.tabaci 11528. However, NCPPB 3335 contains twelvevariable genomic regions, which are absent in all pre-viously sequenced P. syringae strains. Various fea-tures that could contribute to the ability of this strainto survive in a woody host were identified, includingbroad catabolic and transport capabilities for degrad-ing plant-derived aromatic compounds, the duplica-tion of sequences related to the biosynthesis of thephytohormone indoleacetic acid (iaaM, iaaH) and itsamino acid conjugate indoleacetic acid-lysine (iaaLgene), and the repertoire of strain-specific putativetype III secretion system effectors. Access to thisseventh genome sequence belonging to the ‘P. syrin-gae complex’ allowed us to identify 73 predictedcoding genes that are NCPPB 3335-specific. Resultsshown here provide the basis for detailed functionalanalysis of a tumour-inducing pathogen of woodyhosts and for the study of specific adaptations of a P.savastanoi pathovar