Open Access
Knots untie: molecular determinants involved in knot formation Induced by Pseudomonas savastanoi in woody hosts
(Frontiers Media, 2017) Caballo Ponce, Eloy; Murillo Martínez, Jesús; Martínez Gil, Marta; Moreno Pérez, Alba; Pintado, Adrián; Ramos, Cayo; Producción Agraria; Nekazaritza Ekoizpena
The study of the molecular basis of tree diseases is lately receiving a renewed attention, especially with the emerging perception that pathogens require specific pathogenicity and virulence factors to successfully colonize woody hosts. Pathosystems involving woody plants are notoriously difficult to study, although the use of model bacterial strains together with genetically homogeneous micropropagated plant material is providing a significant impetus to our understanding of the molecular determinants leading to disease. The gammaproteobacterium Pseudomonas savastanoi belongs to the intensively studied Pseudomonas syringae complex, and includes three pathogenic lineages causing tumorous overgrowths (knots) in diverse economically relevant trees and shrubs. As it occurs with many other bacteria, pathogenicity of P. savastanoi is dependent on a type III secretion system, which is accompanied by a core set of at least 20 effector genes shared among strains isolated from olive, oleander, and ash. The induction of knots of wild-type size requires that the pathogen maintains adequate levels of diverse metabolites, including the phytohormones indole-3-acetic acid and cytokinins, as well as cyclic-di-GMP, some of which can also regulate the expression of other pathogenicity and virulence genes and participate in bacterial competitiveness. In a remarkable example of social networking, quorum sensing molecules allow for the communication among P. savastanoi and other members of the knot microbiome, while at the same time are essential for tumor formation. Additionally, a distinguishing feature of bacteria from the P. syringae complex isolated from woody organs is the possession of a 15 kb genomic island (WHOP) carrying four operons and three other genes involved in degradation of phenolic compounds. Two of these operons mediate the catabolism of anthranilate and catechol and, together with another operon, are required for the induction of full-size tumors in woody hosts, but not in non-woody micropropagated plants. The use of transposon mutagenesis also uncovered a treasure trove of additional P. savastanoi genes affecting virulence and participating in diverse bacterial processes. Although there is still much to be learned on what makes a bacterium a successful pathogen of trees, we are already untying the knots.
Open Access
Draft genome sequences of two bacillus thuringiensis strains and characterization of a putative 41.9-kDa insecticidal toxin
(MDPI, 2014) Palma Dovis, Leopoldo; Muñoz Labiano, Delia; Berry, Colin; Murillo Martínez, Jesús; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 μg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins.
Open Access
The Pbo cluster from Pseudomonas syringae pv. phaseolicola NPS3121 is thermoregulated and required for phaseolotoxin biosynthesis
(MDPI, 2021) Guardado-Valdivia, Lizeth; Chacón-López, Alejandra; Murillo Martínez, Jesús; Poveda Arias, Jorge; Hernández Flores, José Luis; Xoca-Orozco, Luis; Aguilera, Selene; Institute for Multidisciplinary Research in Applied Biology - IMAB
The bean (Phaseolus vulgaris) pathogen Pseudomonas syringae pv. phaseolicola NPS3121 synthe-sizes phaseolotoxin in a thermoregulated way, with optimum production at 18 °C. Gene PSPPH_4550 was previously shown to be thermoregulated and required for phaseolotoxin bio-synthesis. Here, we established that PSPPH_4550 is part of a cluster of 16 genes, the Pbo cluster, included in a genomic island with a limited distribution in P. syringae and unrelated to the posses-sion of the phaseolotoxin biosynthesis cluster. We identified typical non-ribosomal peptide syn-thetase, and polyketide synthetase domains in several of the pbo deduced products. RT-PCR and the analysis of polar mutants showed that the Pbo cluster is organized in four transcriptional units, including one monocistronic and three polycistronic. Operons pboA and pboO are both es-sential for phaseolotoxin biosynthesis, while pboK and pboJ only influence the amount of toxin produced. The three polycistronic units were transcribed at high levels at 18 °C but not at 28 °C, whereas gene pboJ was constitutively expressed. Together, our data suggest that the Pbo cluster synthesizes secondary metabolite(s), which could participate in the regulation of phaseolotoxin biosynthesis.
Open Access
Four genes essential for recombination define GInts, a new type of mobile genomic island widespread in bacteria
(Nature Publishing Group, 2017) Bardají Goikoetxea, Leire; Echeverría Ancín, Myriam; Rodríguez Palenzuela, Pablo; Martínez García, Pedro M.; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
Integrases are a family of tyrosine recombinases that are highly abundant in bacterial genomes, actively disseminating adaptive characters such as pathogenicity determinants and antibiotics resistance. Using comparative genomics and functional assays, we identified a novel type of mobile genetic element, the GInt, in many diverse bacterial groups but not in archaea. Integrated as genomic islands, GInts show a tripartite structure consisting of the ginABCD operon, a cargo DNA region from 2.5 to at least 70 kb, and a short AT-rich 3′ end. The gin operon is characteristic of GInts and codes for three putative integrases and a small putative helix-loop-helix protein, all of which are essential for integration and excision of the element. Genes in the cargo DNA are acquired mostly from phylogenetically related bacteria and often code for traits that might increase fitness, such as resistance to antimicrobials or virulence. GInts also tend to capture clusters of genes involved in complex processes, such as the biosynthesis of phaseolotoxin by Pseudomonas syringae. GInts integrate site-specifically, generating two flanking direct imperfect repeats, and excise forming circular molecules. The excision process generates sequence variants at the element attachment site, which can increase frequency of integration and drive target specificity.
Open Access
Host range determinants of Pseudomonas savastanoi pathovars of woody hosts revealed by comparative genomics and cross-pathogenicity tests
(Frontiers Media, 2020) Moreno Pérez, Alba; Pintado, Adrián; Murillo Martínez, Jesús; Caballo Ponce, Eloy; Tegli, Stefania; Moretti, Chiaraluce; Rodríguez Palenzuela, Pablo; Ramos, Cayo; Institute for Multidisciplinary Research in Applied Biology - IMAB
The study of host range determinants within the Pseudomonas syringae complex is gaining renewed attention due to its widespread distribution in non-agricultural environments, evidence of large variability in intra-pathovar host range, and the emergence of new epidemic diseases. This requires the establishment of appropriate model pathosystems facilitating integration of phenotypic, genomic and evolutionary data. Pseudomonas savastanoi pv. savastanoi is a model pathogen of the olive tree, and here we report a closed genome of strain NCPPB 3335, plus draft genome sequences of three strains isolated from oleander (pv. nerii), ash (pv. fraxini) and broom plants (pv. retacarpa). We then conducted a comparative genomic analysis of these four new genomes plus 16 publicly available genomes, representing 20 strains of these four P. savastanoi pathovars of woody hosts. Despite overlapping host ranges, cross-pathogenicity tests using four plant hosts clearly separated these pathovars and lead to pathovar reassignment of two strains. Critically, these functional assays were pivotal to reconcile phylogeny with host range and to define pathovar-specific genes repertoires. We report a pan-genome of 7,953 ortholog gene families and a total of 45 type III secretion system effector genes, including 24 core genes, four genes exclusive of pv. retacarpa and several genes encoding pathovar-specific truncations. Noticeably, the four pathovars corresponded with well-defined genetic lineages, with core genome phylogeny and hierarchical clustering of effector genes closely correlating with pathogenic specialization. Knot-inducing pathovars encode genes absent in the canker-inducing pv. fraxini, such as those related to indole acetic acid, cytokinins, rhizobitoxine, and a bacteriophytochrome. Other pathovar-exclusive genes encode type I, type II, type IV, and type VI secretion system proteins, the phytotoxine phevamine A, a siderophore, c-di-GMP-related proteins, methyl chemotaxis proteins, and a broad collection of transcriptional regulators and transporters of eight different superfamilies. Our combination of pathogenicity analyses and genomics tools allowed us to correctly assign strains to pathovars and to propose a repertoire of host range-related genes in the P. syringae complex.
Open Access
Pseudomonas savastanoi pv. savastanoi: some like it knot
(Wiley, 2012) Ramos, Cayo; Matas Casado, Isabel María; Bardají Goikoetxea, Leire; Aragón, Isabel M.; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
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 has witnessed a good deal 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 left the pathogen out of the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A series of studies in the last 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 non-pathogenic 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, that 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 get insights into why some pathovars like it necrotic and why some like it knot. Synonyms: Pseudomonas syringae pv. savastanoi Taxonomy: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; included in genomospecies 2 together with at least P. amygdali, P. ficuserectae, P. meliae and 16 other pathovars from the P. syringae complex (aesculi, ciccaronei, dendropanacis, eriobotryae, glycinea, hibisci, mellea, mori, myricae, phaseolicola, photiniae, sesami, tabaci, ulmi, and certain strains of lachrymans and morsprunorum); when a formal proposal is made for the unification of these bacteria, the species name P. amygdali would take priority over P. savastanoi. Microbiological properties: Gram-negative rods, 0.4-0.8 by 1.0-3.0 µm, aerobic. Motile by one to four polar flagella, rather slow growing, optimal temperatures for growth of 25–30 °C, oxidase negative, arginine dihydrolase negative, elicits the hypersensitive response on tobacco, most isolates are fluorescent and levan negative although some isolates are non-fluorescent and levan positive. Host range: P. savastanoi pv. savastanoi causes tumours in cultivated and wild olive and ash (Fraxinus excelsior). Although strains from olive were reported to infect oleander (Nerium oleander), this is generally not the case; however, strains of P. savastanoi pv. nerii can infect olive. Pathovars fraxini and nerii differentiate from pv. savastanoi mostly in their host range, and were not formally recognized until 1996. Literature previous to about 1996 generally name strains of the three pathovars as P. syringae subsp. savastanoi or P. savastanoi subsp. savastanoi, contributing to confusion about host range and biological properties. Disease symptoms: Symptoms of infected trees include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits. Epidemiology: The pathogen can survive and multiply on aerial plant surfaces, as well as in knots, from where it can be dispersed by rain, wind, insects and human activities, entering the plant through wounds. Populations are very unevenly distributed in the plant, and suffer drastic fluctuations throughout the year, with maximum numbers of bacteria occurring during rainy and warm months. Populations of P. savastanoi pv. savastanoi are normally associated to non-pathogenic bacteria, both epiphytically and endophytically, and were demonstrated to form mutualistic consortia with Erwinia toletana and Pantoea agglomerans that could result in increased bacterial populations and disease symptoms. Disease control: Based on preventive measures, mostly sanitary and cultural practices. Integrated control programs benefit from regular applications of copper formulations, which should be maintained at least a few years for maximum benefit. Olive cultivars vary in their susceptibility to olive knot, but there are no known cultivars with full resistance to the pathogen. Useful websites: http://www.pseudomonas-syringae.org/; http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; ASAP access to the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence https://asap.ahabs.wisc.edu/asap/logon.php.
Open Access
The toxic guardians: multiple toxin-antitoxin systems provide stability, avoid deletions and maintain virulence genes of Pseudomonas syringae virulence plasmids
(BMC, 2019) Bardají Goikoetxea, Leire; Añorga García, Maite; Echeverría Ancín, Myriam; Ramos, Cayo; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
Background: Pseudomonas syringae is a y-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 x 10-3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 x 1 0- 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.
Open Access
Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production
(BioMed Central, 2012) Arrebola, Eva; Carrión, Víctor J.; Cazorla, Francisco M.; Pérez García, Alejandro; Murillo Martínez, Jesús; Vicente, Antonio de; Producción Agraria; Nekazaritza Ekoizpena
Background: Mangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (mgoA) in mangotoxin production and virulence has been reported. Results: In the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts. Conclusions: The results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production.
Open Access
Detection by multiplex PCR and characterization of nontoxigenic strains of Pseudomonas syringae pv. phaseolicola from different places in Spain. Short communication
(Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), 2006) Rico, A.; Erdozáin García, María; Ortiz Barredo, Amaia; Ruiz de Galarreta, José Ignacio; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
El control eficiente de la grasa de la judía causada por Pseudomonas syringae pv. phaseolicola se basa principalmente en la utilización de semilla libre del patógeno. La detección del patógeno en semilla se efectúa mediante métodos altamente sensibles basados en la detección por PCR de los genes responsables de la biosíntesis de la faseolotoxina, la cual, hasta ahora, se consideraba que era sintetizada por todas las cepas del patógeno con importancia epidemiológica. Sin embargo, en la Comunidad de Castilla y León, España, las epidemias de grasa de la judía en campo se asocian frecuentemente con cepas no toxigénicas de P. syringae pv. phaseolicola, que no pueden ser detectadas con los métodos moleculares y serológicos actuales. Los resultados presentados en este trabajo demuestran la existencia de aislados no toxigénicos de P. syringae pv. phaseolicola en zonas distintas de Castilla y León, lo que implica la necesidad de establecer una metodología fiable para la certificación de semillas de judía. Con este propósito, se presenta un sencillo protocolo en dos fases que permite la identificación de los dos tipos de aislados, y que se basa en una PCR multiplex con enriquecimiento a partir de extractos de semilla y en ensayos de patogenicidad.
Open Access
Plasmid replicons from Pseudomonas are natural chimeras of functional, exchangeable modules
(Frontiers Media, 2017) Bardají Goikoetxea, Leire; Añorga García, Maite; Ruiz Masó, José A.; Solar, Gloria del; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
Plasmids 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.