Open Access
Biología de plásmidos y dinámica génica en el complejo Pseudomonas syringae
(2017) Añorga García, Maite; Murillo Martínez, Jesús; Bardají Goikoetxea, Leire; Producción Agraria; Nekazaritza Ekoizpena
La mayoría de las cepas de Pseudomonas syringae posee uno o más plásmidos nativos tipo PFP, que suelen albergar genes adaptativos y que facilitan el intercambio de los mismos entre poblaciones bacterianas. P. syringae pv. savastanoi NCPPB 3335 causa la tuberculosis del olivo (Olea europaea) y es un patógeno modelo para el estudio de la patogénesis bacteriana en plantas leñosas. Esta cepa contiene tres plásmidos PFP estables que han sido completamente secuenciados —pPsv48A, 80 kb; pPsv48B, 45 kb, y pPsv48C, 42 kb— y que portan varios posibles genes de virulencia. A pesar de la importancia de los plásmidos PFP en el ciclo de vida de P. syringae, todavía se desconocen muchos aspectos de su genética y biología, por lo que en esta Tesis nos hemos propuesto los siguientes objetivos: 1) identificación y caracterización funcional de un segundo origen de replicación presente en el plásmido nativo pPsv48C de Ps pv. savastanoi NCPPB 3335; 2) identificación de determinantes de mantenimiento presentes en los plásmidos de la cepa NCPPB 3335 y evaluación de su contribución al mantenimiento de dichos plásmidos, y 3) evaluación del papel del plásmido pPsv48C en la patogenicidad y virulencia de la cepa NCPPB 3335 en su interacción con su planta huésped, olivo. Hemos identificado un segundo replicón en pPsv48C, que es una quimera por compartir su región de control y promotor con el replicón no homólogo RepA-PFP. Hemos demostrado que este tipo de quimeras es frecuente en al menos cuatro familias de replicasas no homólogas de Pseudomonas, incluyendo bacterias patógenas de plantas, animales y humanos. Estos replicones constan de dos módulos funcionales e independientes correspondientes a las regiones de control (módulo REx-C), que actúa como determinante de incompatibilidad, y de replicación (módulo REx-R). Mediante ensayos funcionales, hemos determinado que los tres plásmidos de NCPPB 3335 utilizan diversas estrategias y mecanismos para aumentar su estabilidad. Así, la presencia de estos dos replicones, y especialmente del replicón RepJ, confiere una alta estabilidad a pPsv48C. Además, hemos documentado que los elementos repetitivos IS801 y MITEPsy2 generan frecuentes deleciones y reorganizaciones plasmídicas, cuya frecuencia se reduce por un factor entre 3 y 50 debido a la acción de tres sistemas toxina-antitoxina codificados en pPsv48A y otros tres codificados en pPsv48C. Asimismo, estos sistemas reducen en dos órdenes de magnitud la frecuencia de pérdida espontánea del plásmido pPsv48A y contribuyen al mantenimiento de los genes de virulencia ptz (en pPsv48A) e idi (en pPsv48C). Mediante inactivación funcional de los sistemas de mantenimiento de pPsv48C, hemos obtenido un derivado de NCPPB 3335 curado de sus tres plásmidos nativos (cepa UPN912), que no produce tumores en olivo. Mediante inoculaciones con esta cepa y con mutantes específicos hemos demostrado que el gen idi —que codifica una posible isopentenil difosfato isomersa y éstá probablemente implicado en la biosíntesis de citoquininas— es esencial para la producción de tumores de tamaño silvestre tanto en plantas micropropagadas como en olivos lignificados. En conjunto, nuestros resultados indican que la cepa NCPPB 3335 contiene tres plásmidos nativos de virulencia, que están expuestos a frecuentes eventos de reorganización genética cuyo efecto se ve contrarrestado por la existencia de múltiples determinantes plasmídicos de estabilidad, que a la vez contribuyen a disminuir la pérdida espontánea de los plásmidos y al mantenimiento de genes de virulencia durante el ciclo de vida de la bacteria.
Open Access
Evaluación del efecto de las condiciones ambientales en la transposición de elementos móviles en la bacteria fitopatógena Pseudomonas syringae
(2011) Añorga García, Maite; Murillo Martínez, Jesús; Bardají Goikoetxea, Leire; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza Ekoizpena
Los elementos genéticos móviles están muy extendidos en Pseudomonas syringae , y a menudo están asociados con genes de virulencia. Se han identificado diecisiete tipos de secuencias de inserción y dos “miniature inverted repeat transposable elements” (elementos transponibles miniatura con repeticiones invertidas) MITEs, en P. syringae pv. phaseolicola (Pph) 1448A. Para evaluar si alguno de estos elementos genéticos móviles se activa dentro del genoma, hemos empleado un vector trampa que contiene el gen sacB , que permite la selección de inserciones que inactiven sacB en medios con sacarosa. La frecuencia de transposición en Pph 1448A osciló entre 2,6 × 10 H5 y 1,1 × 10 H6 , dependiendo del clon, aunque se mantuvo estable para cada clon después de varias transferencias consecutivas en medios de cultivo. La frecuencia de transposición fue similar en bacterias cultivadas en medio rico y mínimo, así como en células recuperadas de plantas hospedadoras compatibles e incompatibles. Igualmente no se observaron variaciones en la frecuencia de transposición en las cepas 1448A y 1302A de P. syringae pv. phaseolicola y P. syringae pv. syringae B728a en respuesta a un choque térmico. Estos resultados sugieren que las condiciones de crecimiento y las condiciones de estrés no influyen en la frecuencia de transposición de elementos móviles en cepas de P. syringae . Un 65% de las inserciones atrapadas en sacB en P. syringae pv. phaseolicola 1448A contenían una copia completa de IS 801 , mientras que las inserciones restantes correspondían a las secuencias más pequeñas que cualquiera de los elementos transponibles identificados en la cepa 1448A, y colectivamente identificados como secuencias en miniatura. La mayoría de las secuencias miniatura fueron fragmentos de 229 (8,3%), 360 (0,5%) y 679 nt (16,9%) de la parte derecha de IS 801 . Estos tres tipos de fragmentos se originan en el extremo 3’ de IS 801 y terminan en un tetranucleótido con similitud al extremo 5’ del elemento, por lo que probablemente son el resultado de una transposición terminal (one-ended transposition) de IS 801 . Un promedio de 0,7% de las inserciones analizadas contenían una copia completa de IS 801 unida a un fragmento de tamaño variable de los genes PSPPH_0008/PSPPH_0017, demostrando que este elemento puede movilizar ADN adyacente in vivo . Igualmente, hemos demostrado la movilidad de una secuencia de 100 nt que previamente se ha encontrado insertada en genes de virulencia alterando la especificidad de huésped. Esta secuencia miniatura, designada MITE Psy1 representa un promedio del 2,4% del total de inserciones, y es el primer MITE cuya movilidad se ha demostrado in vivo en bacterias.
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
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.
Open Access
Genes ptz and idi, coding for cytokinin biosynthesis enzymes, are essential for tumorigenesis and in planta growth by P. syringae pv. savastanoi NCPPB 3335
(Frontiers Media, 2020) Añorga García, Maite; Pintado, Adrián; Ramos, Cayo; Diego, Nuria de; Ugena, Lydia; Novák, Ondrej; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
The phytopathogenic bacterium Pseudomonas syringae pv. savastanoi elicits aerial tumors on olive plants and is also able to synthesize large amounts of auxins and cytokinins. The auxin indoleacetic acid was shown to be required for tumorigenesis, but there is only correlational evidence suggesting a role for cytokinins. The model strain NCPPB 3335 contains two plasmid-borne genes coding for cytokinin biosynthesis enzymes: ptz, for an isopentenyl transferase and idi, for an isopentenyl-diphosphate delta-isomerase. Phylogenetic analyses showed that carriage of ptz and idi is not strictly associated with tumorigenic bacteria, that both genes were linked when first acquired by P. syringae, and that a different allele of ptz has been independently acquired by P. syringae pv. savastanoi and closely related bacteria. We generated mutant derivatives of NCPPB 3335 cured of virulence plasmids or with site-specific deletions of genes ptz and/or idi and evaluated their virulence in lignified and micropropagated olive plants. Strains lacking ptz, idi, or both produced tumors with average volumes up to 29 times smaller and reached populations up to two orders of magnitude lower than those induced by strain NCPPB 3335; these phenotypes reverted by complementation with the cloned genes. Trans-zeatin was the most abundant cytokinin in culture filtrates of NCPPB 3335. Deletion of gene ptz abolished biosynthesis of trans-zeatin and dihydrozeatin, whereas a reduced but significant amount of isopentenyladenine was still detected in the medium, suggesting the existence of other genes contributing to cytokinin biosynthesis in P. syringae. Conversely, extracts from strains lacking gene idi contained significantly higher amounts of trans-zeatin than extracts from the wild-type strain but similar amounts of the other cytokinins. This suggests that Idi might promote tumorigenesis by ensuring the biosynthesis of the most active cytokinin forms, their correct balance in planta, or by regulating the expression of other virulence genes. Therefore, gene ptz, but not gene idi, is essential for the biosynthesis of high amounts of cytokinins in culture; however, both ptz and idi are individually essential for the adequate development of tumors on olive plants by Psv NCPPB 3335.
Open Access
Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola
(Public Library of Science, 2011) Bardají Goikoetxea, Leire; Añorga García, Maite; Jackson, Robert W.; Martínez Bilbao, Alejandro; Yanguas Casas, Natalia; Murillo Martínez, Jesús; Producción Agraria; Nekazaritza Ekoizpena
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 2.6 x 10(-5) and 1.1 x 10(-6), 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.
Open Access
Multiple relaxases contribute to the horizontal transfer of the virulence plasmids from the tumorigenic bacterium Pseudomonas syringae pv. savastanoi NCPPB 3335
(Frontiers Media, 2022) Añorga García, Maite; Urriza Leoz, Miriam; Ramos, Cayo; Murillo Martínez, Jesús; Institute for Multidisciplinary Research in Applied Biology - IMAB
Pseudomonas syringae pv. savastanoi NCPPB 3335 is the causal agent of olive knot disease and contains three virulence plasmids: pPsv48A (pA), 80 kb; pPsv48B (pB), 45 kb, and pPsv48C (pC), 42 kb. Here we show that pB contains a complete MPFT (previously type IVA secretion system) and a functional origin of conjugational transfer adjacent to a relaxase of the MOBP family; pC also contains a functional oriT-MOBP array, whereas pA contains an incomplete MPFI (previously type IVB secretion system), but not a recognizable oriT. Plasmid transfer occurred on solid and in liquid media, and on leaf surfaces of a non-host plant (Phaseolus vulgaris) with high (pB) or moderate frequency (pC); pA was transferred only occasionally after cointegration with pB. We found three plasmid-borne and three chromosomal relaxase genes, although the chromosomal relaxases did not contribute to plasmid dissemination. The MOBP relaxase genes of pB and pC were functionally interchangeable, although with di ering eciencies. We also identified a functional MOBQ mobilization region in pC, which could only mobilize this plasmid. Plasmid pB could be eciently transferred to strains of six phylogroups of P. syringae sensu lato, whereas pC could only be mobilized to two strains of phylogroup 3 (genomospecies 2). In two of the recipient strains, pB was stably maintained after 21 subcultures in liquid medium. The carriage of several relaxases by the native plasmids of P. syringae impacts their transfer frequency and, by providing functional diversity and redundancy, adds robustness to the conjugation system.