Evaluación del efecto de las condiciones ambientales en la transposición de elementos móviles en la bacteria fitopatógena Pseudomonas syringae

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.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) in P. syringae pv. phaseolicola (Pph) 1448A. To evaluate the mobilization of these mobile elements, we employed an entrapment vector containing sacB . We estimated that transposition frequency in Pph 1448A oscillated between 2.6 x 10 H5 and 1.1 x 10 H6 , 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. Likewise, no variations were observed in the frequency of transposition in strains 1448A and 1302A P. syringae pv. phaseolicola and P. syringae pv. syringae B728a in response to a thermal shock. These results suggest that growth conditions and stress conditions do not influence the frequency of transposition of mobile elements in strains of P. syringae . The majority of the entrapped insertions (65%) contained a full-length IS 801 element, with the remaining insertions corresponding to sequences smaller than any of the transposable elements identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229 (8,3%), 360 (0,5%) and 679Hnt (16,9%) of the right end of IS 801 ended in a consensus tetranucleotide and likely resulted from oneHended transposition of IS 801 . An average 0.7% of the insertions analyzed consisted of IS 801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS 801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS 801 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 MITE Psy1 (100Hnt), represented an average 2.4% of the total number of insertions, demonstrating for the first time the mobilization of a MITE in bacteria.Ingeniería AgronómicaNekazaritza Ingeniaritz

Multiple relaxases contribute to the horizontal transfer of the virulence plasmids from the tumorigenic bacterium Pseudomonas syringae pv. savastanoi NCPPB 3335

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.This work was funded by the Spanish Plan Nacional I+D+i grants PID2020-115177RB-C21 and PID2020- 115177RB-C22 financed by the Ministerio de Ciencia e Innovación and the Agencia Estatal de Investigación, MCIN/AEI/10.13039/501100011033/, Spain