BUSCADOR RECOLECTA

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., This work was supported by the Spanish Plan Nacional I+D+i grants AGL2014-53242-C2-1-R and AGL2014-53242-C2-2-R from the Spanish Ministerio de Economía y Competitividad (MINECO) and was co-financed by FEDER. AP was supported by a FPU Ph.D. fellowship and AM-P and EC-P by FPI Ph.D. fellowships from the Spanish Ministerio de Educación, Cultura y Deporte (MECD) and MINECO.

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., This work was funded by the Spanish Plan Nacional I + D + I grants
AGL2014–53242-C2–1-R, AGL2014–53242-C2–2-R, AGL2017-82492-C2-1-
R, and AGL2017-82492-C2-2-R from the Ministerio de Economía y
Competitividad (MINECO), co-financed by the Fondo Europeo de Desarrollo
Regional (FEDER).

Incluye 9 ficheros de datos, 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., This work was funded by the Spanish Plan Nacional I+ D+ i grants AGL2011-30343-C02-02 and AGL2014-
53242-C2-2-R, from the Ministerio de Economía y Competitividad (MINECO), co-financed by the Fondo
Europeo de Desarrollo Regional (FEDER).

Incluye ficheros de datos, 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., This work was funded by the Spanish Plan Nacional I+D+i
grant AGL2014-53242-C2-2-R, from the 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).

Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent
manner, being optimally synthesized between 18ºC and 20ºC, while no detectable
amounts are present above 28ºC. The Pht cluster, involved in the biosynthesis of phaseolotoxin,
contains 23 genes that are organized in five transcriptional units. The function of most
of the genes from the Pht cluster is still unknown and little information about the regulatory
circuitry leading to expression of these genes has been reported. The purpose of the present
study was to investigate the participation of pht genes in the regulation of the operons
coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription-
PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This
allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are
essential to prevent their own expression at 28ºC, a temperature at which no detectable
amounts of the toxin are present. We obtained evidence that the phtABC genes also participate
in the regulation of the phtD, phtM and phtL operons. According to our results, we propose
that PhtABC and other Pht product activities could be involved in the synthesis of the
sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the
transcriptional regulation of the phtA operon., This work was funded by grants from the Consejo Nacional de Ciencia y Tecnología (CONACyT), research grant CB-2015-01-255155 to SA, and from the Spanish Plan Nacional I + D + i grant AGL2014-53242-C2-2-R, from the Ministerio de Economía y Competitividad, co-financed by the Fondo Europeo de Desarrollo Regional (FEDER) to JM.

Incluye 4 ficheros de datos, Background: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.
Results: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants.
Conclusions: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence., This research was supported by the Spanish Plan Nacional I+D+i grants AGL2011-30343-CO2-01, AGL2014-53242-C2-1-R and BIO2007-67874-C02-02 as well as by grants ref. P08-CVI-03475 and P12-AGR-0370 from the Junta de Andalucía (Spain).