Resumen de Mobile genetic elements and the evolution of the phaseolotoxin biosynthesis cluster in pseudomonas syringae
This Thesis studies diverse mobile genetic elements from the model bacterium P. syringae pv. phaseolicola 1448A, the causal agent of halo blight of beans. Insertion sequences (ISs) are small mobile genetic elements that often associate to genes with agricultural importance, favouring their mobility either by generating regions of recombination or by mediating the translocation of DNA throughout the genome. ISs, and associated DNA, are often recruited by other, more complex mobile elements, such as genomic islands, with the ability to horizontally transfer to other bacteria. The horizontal transfer of genomic islands favours the quantum leap evolution of bacteria through the acquisition of complex phenotypes and their adaptation to new ecological niches. Genome reannotation and a functional analysis to detect transposition, using an entrapment vector based on sacB, allowed us to define the complement of ISs from P. syringae pv. phaseolicola 1448A. This strain contains at least 17 types of ISs and two of miniature inverted-repeat transposable elements (MITE), representing a 3.3% of the genome. Transposition frequency oscillated between 2.6×10¿5 and 1.1×10¿6, and was not affected by growing conditions in vitro or in planta. A 97.6% of the entrapped insertions corresponded to IS801, from which 26% contained partial fragments resulting from one-ended transpositions and 0.7% consisted of IS801 carrying adjacent DNA. 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. The miniature element MITEPsy1 was identified in the remaining 2.4% of the insertions entrapped in sacB, demonstrating for the first time the in vivo mobilization of a MITE in bacteria.
In P. syringae pv. phaseolicola 1448A, the phaseolotoxin biosynthesis region (Pht cluster) is included in a 38 kb putative genomic island (Pht-PAI), which is postulated to have originated in a bacterium phylogenetically unrelated to P. syringae and to have been recently acquired by horizontal transfer. Southern hybridization, PCR and sequencing experiments showed that the Pht-PAI is highly conserved in structure, genetic content and sequence among strains of pathovars phaseolicola and actinidiae. The nucleotide sequence of the Pht cluster from P. syringae pv. syringae CFBP 3388 is, however, only 82.7% identical to that of strain 1448A. As it occurs in pathovars phaseolicola and actinidiae, biosynthesis of phaseolotoxin in strain CFBP 3388 is dependent on the activity of genes phtL and gacA, although it is not regulated by temperature. Additionally, the Pht cluster from strain CFBP 3388 is integrated in a different genomic region and is not associated to integrases nor obviously included within a genomic island. A phylogenetic analysis of the Pht cluster showed that the closest homologues of 15 out of its 23 genes are found to constitute the ¿pseudophaseolotoxin cluster¿, which appears to be ancestral to P. syringae. The search and comparison of sequences homologous to the Pht-PAI allowed us to define a new family of mobile genetic elements, designated here as GInts. These are characterized by the gin operon, consisting of four CDSs showing homology to integrases, in their 5¿ end and a region of cargo DNA of variable size and genetic content. GInts are integrated site-specifically and excise forming circular, episome-like intermediates. A phylogenetic analysis of the gin operon and the analysis of the DNA cargo from diverse GInts indicate that these elements preferentially capture DNA from species of Pseudomonas and that they are actively transferred horizontally within this bacterial genus. Together, the evidences obtained in this Thesis suggest that the Pht cluster was not acquired as such, but rather assembled from within P. syringae and then recently captured by a GInt, generating the Pht-PAI and acquiring the ability to transfer horizontally among strains of pathovars phaseolicola and actinidiae.
