Miniature transposable sequences are frequently mobilized in the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola

Abstract

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 2.6×10(-5) and 1.1×10(-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.

Figure 1. Conservation of the terminal repeats of MITEPsy1 and MITEPsy2.

A black background indicates conservation of each nucleotide in at least three quarters of the sequences. The ends of the six copies of MITEPsy1 in strain 1448A are identical; for MITEPsy2, the ends of the chromosomal copy (MITEPsy2.1) and of the copy in plasmid p1448A-A (MITEPsy2.2) are shown.

Figure 2. Structure and terminal ends in IS801 and miniature derivatives found to actively transpose in P. syringae pv. phaseolicola 1448A.

Grey boxes indicate the wild type IS801 (1512 nt) or miniature sequences derived from the complete element, with their size indicated inside the box; all of them share the same right terminus and continue without gaps as indicated by their relative position. The sequences indicate the tetranucleotides marking their left and right ends. The broken white box indicates the three different fragments of gene PSPPH_008/PSPPH_0017 that were mobilized by IS801 in three different experiments, with an indication of their sizes. Drawings are to scale.