Transposon Mutagenesis of Xylella fastidiosa by Electroporation of Tn5 Synaptic Complexes

Abstract

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

Upstream Migration ofXylella fastidiosavia Pilus-Driven Twitching Motility

“…The EZ::TN transposome system (7) was used to generate X. fastidiosa mutants. Electrocompetent cells were prepared according to published procedures (7). Xylella fastidiosa was cultured in modified PW liquid medium at 28°C on a rotary shaker (180 rpm) for 7 days.…”

Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell–cell aggregation

“…The EZ : : TN Transposome system <Kan-2> kit (Epicentre) was used to generate X. fastidiosa mutants. Electrocompetent cells were prepared as described by Guilhabert et al (2001). Electroporation was conducted at 2500 V, 200 V and 25 mF for 5 ms, and the electroporated cells were plated on modified PW agar plus kanamycin.…”

Characterization of Regulatory Pathways in Xylella fastidiosa : Genes and Phenotypes Controlled by gacA

“…Two different asymmetric PCRs were performed to generate fragments to the left side (primers GacAA and GacAB) and right side (primers GacAC and GacAD) of the gacA ORF (PD1984) (see Table S1 in the supplemental material). The left and right PCR fragments were mixed, denatured at 95°C for 5 min, and annealed at overlapping barcode regions (indicated with italics in Table S1 (7) were prepared according to published procedures (15). One to two micrograms of pUC19842 DNA in a volume of 5 l was electroporated into the cells under the conditions described earlier.…”