title: Site-directed gene disruption in Xylella fastidiosa.
abstract: As a first approach to generate mutations by DNA insertion, we have developed a shuttle vector, called pSP3, which replicates both in Escherichia coli and in Xylella. Vector pSP3 was constructed by ligating to the E. coli plasmid pBluescript, a kanamycin resistance gene under the control of the Xylella 16S rRNA promoter and the indigenous Xylella plasmid pXF1.3. Transformation experiments have shown that pSP3 replicates stably in Xylella. When a DNA fragment encompassing part of the Xylella xpsD gene was cloned into pSP3, specific integration of the construct into this gene was observed in 10% of the transformants, as early as after two passages of the culture. These results indicate that this vector can be used to generate site-specific gene disruption by homologous recombination in Xylella fastidiosa.
authors.affiliation: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-900, SP, Brazil.
authors.forename: José F
authors.initials: JF
authors.lastname: da Silva Neto
authors.forename: Tie
authors.initials: T
authors.lastname: Koide
authors.forename: Suely L
authors.initials: SL
authors.lastname: Gomes
authors.forename: Marilis V
authors.initials: MV
authors.lastname: Marques
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000235:genetics; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D015183:Restriction Mapping; D014974:Xanthomonas / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S