Site-directed gene disruption in Xylella fastidiosa.
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.
da Silva Neto
José F
JF
Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-900, SP, Brazil.
Koide
Tie
T
Gomes
Suely L
SL
Marques
Marilis V
MV
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*
D017931:DNA Primers; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S