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Journal of Bacteriology, December 1999, p. 7449-7456, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Gene Disruption through Homologous Recombination in
Spiroplasma citri: an scm1-Disrupted Motility
Mutant Is Pathogenic
Sybille
Duret,
Jean-Luc
Danet,
Monique
Garnier, and
Joël
Renaudin*
Laboratoire de Biologie Cellulaire et
Moléculaire, INRA et Université Victor Segalen Bordeaux
2, 33883 Villenave d'Ornon Cedex, France
Received 10 June 1999/Accepted 27 September 1999
To determine whether homologous recombination could be used to
inactivate selected genes in Spiroplasma citri, plasmid
constructs were designed to disrupt the motility gene scm1.
An internal scm1 gene fragment was inserted into plasmid
pKT1, which replicates in Escherichia coli but not in
S. citri, and into the S. citri oriC plasmid
pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the
nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In
contrast, spiroplasmal transformants were obtained with the
replicative, pBOT1-derived plasmid pCJ32. During passaging of the
transformants, the plasmid was found to integrate into the chromosome
by homologous recombination either at the oriC region or at
the scm1 gene. In the latter case, plasmid integration by a
single crossover between the scm1 gene fragment carried by
the plasmid and the full-length scm1 gene carried by the
chromosome led to a nonmotile phenotype. Transmission of the
scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector
(Circulifer haematoceps) showed that the motility mutant
multiplied in the insects and was efficiently transmitted to plants, in
which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal
motility may not be essential for pathogenicity and that, more broadly,
the S. citri oriC plasmids can be considered promising
tools for specific gene disruption by promoting homologous
recombination in S. citri, a mollicute which probably lacks
a functional RecA protein.
*
Corresponding author. Mailing address: Laboratoire de
Biologie Cellulaire et Moléculaire, INRA et Université
Victor Segalen Bordeaux 2, B.P. 81, 33883 Villenave d'Ornon Cedex,
France. Phone: (33) 5.56.84.31.51. Fax: (33) 5.56.84.31.59. E-mail:
renaudin{at}bordeaux.inra.fr.
Journal of Bacteriology, December 1999, p. 7449-7456, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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