Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is

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S Duret1, J L Danet, M Garnier

  • 1Laboratoire de Biologie Cellulaire et Moléculaire, INRA et Université Victor Segalen Bordeaux 2, 33883 Villenave d'Ornon Cedex, France.

Journal of Bacteriology
|December 22, 1999
PubMed

Insights

Homologous recombination successfully inactivated the Spiroplasma citri motility gene (scm1) using a replicative plasmid. This gene disruption did not affect pathogenicity, suggesting motility is non-essential for this bacterium.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Spiroplasma citri is a mollicute with potential gene disruption challenges.
  • Homologous recombination is a key mechanism for genetic manipulation.
  • Understanding gene function requires effective gene inactivation strategies.

Purpose of the Study:

  • To investigate the feasibility of homologous recombination for targeted gene inactivation in Spiroplasma citri.
  • To assess the role of the scm1 gene in Spiroplasma citri motility and pathogenicity.
  • To evaluate the utility of Spiroplasma citri oriC plasmids as tools for genetic manipulation.

Main Methods:

  • Construction of recombinant plasmids containing an internal scm1 gene fragment.
  • Electrotransformation of Spiroplasma citri with replicative and non-replicative plasmids.
  • Analysis of plasmid integration into the host chromosome via homologous recombination.
  • Phenotypic characterization of gene-disrupted mutants, including motility and pathogenicity assays.

Main Results:

  • A replicative plasmid (pCJ32) derived from Spiroplasma citri oriC plasmid pBOT1 enabled successful transformation of S. citri.
  • Plasmid integration occurred through homologous recombination at the oriC region or the scm1 gene.
  • Disruption of the scm1 gene resulted in a non-motile Spiroplasma citri phenotype.
  • The scm1-disrupted mutant retained pathogenicity and was efficiently transmitted to plants.

Conclusions:

  • Spiroplasma citri oriC plasmids are effective tools for promoting homologous recombination and specific gene disruption.
  • Motility is not essential for Spiroplasma citri pathogenicity.
  • This study provides a novel method for genetic manipulation in Spiroplasma citri, potentially applicable to other mollicutes lacking functional RecA protein.

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