Related Experiment Videos
Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is
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.
Abstract:
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.
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.