Transient Transformation of Frankia by Fusion Marker Genes in Liquid Culture

Abstract

Frankia is a nitrogen-fixing actinobacterium that establishes root nodule symbiosis with actinorhizal plants. The molecular basis of the symbiosis is largely unknown because genetic manipulation of Frankia has not been feasible. In this study we made novel technical attempts to transform Frankia strain CcI3. We generated fusion marker genes consisting of a tetracycline resistance gene with a high codon usage similarity to Frankia's and promoters of the strain's translation initiation factor 3 gene. We flanked the fusion genes with genomic sequences from strain CcI3 in the expectation that they would be integrated into the targeted site by homologous recombination. We introduced the transformation constructs into Frankia cells by electroporation and selected transformants in liquid media. The growth of antibiotic resistant cells was dependent on the presence of construct DNA. PCR analysis of the genome and reverse transcription-PCR analysis confirmed that the marker genes were introduced into the cells. Integration of the marker genes into the chromosome by homologous recombination did occur, but at a low frequency. Most of the constructs were not integrated into the chromosome and existed as degraded molecules in the cells. Marker genes declined in the transformant population during maintenance, showing that the transformation was unstable.

Isolation of Mutants of the Nitrogen-Fixing Actinomycete <i>Frankia</i>

“…Frankia CcI3 cells grown in BAP-T medium (14) were collected from 80 mL culture by centrifugation at 25,000× g and resuspended in 7 mL BAP-T. Hyphae were homogenized by forced passage through a 21G needle (TERUMO, Tokyo, Japan) six times. We added ethyl methanesulfonate (EMS) to 1 mL cell suspensions to final concentrations of 1, 2, 4 or 8% and incubated them for 9 min.…”

The Determinants of the Actinorhizal Symbiosis

“…Transformation system is essential for both strategies. In spite of numerous trials over time (9,24,49,69,70,95), nobody has succeeded in transforming Frankia so far, which remains a major hurdle to identify nodulation genes in Frankia.…”

“…However, it is not clear whether they were true transformants because the resistant strains were not characterized at the molecular level. Recently, Kucho et al employed several new technical attempts to transform Frankia strain CcI3 (49). They assumed that antibiotic resistance genes generally used in bacterial transformation are not expressed efficiently in Frankia cells because of differences in promoter sequences and/or codon usage frequency since Frankia genomes are extremely rich in GC (79).…”

Identification by Suppression Subtractive Hybridization of Frankia Genes Induced under Nitrogen-Fixing Conditions

“…1B). We isolated total RNA from the 6-dat cells by the cetyltrimethylammonium bromide method (12) and analyzed the expression level of the nifH gene encoding dinitrogenase reductase by quantitative reverse transcription-PCR (qRT-PCR; see Table S1 in the supplemental material for the primers used) using SYBR Premix Ex Taq II (Perfect Real Time; Takara Bio, Shiga, Japan) and a 7300 real-time PCR system (Applied Biosystems, Foster City, CA). We observed drastic upregulation of the nifH gene under the N Ϫ condition (Fig.…”

“…For a better understanding of nitrogen fixation, the function of the genes we identified here should be analyzed by disruption by the transformation method currently being developed in our laboratory (12).…”