Stable Transformation of the ActinobacteriaFrankiaspp.

ABSTRACTA stable and efficient plasmid transfer system was developed for nitrogen-fixing symbiotic actinobacteria of the genusFrankia, a key first step in developing a genetic system. Four derivatives of the broad-host-range cloning vector pBBR1MCS were successfully introduced into differentFrankiastrains by a filter mating withEscherichia colistrain BW29427. Initially, plasmid pHKT1 that expresses green fluorescent protein (GFP) was introduced intoFrankia casuarinaestrain CcI3 at a frequency of 4.0 × 10−3, resulting in transformants that were tetracycline resistant and exhibited GFP fluorescence. The presence of the plasmid was confirmed by molecular approaches, including visualization on agarose gel and PCR. Several other pBBR1MCS plasmids were also introduced intoF. casuarinaestrain CcI3 and otherFrankiastrains at frequencies ranging from 10−2to 10−4, and the presence of the plasmids was confirmed by PCR. The plasmids were stably maintained for over 2 years and through passage in a plant host. As a proof of concept, a salt tolerance candidate gene from the highly salt-tolerantFrankiasp. strain CcI6 was cloned into pBBR1MCS-3. The resulting construct was introduced into the salt-sensitiveF. casuarinaestrain CcI3. Endpoint reverse transcriptase PCR (RT-PCR) showed that the gene was expressed inF. casuarinaestrain CcI3. The expression provided an increased level of salt tolerance for the transformant. These results represent stable plasmid transfer and exogenous gene expression inFrankiaspp., overcoming a major hurdle in the field. This step in the development of genetic tools inFrankiaspp. will open up new avenues for research on actinorhizal symbiosis.IMPORTANCEThe absence of genetic tools forFrankiaresearch has been a major hindrance to the associated field of actinorhizal symbiosis and the use of the nitrogen-fixing actinobacteria. This study reports on the introduction of plasmids intoFrankiaspp. and their functional expression of green fluorescent protein and a cloned gene. As the first step in developing genetic tools, this technique opens up the field to a wide array of approaches in an organism with great importance to and potential in the environment.