Stable electrotransformation of symboint candidate diazotrophic bacterium with plasmids carrying selectable and screenable marker genes

Abstract

Nitrogen-fixing symbioses had been established between the originally asymbiotic soil bacterium Azotobacter vinelandii CCM289 and different lower and higher plant species. Better characterization and further development of such artificial systems require a reliable genetic transformation method for the introduction of marker genes into symbiont candidates. The performance of electroporation was evaluated using pJB3 (4.8 kb), pBl121 (12.8 kb) and pFAJ31.2 (24 kb) plasmid DNAs containing selectable (Ap, Km, Tc) and screenable ( gusA, lacZ) marker genes. The adapted methods for the preparation of transformation-competent azotobacters and their electroporation (18 kV/cm electric field strength, 5 ms time constant, 0°C) provided up to 6.8 × 10 5 transformants per μg plasmid DNA, which is about 10 3 times the transformation efficiency achieved in control experiments. No electrotransformants were obtained with the 24-kb pFAJ31.2. The size of plasmid DNA did not significantly affect the efficiency of transformation. Transformants were able to grow at antibiotic concentrations that were 100–200 times greater than the lowest amounts that completely inhibited the growth of wild-type bacteria. A constitutive expression of gusA gene was observed in transformants with the CaMV 35S promoter- gusA fusion containing pBl121, while lacZ expression was not detected under the control of the lac promoter in pJB3 transformants. Electroporated plasmids were reisolated from transformants in their original form, while non-transformed bacteria did not contain indigenous plasmids. PCR amplification and Southern DNA blot hybridization showed the integration of plasmid DNA into the host genome as well. Transformants retained their nitrogen-fixing ability and had normal morphological and growth characteristics. Experimental findings proved the stable maintenance of plasmid DNA in azotobacters, making possible the routine transformation and detection of these symbiont candidates.