STABLE ELECTROTRANSFORMATION OF SYMBIONT CANDIDATE DIAZOTROPHIC BACTERIUM WITH PLASMIDS CARRYING SELECTABLE AND SCREENABLE MARKER GENES

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 de velopment of such artificial systems require a reliable genetic transf ormation method for the introduction of marker genes into symbiont can didates. The performance of electroporation was evaluated using pJB3 ( 4.8 kb), pBI121 (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 azoto bacters and their electroporation (18 kV/cm electric field strength, 5 ms time constant, 0 degrees C) provided up to 6.8x10(5) transformants per mu g plasmid DNA, which is about 10(3) times the transformation e fficiency achieved in control experiments. No electrotransformants wer e obtained with the 24-kb pFAJ31.2. The size of plasmid DNA did not si gnificantly affect the efficiency of transformation. Transformants wer e able to grow at antibiotic concentrations that were 100-200 times gr eater than the lowest amounts that completely inhibited the growth of wild-type bacteria. A constitutive expression of gusA gene was observe d in transformants with the CaMV 35S promoter-gusA fusion containing p BI121, while lacZ expression was not detected under the control of the lac promoter in pJB3 transformants. Electroporated plasmids were reis olated from transformants in their original form, while non-transforme d 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-fi xing 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.