Strain Level Identification of Pseudomonads Using Denaturing Gradient Gel Electrophoresis of 16S-23S Spacer Region rDNA

Abstract

Specific Pseudomonas strains were detected by PCR amplification of the 16s-23s rDNA spacer region followed by denaturing gradient gel electrophoresis (DGGE) to generate DNA banding profiles. In initial studies, two diverse sequence areas within the 165-23s rDNA spacer region were located in five closely related Pseudomonas Ruorescens and P. putida strains. DNA banding profiles of 16 different pseudomonads were generated using PCR primers flanking this region, followed by DGGE of the PCR products. Distinct banding profiles were observed for each strain, and specificity could be increased by designing additional primers within the spacer region. A specific primer (613-1) was used to selectively amplify and detect a plant-disease suppressive bacterium, P. Ruorescens strain 613, in soil. Six field soils from different locations were used with the 613-1 primer to test the specificity of this technique. Five soils did not yield any gel bands, but one soil led to a faint 260-bp band, similar in size to that of P. Ruorescens 613. Resolution of strain 513 from the indigenous strain in this soil was achieved by DGGE of the amplified DNA fragments. The results therefore demonstrate the utility of PCR-DGGE analysis for strain-specific identification of pseudomonads in environmental samples. (Received January 17, 2000 ; Accepted May 10, 2000)