Exploitation of Denaturing Gradient Gel Electrophoresis in Analysis of Microbial Diversity

Abstract

An Application of denaturing gradient gel electrophoresis of PCR-amplified hydrogenase gene fragments were its best to determined the genetic diversity of Desulfovibrio species in environmental samples. Comparative analysis of [NiFe] hydrogenase gene sequences were designed with five different PCR primers. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. For Desulfovibrio species only one primer pair was found to be specific, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.