Use of a microchip electrophoresis system for estimation of bacterial phylogeny and analysis of NO3- reducing bacterial flora in field soils

Abstract

Phylogenetic estimation method without determination of DNA sequence was developed. By this method, fragment length polymorphism separately digested with multiple restriction enzymes was measured using microchip electrophoresis and affiliated with those calculated from corresponding DNA sequence in the theoretical database. The phylogenies of 129 NO3- reducing bacteria newly isolated from field soils were estimated by this method, and were compared to those by carbon source utilization profiles and by comparative sequence analysis of 16S rDNA. Various bacteria such as Micrococcus sp. (one isolate), Acidovorax delofieldii (one isolate), Cupriavidus necator (one isolate), Burkholderia sp., (seven isolates), Commamonas acidovorans (two isolates), Herbaspirillum seropedicae (two isolates), Ralstonia sp. (six isolates), Pseudomonas spp. (14 isolates), and Acinetobacter spp. (one isolate), were affiliated as similar to those by sequence analysis of 16S rDNA, while exact affiliation of genus was difficult for those belonging to Enterobacteriaceae.