Newly developed system based on multiple enzyme restriction fragment length polymorphism – an application to proteolytic bacterial flora analysis

Abstract

To assist assignment of bacterial phylogeny based on multiple enzyme restriction fragment length polymorphism (MERFLP) analysis, new programs were developed; one program was used to construct reference database of theoretical multiple enzyme restriction fragment (MERF) data, which were edited from DNA sequence of various kinds of bacteria; the other program was used to search the theoretical MERF having the highest similarity to the measured MERF based on pairwise distance according to Nei and Li. Using proteolytic bacteria (96 strains) isolated from various field soils and 25 reference strains, the factors that affected accurate phylogenetic estimation were evaluated. Bacterial strains from the genus Bacillus were successfully separated into species using the MERF with HaeIII and HhaI as the restriction enzymes. Genera Xanthomonas, Pseudomonas, and Alcaligenes were affiliated with the corresponding genus, but precise affiliation under genus level was not always possible. Differentiation among genera in the Enterobacteriaceae may be possible using the other restriction enzymes (HhaI, RsaI and ScrF1). The method was found to be useful for the analysis of bacterial flora because the differentiation, grouping and phylogeny assignment of bacteria, including various genera, were easily accomplished without preliminary information. Most (97.8%) of the proteolytic bacteria isolated from field soils applied with chemical fertilizer or no fertilizer were affiliated with Bacillus spp., whereas the ratios were low in those isolated from field soils applied with liquid livestock feces.