A COMPUTER-SIMULATED RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS OF BACTERIAL SMALL-SUBUNIT RIBOSOMAL-RNA GENES - EFFICACY OF SELECTED TETRAMERIC RESTRICTION ENZYMES FOR STUDIES OF MICROBIAL DIVERSITY INNATURE

An assessment of 10 tetrameric restriction enzymes (TREs) was conducte d by using a computer-simulated restriction fragment length polymorphi sm (RFLP) analysis for over 100 proximally and distally related bacter ial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logist ic simplicity, commercial availability, and economy. However, the rati onale for selecting the type and number of TREs has not been systemati cally evaluated. Our objective nas to identify the optimal combination of TREs for RnP screening of cloned SSU rRNA genes from undefined bac terial clone libraries. After computer-simulated TRE digestion, the re sultant fragments were categorized on the basis of the frequency of di fferent restriction fragment size classes. Three groups of distributio n patterns for the TREs were determined and further examined via graph ical exploratory data analysis. The RFLP size-frequency distribution d ata for each group of enzymes were then used to infer phylogenetic rel ationships via the neighbor-joining method. The resulting bootstrap va lues and the correct placement of node bifurcations were used as addit ional criteria to evaluate the efficacy of the selected TREs. These RF LP data were compared with known phylogenetic relationships based on S SU rRNA sequence analysis as defined by the Ribosomal Database Project . A heuristic approach testing random combinations of TREs showed that three or more TRE combinations detected >99% of the operational taxon omic units (OTUs) within the model data set. OTUs that remained undete cted after three TRE treatments had a median sequence similarity of 96 .1%. Of the 10 restriction enzymes examined, HhaI, RsaI, and BstUI (gr oup 3) were the most efficacious at detecting and differentiating bact erial SSU rRNA genes on the basis of their ability to correctly classi fy OTUs. Group 3 TREs are therefore recommended for screening in studi es using bacterial SSU rRNA genes as descriptors of in situ microbial diversity.